'COPYRIGHTED •
by
PAUL FUGAZZOTTO

1947

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

:J.%LOLCGIÜ

ÜF LlBClLiTUIcY

bv

PAUL FUCb/^LOTTO

YLÜlo
S üb mi Lt ed t o t h e J c h c o l o f ü r a d u a t o s t u d i e s o f L i c h i ^ a n
S t a t e C o lleg e o f A g r i c u l t u r e and A pplied S cience
I n P a r t i a l F u l f i l l m e n t o f t h e irequirements
i o i ' t h e DefU'ce o f

DOCTOR OF PHILLSOPHY
D e p a r t m e n t o f B a c t e r i o l o g y and f u b l i c H e a l t h

- 1947 -

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

The w r i t e r i s d e e p l y g r a t e f u l

to the

ic h ip a n d ep ai'tao n t o f H ealth

L a b o r a t o r i e s f o r p l a c i n g i t s lomy f a c i l i t i e s a t h i s d i s p o s a l ^ i n g e n e r ­
o u s s u p p o r t , o f a i s w o r k ; an d Lo h i s maiiy a s s o c i a two Lli.^re f o r t h e i r c n couraging cooperation in ob tain in g the

to r i a l s

reouired.

E specially ^

i s t h e w r i t e r g r a t e f u l t o D r , H. L, a e n d r i c k ( u n d e r whose d i r e c t i o n
i.oich o f t h i s i n v e s t i g a t i o n was c a r r i e d o u t ) a n d t o D r , G. h i d e r i n g ,
both a t the

. e s c o r n i . i c h i g a n D i v i s i o n o f t h e ..Michigan D é p a r t i r e n t o f

H ealth L a b o ra to rie s,

f o r t h e i r i n t e r e s t i n t h e p r o b l e i a an d f o r t h e i r

s u g g e s t i o n s a.nd c r i t i c i s e s

re;-ardinp th e u n n u s c r i p t .

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CONTiiUTS
Page

1.

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3.
3.
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HISTUlilO/iL 3 AoI 3 PCP TE3 STUOY
G eneral divergence of opinion i n th e e n t i r e f i e l d
I n c o n s i s t e n c i e s ajaong r e p o r t s r e g a r d i n g h . per t u s s i s
i n c o m p le m e n t ";Lo:ition

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h e a g e n ts , and t i t r a t i o n s
P r o c e d u r e f o r s e l e c t i o n o f t h e " p r e l i m i n a r y " comislement
P r o c e d u r e f o r s e l e c t i o n o f t h e " s t a n d a r d " com ple me nt
T e c h n i c o f t h e " r o u t i n e " complement f i x a t i o n t e s t
The c o n t r o l s o f t h e com ple me nt f i x a t i o n s y s t e m

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A p p l i c a t i o n o f t h e co m p lem en t s e l e c t i o n p r o c e d u r e s
R e l a t i v e s e n s i t i v i t y o f t h e " p r e l i m i n a i g ' " an d " s t a n d a r d " p r o ­
cedures
E f f e c t o f s e l e c t e d co m b le m e n t on t h e comp leme nt f i l i a t i o n s y s te m
The " a n tic o m p le m e n ta r y '' e f f e c t o f a t y j i c a l a n t i g e n on d i f f e r ­
e n t l o t s o f com pl em e n t,
I f f c e t o f a n t i a e n c o n c e n t r a t i o n on s e n s i t i v i t y o f t h e complement
fix atio n te s t.
s e n s i t i v i t y o f com ple me nt f i x a t i o n co m p a r e d t o a g g l u t i n a t i o n
i n t e s t s on a n i m a l s e r a
T e s t s on l a b o r a t o r y a n im a ls o t h e r th a n g u i n e a p i g s

Z1.5.

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rr.;'LïOüUGTiŒT

The in v e s f c i p j' a ti o n s l e a d i n g t o t h e

u r e v e n t r e p o r t were b e p u n i n t h e

e a r l y p a r t o f 1939 vjitli t h e p u r p o s e o f s tu d y in g ; iimaune r e s p o n s e s t o
rieiiLüuiiilu^ p e r t u s s i s hy üiêaiiS o f cGvoplcrocnt f i x a t i o n .

f J T t c r o n l y a fev;

w ee ks o f p r e l i i a i n a i y w ork , i t was r e a l i s e d t h a t " s t a n d a r d i s e d ” c o n d i t i o n s
co u ld n o t e a s i l y he m a in ta i n e d from tim e t o ti m e , le n d i n g added s i g ­
n i f i c a n c e t o t h e s t a t e m e n t made a b o u t two y e a r s l a t e r by F l o s d o r f e t a l ,
(24) w i t h r e g a r d t o co m l e m e n t f i x a t i o n i n p e r t u s s i s :
s u c h a t e s t h a s n o t b e e n d e v e l o p e d . ” : a n d by Lapin. (5 2)

t i i a t ”-----------in 1943, t h a t

t h e c o n t r a d i c t i o n aiaong t h e a u t h o r s w i t h r e g a r d t o s e r o l o g i c t e s t i n g
"

m e r e l y s e r v e s t o e m p h a size t h e n e c e s s i t y f o r s t a n d a r d i z a t i o n o f

p r o c e d u r e s i n a g g l u t i n a t i o n a n d com ple me nt f i x a t i o n . "
couraging is

h v e n more d i s ­

t h e f a c t t h a t t h e s e s t a t e m e n t s - t o t h e w r i t e r ' s k n o w le d g e -

are s t i l l tru e ,

some 40 y e a r s a f t e r 3 o r d e t a n d Gengou (5) a n n o u n c e d d i s ­

c o v e r y o f t h e c a u s a t i v e a g e n t o f w ho opi ng c o u g h .
J u d g i n g f r o m the r e p o r t s i n t h e l i t e r a t u r e ,
perience,

an d f r o m p e r s o n a l e x ­

i t was a p p a r e n t t o t h e w r i t e r t h a t e f f o r t s d i r e c t e d t o w a r d t h e

s t a n d a r d i z a t i o n o f a coi.iplement f i x a t i o n

p r o c e d u r e h a d b e e n b l o c k e d by

a n i n t e r f e r i n / ; p r i n c i p l e c o m p l e t e l y h id d e n among t h e i n t r i c a c i e s

of the

s y s t e m ; end i t was c e r t a i n t h a t no p r o g r e s s c o u l d p o s s i b l y be e x p e c t e d
b e f o r e t h i s p r i n c i p l e c o u l d be b e t t e r u n d e r s t o o d a n d c o n t r o l l e d .

In

f o ll o w i n g th e o n ly p a t h s o f tijought opened by t h e d a t a a l r e a d y acc u m u la te d ,
an d t h e a t t i t u d e s o f a p p a r e n t l y w e l l i n f o r m e d i n v e s t i g a t o r s , a f u r t h e r
Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

“ 2 —

s c o u r i n g o f t h e l i t e r a t u r e se eme d p r a c t i c a l l y s u p e r f l u o u s s i n c e i t
l e n g t h e n e d th e l i s t

o f c o n f l i c t i n g r e p o r t s , w ith o u t o f f e r i n g any d e f ­

i n i t e c l u e s f o r th e s o l u t i o n of th e proble;as In v o lv e d .
g r e a t volu m e o f p u b l i c a t i o n s ,

In th e a l r e a d y

e s p e c i a l l y on l a b o r a t o r y s t u d i e s w i t h

11. p e r t u s s i s , t h e r e was ;

1.

A marked d i v e r s i t y in r e s u l t s

o b t a i n e d by v a r i o u s i n v e s t i g a t o r s

e m p l o y i n g t h e same or s i m i l a r p r o c e d u r e s f o r t h e s t u d y o f t e s t
m a t e r i a l ( s e r a and a n t ig e n s ) .
2.

^-1 l a c k o f c o r r e l a t i o n among t h e r e s u l t s o f s e v e r a l t e s t p r o ­
c e d u r e s em p lo y eu i n i n d i v i d u a l i n v e s t i g a t i u u a .

3.

A l a c k o f u n i f o r i i i i t y i n t h e u s e o f one p r o c e d u r e by i n d i v i d u a l
i n v e s t i g a t o r s o v e r a p e r i o d o f t im e.

It

i s e v i d e n t f r o m t h e a b ov e t h a t t h e e f f e c t o f a n unknown p r i n c i p l e

was i n v o l v e d n o t o n l y i n t h e s e r o l o g y o f p e r t u s s i s ,
t h a n t h o s e o f a p u rely s e r o l o g i c a l n a tu r e .
fore,

but a ls o in phases o th e r

I t se em e d t o t h e w r i t e r ,

th ere­

t h a t th e m a tte r of prim ary im portance to th e serolo;;y o f p e r t u s s i s

i n p a r t i c u l a r , a n d t o t h e p e r t u s s i s p r o b l e m in g e n e r a l , was n o t t h e p u b ­
l i c a t i o n o f a d d i t i o n a l d a t a on immune r e s p o n s e s , b u t r a t h e r a n e x p l o r a ­
t i o n i n t o t h e .meohanisriis o r p r i n c i p l e s r e s p o n s i b l e f o r t h e s e i n c o n g r u i t i e s ,
f f f o r t s d i r e c t e d t o w a r d t r i i s en d w e r e f i n a l l y r e w a r d e d i n t h e a c c u m u la t io n
o f t h e d a t a t o be p r e s e n t e d h e r e ; d a t a w h ic h s u g g e s t e d t h e n e c e s s i t y o f
s u b j e c t i n g to r ig o r o u s s c r u t i n y ,

t h e s e r u m o f a l l a n i m a l s ’' d ii c h v/ould i n

a n y way be in v o l v e d i n s t u d i e s w i t h K. p e r t u s s i s .

S c a tte r e d throughout

a l l t h e p e r t u s s i s l i t e r a t u r e was e v i d e n c e i n s u p p o r t o f t h i s c o n c l u s i o n ,
h id d e n t h e r e by c e r t a i n m a s k i n g f a c t o r s .

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

H iarO RICÆ L P .J 3 I S FOR TPD: STUDY

G e n e r a l d i v e r g e n c e o f o p i n i o n ixi t h e e n t i r e f i e l d .

L o s t o f t h e i n v e s t l e a t o r s s i n c e 1906 h a v e b e e n i n a c c o r d v :i th
B o r d e t a n d h i s co- '.v or ke rs on, some o f t h e b a c t e r i o l o g i c a l a s p e c t s o f
wlioopin,3 - c ough :
T h a t H. p e r t u s s i s
That

is th e e t iologic agent of t h i s d isease,

t h e organism

p a t i e n t s mo st

o fte n in

is

the presence

iso latin g

and o i a i n t a i n i n g

state

(6),

(5),

(8),

p r e s e n t i n t h e s p r a y o f v;hoopin.g-cough

t h e e a r l y vreens o f t h e d i s e a s e , a n d r a r e l y

a f t e r t h e f o u r t h o r f i f t h meek;
That

(3)

of

(3 ),

(9 )

b l o o d i n t h e medium i s v e r y e s s e n t i a l f o r

th e organism in a v i r u l e n t u n d i s s o e i a t e d

(10),

O t h e r p h a s e s i n t h e b a c t e r i o l o g y o f II. p e r t u s s i s , h o w e v e r , h a v e
b een t h e c au se of d iv e r g e n t o p i n i o n .

For i n s t a n c e , G h i e v i t z and iie y e r

( 1 1 ) i n t r o d u c e d t h e c o u g h p l a t e method o f d i a g n o s i s , w h ic h h a s f o u n d a
c o n s i d e r a b l e nuraber o f p r o p o n e n t s s u c h a s Gardner ( 3 1 ) , Go ld & L e l l
G ajza?o & G ottche (2 8 ), Soare & T urai
a n d many o t h e r s ,

( ' ’1 ) , D onald ( I 6 ) , S a u e r (?6 ) ,

e s p e c i a l l y among h e a l t h r o r k e r s who u s e

t h e method d a i l y .

Banks ( 4 ) , and L a c l e a n ( 3 5 ) , h a v e n o t b e e n so w e l l s a t i s f i e d
l e a s t u n d er c e r t a i n c o n d itio n s,
th e use o f n a s a l
I n a d d itio n

(33)»

finderson ( l ) , and F i l l e r

w ith i t ,

at

(61), favored

swabs i n c e r t a i n i n s t a n c e s .
to t h i s ,

co n sid era b le cause for concern

has been found

i n t h e p r e p a r a t i o n o f t h e medium t o b e u s e d f o r c u l t u r e o f t h e o r g a n i s m :

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-

4 -

w h i l e m o s t i n v e s t i o a t o r o hr-vc f a v o r e d t h e u s e o f f r e s h b l o o d s i e d i u r j
S h i v a e t a l ( 7 7 ) , - rurie.-iede e t a l
favored use o f the

(4 7 ) an d - h s h u l o v . e t a l ( 6 ;>) h a v e

" c h o c o la te " ' ' b l o o d l a e l i u m .

u s e d ' . . h a t e v e r b l o o d seemed c o n v e n i e n t
ox, e t c . ) ;

b u t -hshulov; e t a l

;..oct i n v e s t i g a t o r s ha ve

( h o r s e , siuman, r a b b i t , s h e e p ,

(o3) p r e f e r r e d , h o r s e b l o o d t o liuinan ( 6 4 ) ,

v ù : i l e on t h e o t h e r h a n d , ' .'oomey 1 h a h a c s

( 8 7 ) an d ..u e t a l ( 9 2 ) h a v e

s h o .n d e c i d e d oh n re s i n t ' . i e i r o u lu u i ': .s f r o . . us:e o f h o r s e I ' l o o d .
I'hey, t h . a r o f o r e , n r . - . f e r r o d hui,ian o r Sfioen b l o o d .

In Che 3e r o l o . f i c o l and ii;Lüunolo;,;ical a s p e c t s , t h e e x p e r i e n c e s have
b e e n e p u a l l y ci I v o r . s i f l e d , bu
cussed in d iv id u a lly .
a : follov.'S :

_ riefly ,

t h e r e p o r t s a r e to o nu merous t o be d i s ­
t h e s i t u a t i o n w h i c h e x i s t s ca n b e s t a t e d

..■"aile tfie r e s n o n s e s o f humans a n d o t h e r a n i m a l s t o

.. p e r t u s s i s

a n t i g e n s i n one form 0 .’ a n o t h e r , have b e e n s t u d i e d by cor.iple. .e n t f i x a t i o n ,
a g g l u t i n a t i o n , a n i m a l — u s u a l l y , mouse — p r o t e c t i o n , n eut r a i l z a t i o n ,
o p s o n o c y t o p h a r i c , and. s.kin t e s t s , or by t h e a b i l i t y o f th e o rg a n ism t o
cause s ic k n e s s or d e a th ,

t h e r e h a s b e e n no u n a n im ity o f o p i n i o n ajiicuig

i i v o s t i gators as t o th e v a l u e e i t h e r o f th e o e tiio d s , o r of t h e r e s u l t s
o b ta in ed ,

zhi.-'t.hen.'iore, w h i l e some h a v e c la im e d good c o r r e l a t i o n among

t h e r s u i t s o f two or
stand.

:orc o f t h o s e m e th o d s o t h e r s h av e ta k en t h e o p p o s i t e

A s t u d y o f t h e c h r o n o l o g i c a l o r d e r i n : .h ic h t h e v a r i o u s r e p o r t s

hav e a p p e a r e d i.n t h e l i t e r a t u r e i n d i c a t e s t h a t t h e r e hoc b e e n no g e n e r a l
t r e n d toward a c c e p t a n c e o f one method i n p r e f e r e n c e t o a n o t h e r .
seems t o b e no i n d i c a t i o n

There

th a t any one p r o c e d u r e h as become so w e l l

d e v e lo p e d w i t h t h e p a s s a g e o f t i m e , t h a t i t c o u l d c l a i m 'w id er u s a g e th an
e v e r b e f o r e , and
is

_ e r i t h i g h e r th a n any o t h e r s i m i l a r p r o c e d u r e .

ra th e r sur-prising,

in view o f th e f a c t

Th is

t h a t a t t h e v e r y b e g i n n i n g and

i n the e a r l y y e a r s o f t h e m o r k , b o r d e t and h i s co-workoh's gave r a t h e r
co m p r e h e n s iv e a c c o u n t s o f t h e r e s u l t s

t o be e x p e c t e d i n most p h a s e s o f

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

- o s t u d y •.'.Itli d . p e r t u s s i s ;
d i s y s t u d i e d t h e l e t h a l e f f e c t s oT cf.e o r y i n i e m f o r l a b o r a t o r y
ani:iial.s ( 5 ) , .a n d a u r y e s t e d tiio p r e s a n c e o f a p e r t u s s i s t o x i n ( 5 ) ,
( b ) , a h i c h -.;as s e n s i t i v e t o h e a t , and n o t a r . t i . y e n i c ( 7 ) .
They s t u d i e d r e s p o n s e s t o t h e o r y a n is is by a y y l u t i u a t i o n , and
coj'iplei.iont f i x a t i o n

(b) , ( h ) ,

( 1 0 ) , a n d by s k i n t e s t s

f h e y sho'/r ;d t h e p r e s e n c e o f r e a c t i x

(7 ).

s u b s t a n c e s i n tie:-: s er u is o f

n o r n a l h o r s e ; ; , î ' a b b i t s and p u i n e a p i p s

and i n j e c t e d a n i m a l s x l t h

suspensions of orpanisna to produce a n tio e r a fo r th e s tu d y of the
a n t i p end c pr up ^^r ti e.^ 'uf d i f f - r o ^ t o t n i n e
f h e y m e n tio n e d one e f f e c t
infection

(6 ),

(1 0 ) .

o f i n .une serui.i on t h e c o u r s e o f t h e

( 6 ) a s w e l l a s e h e e f f e c t o f .media ( 6 ) ,

(8),

( 1 0 ) a n d immune

s e r u m (6) on t h e organism i t s e l f .
Of t h e many a u t h o r s

.ho have e x e r t e d t h e i r e f f o r t s t o e l a b o r a t e on t h e work

o f t h i s g r o u p , c o m p a r a t iv e ly fev; nr.ve a d d e d a n y t h in .; v i t a l t o a b e t t e r
under^'.tanding^ e s p e c i a l l y o f t h e a n t i g e n i c n a t u r e ,
when u sed i n v i t r o .

\ather,

o f t h e p e r t u s s i s organ ism

t h i . s aocur.iulati.ng l i t e r a t u r e h a s ,

i n i t s .many

d i v e r g e n t o p i n i o n s , p r e s e n t e d many d i s t u r b i n g i n c o n s i s t e n c i e s t o t h e f i e l d .
C e r t a in

a u t h o r s h a v e f e l t t h a t t h e y c o u l d on t h e b a s i s o f t h e i r o..n

work,

explain

some o f t h e i n c o n s i s t e n c i e s w hich h ave a r i s e n ; and i t mould

perhaps

b e i n o r d e r t o mive a f e n e x a m p l e s .
In t h e use o f a n a g g lu t in a t io n t e s t ,

f o r i n s t a n c e , .urnhGim(2) p o i n t e d

o u t t h a t f a l s e negau-ive t e s t s may be due t o a p r o z o n e phenomenon, s i n c e
a g g l u t i n a t i o n i s h i n d e r e d i n lo.-; d i l u t i o n s o f s e r u m ,

f o r d e t & Cle esw yk

IC) a l s o m e n t i o n e d a s i m i l a r phenomenon due t o a n t i - a g g l u t i n i n s .

Auto-

a g g l u t i n a t i o n o f a n t i g e n s h a s b e e n found a d i s t u r b i n g f a c t o r by Odaira
( 6 7 ) , I x c i s b e n s e n ( 46), L a p i n e t a l

(48)

and many o t h e r s .

O d a i r a (6 7)

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-6 -

t r e a t e d t h e a n t i g e n s t o r e d u c e a u u o - a a r l a t i a a t i o n , ' a i l e a a n e e n (3 7)
s u r e s -red t h e n e c e s s i t y o f us l a y o n l y s u s p e n s i o n s . / h i c h do n o t a u t o a g g lu tin a te .

- l o s i o r f et a l

o r - ' a n i s n s ; a n d 41 H e r e t a l
strain s.
tests

(2 4 ) c l a i r . e d b e s t r e s u l t s v : i t h l i v i n g

(62 ) r e q u i r e d a :f i x t u r e o f 3 t o 5 f r e s h

I 'u r t h e n .io r e , s h e t i n e and t e u p e r a s u r e o f i n c u b a t i o n os' t h e

h a v e 3ee:eed t o be i u o r t a n t f a c t o r s d e t e i u i i n i n - t h e s u c c e s s o f t h e

te st.
.is f o r CO sole'::out f i x a t i o n , J l u s t s a i

buttin p er

a t e d a l a r g e nuiaber o f f a c t o r s v:hicli t h e y n a v e f e l t

(69) have eriuise:/-

c o u l d h a v e been,

r e s non s i b l e f o r so.ne o f t h e d i s c r e p a n c i e s , and added t h a t “jln e x p la n ­
a t i o n o f sOi 13 d i s c r e p a n c i e s i s s t i l l
s i d e r e d so.-ie f a i l u r e s

t o be f o u n d ."

G-isse ( 7 2 ) c o n ­

due t o t h e a . a o u n t s o f coippleriieiib a n d a n t i g e n u s e d ,

v r i i l e d- ajzago & G o t t c h e ( 2 3 ) c l a im e d t h a t a r e l i a b l e a n t i g e n i s d i f f i ­
c u lt to obtain,

h is h u lo u et a l

( 6 6 ) a t t r i b u t e d d i s c r e p a n c i e s i n com­

p l e m e n t f i x a t i o n r e s u l t s t o t h e me th od o f t e s t i n g ,

a n a a d m it t e d t h a t

t h e i r o..u m ethod mas su ch a s t o r e s u l t i n a t e s u o f lorn s e n s i t i v i t y .

f h e o p s o n o c y to p h o g ic t o s t h a s b e e n fou n d by 'Glynn 1 Cox ( 3 d ) ,
and oinpsr-...rooks n i . i l l e r
of e rro r;

(7 9)

t h e number o f c e l l s

t o b e c o m p l i c a t e d by s e v e r a l s o u r c e s
counted,

th e age a n d c o n c e n t r a t i o n o f

t h e a n t i g e n s u s p e n s i o n and c e r t a i n p h y s i c a l f a c o o r s i n h a n d l i n g t h e
r e a g e n t s u s e d ; t h e p r e s e n c e o f norm al o p s o n i n s , v a r i a t i o n s i n t h e
le u co cy te consent of th e blood,

fh e sk in t e s t ,

etc.

b o th i n humans and i n l a b o r a t o r y a n i m a l s , v/as c o n ­

s i d e r e d by r a t e r s o n e t a l (71) t o b e l i m i t e d b y unkao’. n f a c t o r s i n t h e
a n t i g e n , s i n c e r e s u l t s seem ed t o v a r y n l t h

the l o t of in j e c t io n m a teria l

used.

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

_

7-

- h e p a s s i v e mouse p r o t e c t i o n t e s t , a c c o r d i n g t o L a p i n ( 4 9 ) , i s
cojipjlicated. by an u n a c c o u n t a b l e v a r i a t i o n in v i r u l e n c e o f th e c u l t u r e s .
'.ishulO'.'.r e t a]. ( 6 6 ) c o n s i d e r e d t h e

intram uscular ro u te fo r ad m in istra­

t i o n o f t h e t e s t s e r u ia a s a n i m p o r t a n t f a c t o r i n a v o i d i n g t h e " n o n ­
sp ecific ' re su lts

r e p o r t e d by I r t h i e s on 1 H a r k

(57)

i:c t e s t s v / i t h

n o r m a l a n d n o n - p e r t u s s i s immune a n i m a l s e r a .

mil e s c a r e o n l y a f e n o f t h e e x p l a n a t i o n s , o f f e r e d b y v a r i o u s i n ­
v estig ato rs,

for the

inconsistencies

s u f f i c i e n t in them selves
all

t]ie i n t r i c a c i e s

in rep o rted r e s u l t s ,

but th e y a re

to em phasize th e f a c t t h a t f u l l r e a l i z a t i o n of

of the v a rio u s t e s t

s y s t e m s h as o f t e n b e e n l a c k i n g .

T h i s i s e s p e c i a l l y t r u e o f t h e com plem ent f i x a t i o n t e s t , b e c a u s e o f i t s
v e r y i n v o l v e d n a t u r e ; and a s r e s u l t o f t h e v ; r i t e r ’ o e x p e r i e n c e w i t h t h i s
system ,

i o i s nov; p o s s i b l e t o o f f e r a n o t h e r e x p l a n a t i o n f o r some o f t h e

d ifficu lties

encountered d uri n : the co u rse of p re v io u s in v e s t ig a t io n s

n o t o n l y i n c o m p le m e n t f i x a t i o n , b u t p erh a p s i n e t h e r f i e l d s a s w e l l .
I n m a r t i a l s u p p o r t o f t h e w r i t e r ’ s sta tem en t i s t h e f a c t t h a t most
in v e s tig a to rs have,

a t some t im e d u r i n g t h e c o u r s e o f t h e i r work

( e s p e c i a l l y i n c o m p le m e n t f i x a t i o n ) ,

considered t h e i r antigens resp o n s­

i b l e f o r th e i n c o n s i s t e n c i e s in t h e i r r e s u l t s .

By s o d o i n g ,

th e em phasis

o f t h e i r w o rk h a s b e e n w e l l p l a c e d , b u t a p p a r e n t l y n o t w i d e l y e no ug h
a p p l i e d s in c e c e r t a i n problem s have s t i l l rem ained u n so lv e d .

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-8 -

l a c o n c i s t e n c i e s euiionç r e p o r t s r e g a r d i n s K. p e r t u s s i s a n u i g e n s i n
com p le m e n t f i x a t i o n .

Of t h e many P u b l i c a t i o n s r p o r t i n g e x p e r i e n c e s w i t h complement
fixation

in p e r tu s s is

s t u d i e s , v e r y few ha ve p i v e n d e s c r i p t i o n s o f

■iiethods anu r e a g e n t s u s e d ; and o f t h e s e ,

no ne c o u l d be c o n s i d e r e d s o

c o m p l e t e a s t o be r e p r o d u c i b l e i n e v e r y r e s p : : c t .

f h is la ck of re p ro ­

d u c i b i l i t y r e s u l t e d i n a s t a t e m e n t made i n 1914 by . . i l l i a m s ( 9 0 ) , w h i c t i ,
it

app ear.'^, i s a t i 11 a p n l i c s b l e t o d a y ;

"

t h a t many o f t h e s e m e t h o d s

do n o t s t a n d t h e t e s t o f s u f f i c i e n t c o n t r o l s . "
buted th i s

to e c c e n t r i c i t i e s

of th e a n t i g e n s ,

She a p p a r e n t l y a t t r i ­
s i n c e s h e m e n t i o n e d " -------

a h y p o t h e t i c ferm en t w h i c h seems t o make t h e a n t i g e n u n s t a b l e . " an d
t r i e d s e v e r a l methods o f p r e p a r a t i o n t o surmount t h i s
also,

gave no ne o f t h e p a r t i c u l a r s ,

obstacle.

She,

however.

The p r e p a r a t i o n o f t h e t e s t a n t i g e n s has a lw a y s b e e n a c a u s e f o r
c o n c e r n ; and o n l y a f ew o f t h e a u t h o r s h a v e a t t e m p t e d t o d e s c r i b e t h e
r e a g e n t w h ic h t h e y h a v e c o n s i d e r e d s a t i s f a c t o r y .
o f i - e i e r ( 5 8 ) , and h o l l s t e i n

A s id e from t h e r e a g e n t

( 9 1 ) , who u s e d , among o t h e r t h i n g s , e x t r a c t s

o f t i s s u e s from f a t a l c a s e s o f w h o o p in g - c o u g h , t h e s e a n t i g e n s may b e c l a s s i ­
f i e d u n d e r two m a i n t y p e s ;
1;

i i a l i n o s u s p e n s i o n s o f v a r i o u s l y t r e a t e d w h o le o r g a n i s m s i n v a r y i n g
concentrations,

and o b t a in e d from growth on v a r i o u s l y p r e p a r e d

m edia.
2,

E x t r a c t s o f w h o le o r g a n i s m s .

I n some c a s e s , t h e a u t h o r s h a v e s p e c i f i e d t h e t e c h n i c s which t h e y h a v e
c o n s i d e r e d e s s e n t i a l to t h e p re p a r a tio n o f a s a t i s f a c t o r y r e a g e n t .

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

Y et,

-9 -

t h e r e lias b e e n no t e n d e n c y anion.;; o t h e r i u v e s t i g a t o r s t o a d o p t one m e t h o d
i n p r e fe r e n c e to sny o th e r .

JOllovi::.g i s a n i l l u s t r a t i o n o f t h i s .

ils r e s u l t o t t h e i r i n v e s t i g a t i o n s ,

tu n n e l & o c h lu te r

(56) a d v o c a t e d

u s e o f a l e c i t h i n i z e d a l c o h o l e x t r a c t o f h . p e r t u s s i s a ; b e i n g more
s p e c i f i c , v i h i l e 01 ah 1 i h i n s a p i (6 {) u s i n g a s u s p e n s i o n o f -.hole o r g a n i s m s ,
c la im e d t h e r e i s no a i v a n ta g e i n s u c h a n e x t r a c t .
oth er hand, re v o n -e d

L an in (fS)

on t h e

t h e c o n c l u s i o n o f Oj.ah ic ^..unsagi (6 :) by s u p p o r t i n g

u s e o f t b o l e c i t h i n i z e d a l c o n o l e x t r a c t a n t i g e n o f n e i c h s e l L D o u g la s
( 89 ) c l a i : .. i n h , a s d i d a l s o dun d e l e t a l

(36) t h a t a s im p le s u s p e n s i o n

o f 11. p e r t u s s i s i.s u n s u i t a b l e b e c a u s e i t ; ; i v e s f r e q u e n t n o n s p e c i f i c
and an t i Go ::ipl ement a r y r e a c t i o n s .

I n p a r t i a l d is a g r e e :,le n t v i t h b o th s i d e s ,

t h e v o r k o f O l m s t e a a - v o v i t s k y ( 7 0 ) i n d i c a t e d t h a t t h e r e i s n o method
v ai ic h c a n d e f i n i t e l y a s s u r e one o f a s a t i n f a c t o r y a n t i g e n ,
is

s i n c e "There

s u c h g r e a t i n d i v i d u a l v a r i a t i o n i n a n t i ;ens o f t h e same s t r a i n made

by t h e same m eth o d , t h a t co m p a riso n o f methods i s d i f f i c u l t . "
s t a t e m e n t , th o u gh c o r r o b o r a t i n g t h a t o f

.illiam s

T h is

(9 0 ) c e r t a i n l y does

' n o t c l e a r t h e c o n f u s i o n ; and t h e p o i n t t o be d e r i v e d f r o m t h i s exam ple
i s t h a t r e g a r d l e s s o f t h e i n d i v i d u a l e x p e r i e n c e s and n r e f s r e n c e s ,
r e p o r t e d in th e l i t e r a t u r e , th e j. a in d i f f i c u l t i e s

e n c o u n te re d i n th e

com plement f i x a t i o n s y s t e m h a v e b e e n i n t h e b e h a v i o r o f t h e t e s t a n t i g e n s ,
f u r t h e r a n a l y s i s of th e so r p orts i n d i c a t e d t e e d i f f i c u l t i e s
some may c o n n e c te d n i t l i t h e a n t i c o u p le m e n t a r y a c t i v i t i e s

to be in

o f tn e^ e a n t i g e n s .

Though most o f t h e r e p o r t s i n t h e l i t e r a t u r e f a i l t o make a n y d e s c r i p t i o n
w h a t s o e v e r o f t h e a n t i ens u s e d , t h o s e o f L o r d e t â G e n g o u ( 3 ) , a n d r a t o n
( 7 2 ) c l a i m t h e i r a n t i g e n s -.rare n o t a n t ic o m p le m c n t a r y .

Dvans & ' a i t l a n d

( 1 7 ) , and ( ? i e s e ( 3 2 ) c o k p o r s a t e d f o r " s l i g h t " a n t ic o m p lsmon t a r y p r o p e r t i e s
by t h e a d d i t i o n o f an e x t r a s m a l l amount o f com p lem en t, v h i l e 01mst e a d

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

—1 0 —

ô; L u t t i n ^ e r ( 6 9 ) , Lonr; « l l i s 2 ( $ 4 ) , L^ishulov/ e t a l
a d ju '.'ted th e system by eiayloyiny l / j t o l / 4
o f a n :ig e n in th e t e s t s .

( 6 $). a n d otliarw

o f t h e a n tic o m p le m s n ta ry dose

O l m s t e a d d P o v l t s k y ( ?0 ) l a t e r

presented the

n o v e l i g e a o f c o m p a r i n g t h e a n t i - c o i . : p l e m c - n t a r y t o t h e c o m p le m e n t f i x i n g
potencies

of an a n t i g e n t o d e te rm in e a n u m e ric a l "Index o f F i x a t i o n "

f o r th 's s u i t a b i l i t y o f an a n t i g e n ,

f l i i s l a t t e r j.iethod h a s a p p a r e n t l y

n e t b e e n n i d e l y a d o p t e d , non e v e r ; an s. , j iid. ;in g Croi.i t h e . .o r ns o f
L f u i i o a d i t e ( p 6 ) , ..- .i lie r ( 6o ) , Lou y & B l i s s

(p4) a n d o t h e r s , t h e p r o ­

c e d u r e g e n e r a l l y e m p lo y ed h a s b e e n t o p r e p a r e t h e a n t i g e n s b y i.ii a t e v e r
m es h oa seem ed s u i t a b l e o r c o n v e n i e n t l.u Lh'- i n v e s t i g m t o r , and t o t i t r a t e
them d a i l y .

Ir

o th e r m ords, t h e s e i n v e s t i g a t o r s have a p p a r e n t l y a c c e p te d

th e a n tic o m p le m e n ta ry p r o p e r t y as sn i n h e r e n t , u n a v o id a b le c h a r a c t e r i s t i c
o f m. p e r t u s s i s a n t i g e n s , a n d h a v e u s e d t h i s c h a r a c t e r i s t i c a s t h e p o i n t
o f r e f e r e n c e f r o m v;hieh t h e s u i t a b i l i t y o f a n a n t i g e n c o u l d b e j u d g e d .
I n Vi e u o f t h e e x p e r i e n c e s i n p r e l i m i n a r y w o r k , t h e w r i t e r rias f e l t ,
hO', e v e r , t h a t s u c h a b a s i s f o r tb,e s e l e c t i o n a n d t i t r a t i o n o f an a n t i g e n
i s n o t .'.el], fo u n d e d .

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-1 1 -

?R.HD.:riLRY .011': o::

i i v .i f i o .:

rj;:.: ii. P ii.-n -siiis

Du r in ' : ühu c o u r s e oi' c o n s i à e r a h l s c r e l i i . i i n a r y s t u d y ,
fix âtio u

s ” cte:s oas p u t t o t

t h e coi.'Lçlenent

o t ou ...ore t h a n 1^00 " e r a f r o i i hui.iaiis o f

v a r y i n : a y e ar.â p e r t u s s i s h i s t o r y .

An a n a l y s i s o ' o one o f t h e r e s u l t s

c b i a i n c d was i n c l u d e i Ir. r e s o r t s l y . . o n d r i o k ( i n ) ,

on one o f t h e

. ' T i t e r v i i t h sosie o f t h e n o t a r i a l r e p a i r e d i n h i s cu.niplei.ient f i x a t i o n
v;crk).

n o t i n d i c a t e d i n ^ o n d r ic k 's r e p o r t s ,

i s th e fa c t th a t th e

CO. y l e i . e n t f i x a t i o n t e s t on s g r a o f iiMUirized s u b j e c t s u a s p o s i t i v e as
lo n .s

as I t

contrast

t o 22 ...onths ( l o n y e s t p e r i o d t e s t e d )

t o t h e r e t o r t s o f Sin-yer-..-.rooks e t a l

(50) , L a p in e t a l

after

i..niunination, in

( 79 ) , -lajzayo et a l

( 29),

( 4 ? ) , L a p i n (50) > ( 5 2 ) , ( 5 3 ) , r.-ho c l a i a e d t h a t t h e

COMp l a n e n t f i x a t i o n t e s t

i^. g e n e r a l l y n e g a t i v e

cy t h e t i . i r d t o t h e e i g h t h

..lonth.

A l s o n o t i n d i c a t e d a r e t h e r e s u l t s o f t e s t s vihich a e r e n o t d i r e c t l y

related

t o L e n i r i c k ' : - -" or k , a n d o f o t h e r s r h i c h n a d t o be d i s c a r d e d b e c a u s e

of sporadic in c o n s is te n c ie s

i n th-.

behavior of the cosiplerent f i x a t i o n system .

-LVidence o f t h e s e i n c o n s i s t e n c i e s l i e

in the fa c t t h a t oftentim es th e re

n a s a n i n c r e a s e i n p o s i t i v i t y o f t h e s rr u- o v;ir,h i n c r e a s e i n s e r u m d i l u t i o n ;
or the r e s u lt s

o f r e p e n t e s t e s t s on i n d i v i d u a l s e r a c o u l d n o t b e d u ; l i c a t e d .

A f t e r r e p e a te d e x p e r ie n c e v;ith t h i s

ty p e o f b e h a v i o r , an a n a l y s i s of

the r e s u l t s rev ealed c lo se c o r r e la tio n b etseen

the occur.'ences o f th e

i n c o n s i s t e n c i e s an d t h e a n t i c o m p l e m e n t a r y a c t i v i t y

of the a n tig e n s .

Lot

o n l y x e r e suspensions of v a r i o u s l y t r e a t e d organisms o r t h e i r e x t r a c t s a t t i m e s
m a r k e d l y anticO i. wls m ent ar; !/'; t h e a n t i c o n . . l e m e n t a J . - y p r o p e r t y of a n y g i v e n l o t
of antigen seemed t o f l u c t u a t e froi.i time t o tinie.

Lome o f t h e a n t i g e n s

made a v a i l a b l e t l i r o u y h t h e kindness of mrs. Kendrick and

Ideriny a t t h e

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

"12—

W e s t e r n ü i c h i':&n d i v i s i o n oi' t h e i . d c h i a n .Liepartmont oi' H e a l t h L a b o r a t o r i e s
i n Uranii / r a p i n e , Lao b e e n i n t h e r e f r i g e r a t o r f o r a s Ion/', a s 14 y e a r s ;
a n d i t seeziied t o t h e w r i t e r t h a t i f f l u c t u a t i o n s i n a n t i c o n u l c.iientary
a c t i v i t y v/ere clue t n c h a n g e s i n t h e a n t i p e n i t s e l f ,

th ese suspensions

certain ly ,

Y et, th e ir a n t i -

had had a/cple t i n e

GO’r c l e ; i i e n t a r y a c t i v i t y

to becone s t a b l i z e d .

f e l l one d t h e ea.ee u n d u l a t i o n s a s f r e s h l y p r e p a r e d

H c i t e r i a l , v ; i t h no p e n e r a l tr e n d i n a n y p a r t i c u l a r d i r e c t i o n .
t h i s a c t i v i t y - f a i l e d t o f i t any l o g i c a l p a t t e r n ;

I n so d o i n y ,

and t h e r e f o r e , c o u ld n o t

i n i t s e l f be c o n s i d e r e d a s a c a u s e o f t h e d i s t u r b a n c e s i n t h e a n t i g e n s .
It

seeitiod r a t h e r to b e a n e f f e c t , t h e c a u s e o f w h ich m u s t n e c e s s a r i l y l i e

e l s e w h e r e i n t h e complement f i x a t i o n s y s t e m .

I n t h a t c a s e , t h e o n ly hop e

f o r t'ne f u t u r e o f t h i s p roblem w o ul d c o n s i s t i n d e t e r m in in g t h e c a u s e ,
r a t h e r th a n a t t e m p t i n g t o m in im iz e i t s
in clin atio n .

To t h a t e n d , i t

e f f e c t , a s had been t h e g e n e r a l

seemed n e c e s s a r y f i r s t o f a l l t o e s t a b l i s h

a s t a b i l i z e d s t a r t i n g p o i n t and s i n c e t h e oor-pl-.Uiient a c t i v i t y o f f r e s h
g u i n e a pi; : serum was known t o d e p r e c i a t e c o n s i d e r a b l y i n a fev; d a y s , i t
seamed l o g i c a l t o b e g i n w ith t h i s r e a g e n t .
A r e p o r t on a s t u d y o f p r e s e r v e d complement b a s e d on t h e work o f
G i n s b u r g & î î a ,l i n i n (26 a) h a s a l r e a d y ap p eared i n t h e l i t e r a t u r e
and i n t h i s

(26);

s t u d y i t was f o u n d t h a t g u i n e a p i g serum , t o w h i c h 80 mg. o f

sodium c h l o r i d e a n d 40 mg. o f b o r ic a c i d c r y s t a l s ha d b een added f o r e a c h
uil . o f v o l u m e , r e t a i n e d a s a t i s f a c t o r y c o m p le m e nt a c t i v i t y f o r a s lo n g
a s t h r e e to fo u r /lo n th s.

'The u s e o f g u i n e a p ig se ru m s t a b i l i z e d i n t h i s

manner was a d o p te d f o r t h e w o rk w i t h H. p e r t u s s i s ,

b / i t h s u c h a complement

on h and, i t was now p o s s i b l e Lo g a i n a f u l l e r a n d a c l e a r e r c o n c e p t i o n
of th e so c a l l e d

" a n ticom p lem en tary" a c t i v i t y o f th ; a n t i g e n s ; a n d t h e

r e s u l t s o f f u r t h e r t e s t s b r o u g h t out th e f o l l o w i n g f a c t s :

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-1 3 -

1.

I h o antieoiiipleniontarv a c t i v i t y o f a n t i y e a s v a r i e d v:idely v;ith
d ir ’f e r s x i t l o t s o f com plem ent.

2.

j J i ' i e v e n t c u l t u r e s o f H. p e r t u s s i s d i f f e r e d i n a n t i c o m p l e m e n t a r y
a c i t i v i t y t o some e x t e n t , b u t r e t a i n e d t i i e same r e l a t i o n s h i p t o
e a c h o t h e r nh en t e s t e d x i t h d i f f e r e n t l o b s o f coi.iplement.

3.

ill e a n t i g e n s became

o r e and. more a n t i c omul œiien t a r y

.'1t h i n c r e a s e

i l l a g e o f t n e co ii m le m en t,
4.

I t v'as n o t p o s s i b l e t o s a t i s f y t h e a n t i c o . . . i p l e m e n t a r y a c t i v i t y
ox a n t i g e n s uy mixiii;:.; tneiii v.xtii a n àniO'Uiit o f c OiXi )1 Oniont o p u x v a l e n t
to th is a c t i v i t y

( i n a m e th o d s i m i l a r t o t h a t d e s c r i b e d a few

y e a r s l a t e r by f a r a n (84) f o r "'.'.asserman" t e s t s on a n t i c om ul em entary
sera).
5.

In ad d itio n to

(4) a b o v e ,

it

..as found t h a t t h e a n t i c o m p l e m e n t a r y

a c t i v i t y o f a n t i g e n s \ia.s e n h a n c e d by a n i n c r e a s e i n t h e amount o f
co m o lem en t s e r u m u s e l ( w i t h i n d e m o n s tr a b le l i m i t s ) .
6.

The a n t i c o i a p l e r a e n t a r y a c t i v i t y o f a n t i g e n s f o r c o n s t a n t amounts
o f com pl em en t rem ained f a i r l y c o n s t a n t o v e r v e r y w id e r a n g e o f
antigen d ilu tio n s ,

in c o n t r a s t t o th e a c t i v i t y o f a n t ic o m p l e m e n ta r y

s e r a , 'v h ic h d e c r e a s e s v e r y r a p i d l y w i t h i n c r e a s e i n d i l u t i o n .
T h ese r e s u l t s w ere v e r y s u g g e s t i v e o f a n t i g e n - a n t i b o d y r e a c t i o n , and
it

was c o n s i d e r e d p o s s i b l e t h a t t h e a p p a r e n t a n tic o m p le m e n t a r y p r o p e r t i e s

and t h e i n s t a b i l i t y o f a n t i g e n s w ere due r a t h e r t o t h e p r e s e n c e o f a n t i ­
b o d i e s i n t h e g u i n e a p i g comp leme nt s e r u m .

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-1 4 -

A lf^ I D O D Y C O Y C Z iP ? O F llT O I O O i.T L I F F l l A P Y A C T r / I ' f Y

I'he f a c t o r s v.hich m i l i t a t e d a g a i n s t o b t a i n i n g d e f i n i t e d i r e c t
p ro o f fo r t h i s an tib o d y concept of an ticom plem entary a c t i v i t y of
a n t i g e n s mere tw o fo ld :

1,

I n t h e e a i ’l y p a r t o f t h e \/ork, t h e i n i t i a l l a c k o f e x p e r i e n c e

i n t h e f i e l d n e c e s s i t a t e d r e l i a n c e upon t h e r e s u l t s a n d o p i n i o n s o f
Of a l l t h e r e p o r t s t h a t had come t o t h e

p rev io u s i n v e s t i g a t o r s .

w r i t e r ’s a t t e n t i o n , only m a t of : - i i l e r

(dG) m e n t i o n e d co:n l i c a t i o n s

i n t h e co.irolement f i x a t i o n s y s t e m i n v o l v i n g u s e o f g u i n e a p i g comp le ii i e n t c o n t a i n i n g a n t i b o d i e s
the in v e stig a to rs

(fo r B rucella b ro n c h is e p tic a ) . A ll

i n c l u d i n g : A l l e r ha d a p p a r e n t l y a s s u m ed t h e a n t i -

co m p lem entary a c t i v i t y o f p e r t u s s i s a n t i g e n s t o b e a n u n f o r t u n a t e ,
u n p r e d i c t a b l e c h a r a c t e r is t ic , e n t i r e l y s e p a r a t e from o th er a c t i v i t i e s ;
and t h e o n l y m e t h o d s s u g g e s t e d t o c o p e w i t h i t ,

when i t

e x i s t e d , w ere

e i t h e r t o i n c r e a s e t h e am ou nt o f c o r a p l e m e n t , o r t o d e c r e a s e t h e aiaount
of a n tig e n ,

iio on e,

till

t h e p r e s e n t t i m e , h a s s o much a s a t t e m p t e d

to p r e s e n t th e b a s is f o r such an a t t i t u d e to ./ard th e antico m p lem e n tary
a c t i v i t y of a n t i g e n s .
T jriter as a le s s
2.

T h i s was n a t u r a l l y r a t h e r m i s l e a d i n g t o t h e

inform ed w o rk e r.

On t h e o t h e r h and, when i t

had b e e n d e c i d e d by t h e w r i t e r t o

d i s r e g a r d t h e i m p r e s s i o n s l e f t by p r e v i o u s a u t h o r s , and t o d e v e l o p s
th e problem in d e p e n d e n tly ,
t h e g u in ea p i g s .

i t seeme d n e c e s s a r y f i r s t o f a l l t o p r e t e s t

This p resen ted s e r io u s t e c h n i c a l d i f f i c u l t i e s :

I n t h e wo rks o f l o r d e t and 31eesv/yk ( 8 ) , h r u n v / i e d e e t a l ,

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

(47)

-1 3 -

Tooriey 6 Talcacs ( 8 6 ) , j j i e n s t ( 13 ) , ..-.vans cc l . l a i t l a n d
c o u l d be s e e n f a v o r a b l e p o s s i b i l i t i e s
te st;

( I ' i ) a n d . .v an s ( 1 9 )

o f employing: a n a g g l u t i n a t i o n

b u t t h e a g g l u t i n a t i o n phenomenon v;as u n f o r t u n a t e l y f o u n d t o be

d efin itely

i n a d e q u a t e , a s i v i l l b e slio/ai l a t e r ,

I ' r o n t h e v;orks o f o p r u n t e t a l
and I v a n s 1 . l a i t l a n d

(32),

( 8 3 ) , C u lo tta e t a l

(12),

( l o ) ca;;;.e t h e s u g ; ; e s t i o n o f c u l t u r i n g j t a t e r i a l f r o m

t h e a i r p a s s a g e s f o r t h e p r e s e n c e o f I r . b r o n c h i s e p t i c a w h ich - h l l e r
has c o n s id e r e d r e sp o n sib le f o r such a n t i b o d i e s ,
o b je c tio n s
a culture

(6 0 )

H o w ev sr, b e s i d e s o t h e r

( t e c h n i c a l a n a t . n e o r e t i c a l ) , U ie r e i s no e v i d e n c e t h a t s u c h
co u ld g iv e any d e f i n i t e d i r e c t i n f o r m a t i o n r e g a r d i n g th e

p r e s e n c e or ab sen ce o f a n t i b o d i e s

i n th'. a n i r . i a l ' s s e r u m .

f o t e s t t h e g u i n e a p i g s by co ia ple m ent f i x a t i o n seemed t h e m o s t
n l a u s a b l e a l t e r n a t i v e ; b u t i n t h i s c a s e , t h e r e v/as n e e d f o r an " a n t i b o d y f r e e " convole ; e n t v i t h v.'hich t o t e s t t h e g u i n e a p i g s .
of course,

o u ch a cojiipleriient,

is not r e a d i l y a t t a i n a b l e w ith o u t f i r s t p r e t e s t i n g th e anim als

v h ic h a r e t o b e u s e d a s t h e s o u r c e o f s u c h a c o m p l e m e n t ,

dompleviont

f i x a t i o n procedures for p r e t e s t i n g guinea p i g s , w ith o u t use o f a p r e v io u s ly
sta n d a r d iz e d

c o m p l e m e n t , hav e b e e n s t u d i e d a n d d e s c r i b e d f o r t h e T'asserm an"

p r o c e d u r e s by h o l r n e r ( 4 3 ) , H a r r i s ( p P ) , A u s t i n ( 3 ) , G iord ano & C a r l s o n ( 33 ) >
Diamond ( 1 4 ) , and . . a d s w o r t h ( 8 3 ) ,

f h e s e m e t h o d s , h o w e v e r , h a v e th e

fo llo w in g o b je c tio n s :
They em plo y a s y s t e m o f " a u t o - f i x a t i o n " ;

and s i n c e t h e co m p le m e n t

a c t i v i t y o f i n d i v i d u a l guinea p ig s vary g r e a t l y , th e in d i v id u a l sera
a r e n o t t e s t e d un'ier i d . m t i c a l - o r s t a n d a r d i z e d - c o n d i t i o n s ,
ih ir th e r m o r e , t h e r e i s no e v i d e n c e s u p p o r t i n g t h e a s s u m p t io n t h a t
t h e r e a c t in .: s u b s t a n c e ( a n t i b o d y , r 'la g in ) i n any g i v e n g u i n e a p i g
se r m i i s n e c e s s a r i l y ca p a b le o f f i x i n g i t s

hom ologous co.rplem ent

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

-lb -

i ll t h e p r e s e n c e o f t h e

b s t aatif^en.

^11 s o , t h e pj.iount o f co-aplament

' p r e s e n t i n t h e p;uitiea p i p scrum , a t t h e d i l u t i o n s u s e d , J.in;' be so
i i i p h as t o :iin'-;e a p o s i t i v e

(fixation)

r e a c tio n im 'o ssib le , thereby

le a d in y . one t o a f a l s e s e n s e o f c o n f i d e n c e i n t h e s e r a c o n s i d e r e d
s a t i s f ctory.

In vie:.' o f t t e s e ina'-LehuaciGs, o t h e r ..Letliods o f p r e t o s t i n . ; _,ere
s o u , : h t , j,iethods d e v o id a s much a s p o s s i b l e , o f assumed f a c t o r s , a . _d
r i g i d l y c o n t r o l l e d i n e v e r y is s p e c t .
on a s ü ia ll group o f g u in e a p i g s ,

i. r e p o r t on t h e r e s u l t s o f t e s t s

omployii^j an e a r l i e r p r e te s t in g ,, ..istnod

h a s a l r e a d y b e e n iiade by t h e m r i t e r ( 2 ? ) .

f h i s '.vorlc s e r v e d a s im p etu s

t o a o r t . c o n c e n t r a t e d e f f o r t s r h lc h r e s u l t e d i n :
.levelop m on t o f r e p r o d u c i b l e , s t a n d a r d i z e d p r o c e d u r e s b o th f o r
pretestin.

t h e s e r a o f g u in e a yigis t o b e u s e d f o r tiie complement

s u p p l y , and f o r d e t e c t i n g h . p e r t u s s i s a n t i b o d i e s i n r o u t i n e
couple: lent f i x a t i o n t e s t s .
r e a l i z a t i o n o f t h e im p o rta n ce o f u n d e t e c t e d ,

" n o n sp ecific"

a n t i b o d i e s i n l a b o r a t o r y a n i m a l s o t h e r t h a n »;uinoa p i g s , and t h e i r
p o s s i b l e e f f e c t on some o f t h e ivork t h a t has a p p e a r e d i n t h e
lite r a tu r e .

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

—1 7 “

-iquirment and s u p p l i e s

'-esid e s th e u s u a l t e s t t u b e s ,

r a c k s a .id o t h e r e-quiuiiient n e c e s s a r y

f o r p e r f o r m a n c e o f s e r o l o ' i c . a l t e s t s — t h e d.ahn t e s t ,
v:as f o u n d c o n v e n i e n t and m or s a c c u r a t e ,

fo r instance-,

it

ever s in c e the very beginning

o f t h i s '.cork, t o efi' rl oy b.r,i t - t i p p e d p i p e t t e s l i k e t h e o n e d e s c r i b e d a n d
ill.u strated

i n 1943 b;/ k ap p ap o rt & - f e p n a p o r t ( 7 3 ) »

These can e a s i l y

be made b y h e a t i n g t h e t i p o f t h e a p p r o p r i a t e p i p e t t e

(O.Oh m l , , 1 . 0 m l . ,

and 2 . 0 ml) s u f f i c i e n t l y t o a l l o v b e n d in g a t no more t lia n 3 ma. from
th e end.

fh e a n g l e o f th e b e n t t i p

s h o u l d bo a p p r o x im a t e l y a t 4 3 ° n i t h

t h e a x i s o f th e p i p e t t e ; an d f o r t h e r i g h t - h a n d e d o p e r a t o r , d i r e c t e d
t o t h e r i g h t i.nen t h e p i p e t t e i s h e l d i n p o s i t i o n f o r u s e .

. . e a g e n t s and t i t r a t i ons

b io tilled
baline:
A ntigen:

..a t e r k e p t i n th e r e f r i g e r a t o r ,

0 . 9'/: NaCl i n d i s t i l l e d w a t e r .
A s a l i n e s u s p e n s i o n o f a s m o ot h ( " P h a s e I " )

H. p e r t u s s i s grown on e c r d e t - G c n g o u medium.

It

culture of

i s once-washed; k i l l e d

w i t h h e a t , an d em pl o ye d i n a c o n c e n t r a t i o n o f 20 b i l l i o n / i t i l . or l e s s
do s e n d i n g on a n t i g e n i c a c t i v i t y a s d e t e r ; , l i . i e d by t i t r a t i o n .
fm tigen titr a tio n :
of 2 , 4 , 6 , 8 , e t c .,

I n d e t e r m in in g a n t i g e n i c a c t i v i t y ,

suspensions

b i l l i o n / m l are prepared; 0 .1 ml. o f eac h i s added

t o 0 . 0 4 i:il. o f a p o s i t i v e serum i n s e r i a l d i l u t i o n i n Kuhn t e s t t u b e s ;
2 . 0 u n i t s o f " stan d ard " com p lem ent, and 0 . 1 m l. o f s a l i n e a r e added t o
e a c h m i x t u r e , and th e t u b e s a r e p l a c e d I n t h e r e f r i g e r a t o r f o r f i v e h o u rs,
fJ ien , t h e y a r e p l a c e d a t room tei;m erauure f o r 13 m in u t e s ; 0 . 2 m l . o f

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-

se n siü iz e l c e lls

is e i le l;

37° 3 . f o r 15 n i n u t e s

18 -

t h e t u t s a re irL su b atel 1:: Li

r.ri i n s p e c t e d ,

3e

tite r

Liber be.r.;i r.b

s e l e c s e d i s t h e lo v ;e s t

c o n c e n t r a t i o n c a n c b lo o f -inxi.nurn f i x a t i o n , p l u s one b i l l i o n ;
r o r d s , i''" an - b i l l i o n / : : : ! , s u s p e n s i o n and ab ove i s
fix a tio n ,
I t i:

in oth er

csn-'oble o f i.ia%imui'i

o'iS t e s t t i t e r i s a 9 b i l l . i o n / n l . s u s p e n s i o n .
nou l ecesjr^.r;- t o u-r: n -' ' o i ' - y r , i e n t

ban d ha s a l l t h :

: : ii : :t n r s i f

c b r z ' a c n c r i s t i c s o f n Siiootb: (".fnnne I " )

d ie s t r a i n on
culsnre.

jfLso,

i t s e e n s b e s t bo s t o r e t h e s o o s l o f .ar.tijo-n a t 4 - 8 ° 0 i n an;: c o n v e n i e n t
concentration

i,lUU û i ± l i o n / 8 u . o r n o r e j ; t n e r e o u i r s n d i l u t i o n c a n be

c a d e as n e e d e d .
Ooinple:,ie:.t :

fh e

- u i n e a p i p s t o be u s e d f o r c o n n l e i . i o i i t , p r e f e r a b l y

s h o u ld be b e t v 's e : : 3 n e e d s a n d 3 n o n t b s o f a p e ( 1 ,9 0 -3 uO y r a i . i s ) ,

fdey a r e

b l e d t o t h e d e s i r e d e n t e n t , and by t h e : lethod c o n s i d e r e d :.iOst c o n v e n i e n t ,
fh e i n d i v i d u a l speci:;ie is ar:

p l a c e d i n s e s a r a t s c o n n a i.n e r s , l o f t a t

r o o n t e is p e r a t u r e f o r a n h o u r , and a r e th e n t r a n s f e r r e d t o t h e r e f r i y e r a t o r
a t 4 - 8 ° G.
As s o o n as p o s s i b l e

( - s i t u i n 11-1.3 h o u r s )

a f t e r th e g u in e a p i p s a r e

b l e d , t h e s e r a n u 3 t b e i n d i v i d u a l l y t e s t e d by t h e a p p r o p r i a t e c o n o le tio n t
s e l e c t i o n p r o c e d u r e t o be d e scrib ed l a t e r ,

t h e s a t i s f a c t o r y s e r a p o o le d

a n d t h e co: v l e o i o n t o f t h e p o o l e d so rue. s t a b i l i s e d by a d u i t i o i i o f 80 :ny.
sodi'urri c h l o r i d e a n d 40 n;-;. b o r i c a c i d c r y s t a l s f o r each a l . v o l u r i e ( 2 o ) .
Avery p r e c a u tio n i s t a k e n throughout o v e r ^ s t e p t o a v o i d u n n e cessa r y r i s e
i n t h e te ic n e r a t u r o o f t h e s e r a t h a t a r e t o s e r v e a s com p lem ent.

In preparin'^

t h e complement f o r u s e , t h e s t a b i l i z e d ser um i s d i l u t e d 1 : 1 0 v.ût’i d i s t i l l e d
m a t e r , an d f u r t h e r d i l u t i o n s a r c made r i t h
Comple::ieut t i t r a t i o n :

salin e.

A. s e r i e s o f 25 t u b e s i s

a rra n g ed ( i n a l\ahn r a c k )

c o n t a i i - i i r i f i n c r e a s i n g amo un ts o f s a l i n e i n in c r e m e n ts o f 0 . 0 2 m l . ; t h a t i s ,
0 . 0 2 , 0 . 0 4 , 0 . 0 6 , O.OB, e t c . p r o c e d i n g from t u b e 1 t o t u b e 2 5 .
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-1 9 -

lo

e a c h t u b e i'- added 0 . 2 ::il. oh a 1 : 2 0 d i l u t e d s t a b i l i z e d coi.iulenient ( n r e -

p a r e d b;^ z d x i n p 0 . 3 u l .
and 3 .0 m l.
d ilu tio n

of s a l i n e ) ,

o f t h e coiunlement seruin 'v ith 2 . 7 m l . o f d i s t i l l e d v:ate:
.-ifter t h o r o u g h n i x i n g , 0 . 1 ra l. o f t h e r e s u l t i n '

i n each o f t h e 23 t u b e s ,

in a second s e r ie s

is

t r a n s f e r r e d t o a. c o r r e s p o n d i n g t u b e

c o n t a i n i n ' 0 . 2 ml,, o f s a l i n e .

i s added 0 . 2 m l . o f s e n s i t i z e d c e l l s ,
shaken,

a f t e r u h ic h t h e r a c k o f tu b e s i s

an d p l a c e d i n a 37 ° 0 . a a t e r b a th

I'he coi.'iplemt'nt u i t e r i c

f o r 13 m i n u t e s .

i i i o i c a t c ; d oy

vfiioh t h e r e i s c o m p le te h e m o ly sis,

do e a c h o f t h e l a t t e r

tziS m - cn e s' t numzexGu t u b s m

fhe t i t e r -

r e p r e s e n t e d in th e tubes

( 1 - 2 3 ) a r e 1 : 1 1 , 1 : 1 2 , 1 : 1 3 , e t c . t o I : ' 5; and a t t h e t i t e r d i l u t i o n
0 . 1 m l . c o n t a i n s 2 . 0 c o m p le m e n t ' u n i t s .
F or c o n v e n ie n c e in p e rf o rm in g t h e

tests,

t h r e e co m pl em en t d i l u t i o n s

a r e r e q u ir e d .
D ilution I:

0 . 1 nil. c o n t a i n i n ' " 2 u n i t s
( Complement a t

D ilution I I :

D ilu tio n I I I :

its

tite r)

0 .1 ml. c o n ta in in g 1 .3

u n its.

(3 p a r t s o f D i l u t i o n I ,

plus 1

o fsalin e)

0 . 1 m l . c o n ta in in g : 1 . 0 u n i t .
(1 p a r t o f D i l u t i o n I ,

-kaboceptor:

part

plus 1 p a rt of sa lin e )

A n t i s h e e p r e d c e l l r a b i t serum , t i t r a t e d f o r t h e

h i g h e s t s a l i n e d i l u t i o n c a p a b l e o f maximum s e n s i t i z a t i o n o f v;ashed sh eep
red c e l l s .
■Sheep c e l l s :

a 3)4 s a l i n e s u s p e n s i o n p r e p a r e d from a p a c k e d voluise

o f t h r e e t i m e s m a shed s h e e p b l o o d .
S en sitized c e lls :

f h e r e q u i r e d v ol u m e o f t h e a m b o c e p t o r a t i t s

t i t e r i s p o u r e d i n t o a n e q u a l volume o f t h e v;3.shed s h e e p c e l l s u s p e n s i o n .
The m i x t u r e i s a g i t a t e d g e n t l y f o r a f e v s e c o n d s , a n d a ll o w e d t o s t a n d
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-2 0 -

15 m i n u t e s \ ; i e h o c c a s i o n a l a g i t a t i o n b e f o r e u s e .
J e s t serum:

-ne e l s r s e r u n n e a t e t a t 5 6 ° ü . f o r JO .m inutes,

P r o c e d u r e f o r s e l e c t i o n o f t h e ’' r r e l i m i n a r v " c o ; i i~ le s e . a t.

I n s he a b s e n c e o f a s a t i s f a c t o r y
necessary f i r s t

of a ll,

means o f n h i c h t h e s e r a

to o b ta in a
to r the

("g
p r s l i m i n n r y " co: . e l e m e n t , by

''s t a n d a r d " r e a y e n t can be s e l s c t s d .

X--S s o o n a s t h e ssruj.i b e y i n s t o s e p a r a t e f r o m t h e c l o t t e d g u i n e a
p i r b lo o d specim ens o b ta in e d as a l r e a d y d e s c r i b e d

( s e e n o a g e n t s ) , an

a p p r o x im a te ly 0.5 m l. sample o f each i s

tr a n s f e r r e d to o th er tubes,

and c l e a r e d by minimal c e n t r i f u g a t i o n ,

h n a c t l y 0 .1 m l. o f each c l e a r

eeruin i s t r a n s f e r r e d t o a c o r r e s p o n d i n g t u b e i n s e r i e s , an d p l a c e d
iiaiiiO dia te ly i n t h e r e f r i g e r a t o r a t 4 - 3 ° 0 . :
w ith 5 .5 ml. s a l i n e

(1:56)

(th ese a re to be d il u te d

im raediately b e fo re u s e ) .

The r e m a i n d e r o f

e a c h s a m p l e i s h ' a t e d a t 5 6 ° C. f o r 50 m i n u t e s .
F o r e a c h g u i n e a p i g seruia t o be t e s t e d ,

a s e r i e s o f 5 t o 7 Kalin

tu b e s i s a r r a n g e d i n K a h n - t e s t r a c k s , and t h e r e a g e n t s a r e d e l i v e r e d
a s c l o s e l y t o t h e bottom as p o s s i b l e

(bent-tipped p ip e tte ),

in the

o r d e r r i v e n i n Table I .

P r o c e d u r e f o r s e l e c t i o n o f t h e " p r e l i m i n a r y " com ple me nt
Tube
6
4
2
5
7
5
.1 .
~ÜT04:'
~0702T
Ü
0.04
' 0 " "" ' ■"Ü ■ —
H eated g u in e a p i g serum
(ml) Ü
A ntigen su sp e n sio n
15 b i l l i o n / m l .

(ml.) 0

0

0.1

0.1

0.1

0.1

0.0

Ho mologous g u i n e a p i g
seruia ( 1 : 5 6 )

(ml) 0 . 0 5

0.1

0.05

0.1

0.05

0.1

0.1

S aline

( mlj 0 . 2 5

0.2

0.15

0.1

0.15

0.1

0.2

H ots:

Tubes 5 a n d 5 may b e o m i t t e d i f d e s i r e d .

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

—2 1 —

I h e t u b e s a r e t h o r o u ^ r h l y s h a k e n and p l a c e d i n t h e r e f r i g e r a t o r a t
4 - 8 ° C. f o r 3 h o u r s , a f t e r v : f i c h t h e y a r e p l a c e d a t roo m t e m p e r a t u r e f o r

13 m i n u t e s ,

f o e a c h t u b e i s added 0 . 2 ' / I . o f s e n s i t i z e d c e l l s ;

sh a k en a n d p l a c e d i n a 3 7 ° C. w a t e r b a th f o r I p m i n u t e s .
in s p e c t e d ; and t i e

th e tubes are

The t e s t s a r e

s e r a no b e ac e p t e l f o r t h o com plem ent p o o l a r e i n ­

d i c a t e d by t h e t e s t s

d i s p l a y i n p com ulote h em oly sis

more t h a n one+ f i x a t i o n

in e i t h e r t u b e 4 o r 6 .

i n t u b e s 2 and 7 , and no

tube 1 p iv e s in d ic a tio n

o f s pe ci ii ie ns t h a t a r e u n u s u a l l y l o . ; i n com plem ent a c t i v i t y by d i s p l a y i n g
p a r t i a l h e m o ly s is .

ilib e s 3 and 3 g i v e a d d i t i o n a l in f o n p a t io n c o n c e r n in g

t h e " a n t i b o d y ' a c t i v i t y o f th e s e r u m , b u t n eed n o t

be

ta k en in to con­

s i d e r a t i o n i n th e f i n a l a n a l y s i s o f t h e t e s t s .
The s e r a c o n s i d e r e d s a t i s f a c t o r y a r e p o o l e d , a n d ,
b y omni mal c e n t r i f u g a t i o n ,
o f th e s a l t s ,

i'he p o o l i s

i f necessary, cleared

im m e d ia te ly s t a b i l i z e d by a d d i t i o n

and s t o r e d i n t h e r e f r i g e r a t o r .

T h i s " p r e l i m i n a r y " c o m p le m e n t

c a n b e c o n s i d e r e d s e r v i c e a b l e f o r no more t h a n t i ' o v je e k s .

Procedure, f o r s e l e c t i o n o f th e

" s ta n d a r d " com plem ent.

A " s t a n d a r d " commlement ca n n o t be o b t a i n e d w i t h o u t f i r s t h a v i n g on
h a n d a c o m p le m e n t se ru m p r e v i o u s l y fo u n d s a t i s f a c t o r y ,

.i. p r e v i o u s l y

selected

aosen ce, a

" s t a n d a r d " c o m p le m e n t i s

o r e f e r r e d , but in i t s

" p r e l i . m i n a r y " comple;.ae-:u m ust be o b ta in e d and u s e d i n t h e p r o c e d u r e f o r
t h e s e l e c t i o n o f th e " sta n d a rd " c o m p l e m e n t .
S m a ll s a m o le s o f g u in e a p ig s e r a , o b t a in e d a s a lr e a d y d e s c r i b e d , a re
h e a te d a t 3 6 ° G. f o r 30 m in u t e s ; and t e s t s a r e s e t

up on e a ch a s

i n T a b le I I ,

p o s s i b l e t o t h e b ottom

d e l i v e r i n g th e r e a g e n t s a s c l o s e l y a s

o f th e t u b e s , a n d i n t h e O r d e r g i v e n .

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

in d ic a t e d

-2 2 -

II

P r o c e d u r e f o r s e l e c t i o n o f t h e " s t a n d a r d " cgüitjI emeiit.
■ 1
h e a t 5'1 p u i n o a p ip serum (iiil.)
iu itig en :

I f b i l l i n / m l , (nil.)

j

Gompleifient

(units)

m alins

(ml.)

b o te:

2

0.04

0 .0 4

0 .0 4

G .l

0,1

0 ,0

1,0

1.0

0.1

0 .2

1 0.1

I n c lu d e d w ith t h e s e t e s t s
later.

3

i s the s e t o f c o n tr o ls t o be d e s c r i b e d

The t u b e s a r e th o r o u g h ly sh ak en an d p l a c e d i n t h e r e f r i a e r a t o r a t
4 - 3 ° G. f o r 5 h o u r s , a f t e r vniicli t h e y a r e p l a c e d a t room t e m p e r a t u r e f o r
I h m in u t e s ,

f o e a c h tu b e i s a d d e d 0 . 2 a l .

o f s e n s i t i z e d c e l l s , and the

t u b e s a r e sh a k en and p l a c e d in th e 37° G. w a te r b a th f o r 13 m i n u t e s .
t e s t s a r e i n s p e c t ' d , and t h e s e r a s e l e c t e d f o r t h e
p o o l a r e o r d i n a r i l y t h o s e i n d i c a t e d by t h e t e s t s
d i s p l a y c o m p le te h e m o l y s i s .

O ccasionally,

The

"stan d ard " com plem ent

i n u iiich a l l th ree t u b e s

s e r a w ith c o m p l e t e h e m o l y s i s

ill t u b e s 1 a n a 3 , and a J_ o r a one + f i x a t i o n i n t u b e 2 may b e i n c l u d e d ;
b u t i n t h i s c a s e i t i s a good p o l i c y t o p l a n on r e p la c in g su ch a complement
s e r u m w ith a :.:ore s a t i s f a c t o r y one a s s o o n a s p o s s i b l e w it h in one m o n t h .
The s e r a c o n s i d e r e d s a t i s f a c t o r y a r e p o o l e d a n d ,
by m i n i m a l c e n t r i f u g a t i o n .

The p o o l i s

i f necessary, cleared

i m m e d i a t e l y s t a b i l i z e d b y a d d it io n

o f t h e s a l t s , ana s t o r e d in t lie r e f r h r e r a t o r .

i t h an " a n tib o d y -fr e e "

com plem ent on hand, one i s now p rep a red t o p erfom a t h e r o u t in e complement
fix atio n te s t.
J e c h n i c o f t h e " r o u tin e " com plem ent f i x a t i o n t e s t .

r e s u lts

o f g r e a t e s t r e l i a b i l i t y ca n n o t b e e x p e c t e d u n le s s a " standard"

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

co;,iple.7ient, s e l e c t e d
e'dhe t e s t s

es a i r a d y i n d i c a t e d ,

is

available

a r e s e t up a s i n d i c a t e d i n t a b l e I I I ,

I'or t h e t e s t s .

delivering a ll

r e a y s n t s a s c l o s e l y a s p o s s i b l e t o t h e b o tto m s o f t h e t u b e s , a n d i n t h e
o rd er yiveu.
llillh I I I
t h e " r o u t in e ' co;eole;:ient f i x a t i o n t e s t
du ne
1
d o s t sesnut

(ral.)

0 .0 4

0 .0 4

0 .0 4

^m tigen

(mlU)

0.1

0.1

0 .0

Tomplemenb

(units)

2 .0

1.5

1 .5

oaline

(rrlj

0.1

0.1

0 .2

note:

I n c l u d e d vvith t h e t e s t s
t o be d e s c r i b e d b e l ox.

th e t u b e s a r e t h o r o u g h l y oh a h : r.
5 hours,

is the

sy ste m o f c o n t r o l s

p t. ac o d i n t h e r e f r i g e r a t o r f o r

a f t e r u h i c h t h e y a r e p l a c e d a t room t e m p e r a t u r e f o r I t i . i i n u t e s .

t o e a c h t u b e i s a d d e d 0 . 2 m l. o f s e n s i t i s e d

cells;

th e tu b e s a r e shaken

and p l a c e d i n a 37 ° 0 . m a t e r b a t h f o r 15 m i n u t e s .
Upon i n s p e c t i o n o f t h e t e s t s ,
th e sym bols,

+,

c o m p le te f i x a t i o n .
p r i m e ( ’ ) m ark:
3 and 4 ,

etc.

th e tu b e r i a d i n y s a r e r e c o r d e d math

2, 3 , 4 , d e s i y n a t i n y th e d e p r e s s from z e r o to

I n t e r m e d ia t e r e a c t i o n s c a n b e d e o l' n a t e d w ith a

a r e a c t i o n b e ta o e n 2 an d 3 w ou ld be w r i t t e n _ 2 ': b e t w e e n
The f i n a l r e p o r t i s e s t i m a t e d a c c o r d i n g t o T a b l e T(f.

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-24'
,-L'J VT

deport

R eactions in tube
1
C.

7_j

to

-f

-r

—

fr om

+

2

-

to

2

from
iterative

roubtful

1 fr o m
1
to

P o sitiv e

5

-

2

4

4

4-

-

‘J h e c o n t r o l s o:? bke coj^rolcirezit, f i x a t i o n s y s t e ri
T n e s n c o n t r o l s a r e in c lu d e d d a i l y

to

ive in d icatio n of possible

c h r n y e s i n t h e a c t i v i t y o f t h e c one,1 orient a s v e i l a s th e " a n t i comp l e n n e n t a r y ’’ a c t i v i t y

of the p a r t i c u l a r a n tip e n su sp e n sio n .

They c o n s i s t

o f t o o s e r i e s o f s e v e n tu b e s e a c h , c o n t a in in g t h e r e a g e n t s i n d i c a t e d
i n f a b l e T , a n d c a r r ie d t i i r o u ;h t h e S9j,ie m a n ip u la t io n s a s t h e t e s t s
vniich t h e y c o n t r o l .
f .-Lhl d V

f ubos

Oomplsment c o n t r o l s e r i e s
1

2
Ô. Oë

3
0.07

4
o.od

3
0.09

6
0.10

7
0.11

Üornpdement ( iJ 11 .

(m l . ) -

S aline
Im tisen co n tro l se rie s

(m l.)

0.2^

0.24

0.23

0.22

0.21

0.20

0.19

Gomplement 1 D i l . I l l ) ’*' ( r r l . )

0 . 0^

0.06

0.07

0.08

0.09

0.10

0.11

itntiyen

0 .1 0

0.10

0.10

0.10

0.10

0.10

0 . 1c

O.IJ. 0 . 1 2
0,1^ 0.14
S aline
(m l.)
See d e s c r i p t i o n o f con.lejaeint u n d e r hea j e n t s .

0.11

0.10

0.09

(mJ..)

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

-2 5 -

•-L’u ' ; e s 1 t o 7 o i b o t h s e r i e s

c a r r y c o r r e s ' s . o n d i n r d o s e s o f coiüaleiucnt ;

a n d v d i i l e t h e r e s h o u l d be a s m o o th c u r v e o f i n c r e a s i n g h e s i o l ; r s i s fr om
le ft

to ri'd i" ,

/i t h C0i,ii)lete h e m o l y s i s i n t u b e s 6 a n a 7 ,

i n c o r r e s p o n d i n g t u b e s s h o u l d .be i d e n t i c a l .

th e h e m o l y s i s

K o v .ev er, a s l i g h t

difference

i s a lio ,,-a b le ; and t h e systm.i ca n s t i l l b e c o n s i d e r e d as o p e r a t i n g s a t i s ­
f a c t o r i l y p r o v i d e d t h e d e g r e e o f h e m o l y s i s in a n y one t u b e o f t h e
" a n tig e n c o n t r o l"

i s no l e s s t h a n t h a t i n t h e s e c o n d p r e v io u s tu b e o f

t h e "complement c o n t r o l . "

O ther u ses f o r th e system o f c o n t r o l s :

T h i s s y s te m o f c o n t r o l s i s

a n e x t r e m e l y i m p o r t a n t p a r t o f t h e co m p le m e n t f i x a t i o n t e c h n i c ,

o,nd i t

i s u s e d ex e r i m e n t a l l y i n tv.'o vjays:
1.

h'ith a s a t is f a c t c jy

'Oi.e T . i u e n t on h a n d ,

it

i s used t o d e te rm in e

t h e t o l o r e n c e o f t h e c o m p le m e n t f i c z a t i o n s y s te m f o r a p a r t i c u l a r l o t
of antigen.

By " t o l e r e n c e " i s m e a n t :

t h e l a r g e s t amount o f a n t i g e n

xvhich, u n d e r t h e c o n d i t i o n s o f t h e s e c o n t r o l s ,

c a u s e s no s i g n i f i ­

c a n t d e s t r u c t i o n o f v e r y s m a ll am ounts o f complement.

As a n e x am p le

o f j.ts use i n t h i s r e s p e c t see Table X TH .
2.

h i t h a s a t i s f a c t o r y a n t i , g e n on h a n d t h e

system i s used (as in

T a b l e ICEIc) t o d e t e r m i n e t h e s u i t a b i l i t y o f a g i v e n l o t o f com p le m e nt .

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-

26-

A p p l i c a t i o n o f t h e cofiiplejaent s e l e c t i o n p r o c e d u r e s .

O v e r a p e r i o d o f a b o u t 2.': y e a r s , t e s t s vjore made on a t o t a l o f
1 ,0 2 1 .'juinea p i g s and t h e r e s u l t s a r e sumraarized i n T a b le s VI a n d V I I .
The s e r i e s

i n t h e r e s p e c t i v e t a b l e s a r e d iv id e d i n t o o r o u p s . r i v i n g

th e d a te o f b le e d in r ,

uluj n um be r o:b p u i n e a

o f th e f i x a t i o n r e s u l t s

I n ea ch .jr o u n .

a l a s b l e d and an a n a l y s i s

U n le s s o th e r ,s i s e s t a t e d ,

th e

t u b e num bers ; ; i v o n r e f e r to t h e t u b e o f t h e r e s p e c t i v e p r o c e d u r e o u t l i n e d
i n T a b le I o r T a b l e I I .
On t h e 629 g u in e a p i g s t e s t e d by t h e " p r o llis in a r y '

p rocedure f o r

compleiiieiit s e l e c t i o n t h e r e n e r e 503 f o r v h ic h t h e r e s u l t s
b e d i v i d e d i n t o tv;o s e r i e s :

(l)

b a s e d on r e a c t i o n s i n t u b e 6 .
VI a r e r e s u l t s

the

i n t u b e 4 ; and

(2)

n l s o in c lu d e d i n a s e p a r a t e p a r t o f T a b l e

o f t u b e 6 ( o n ly )

sake o f convenience,

b a s e d on r e c t i o n s

( T a b l e VI) c o u l d

on th(/ a d d i t i o n a l 121 g u i n e a p i g s .

I'or

(+) colum n s i n d i c a t e t h e nu mber o f g u i n e a p i g

s e r a v.inich u n d e r t h e c o n d i t i o n s o f t h e t e s t g a v e a 4 + f i x a t i o n ;
c o lu m n s, t h o s e w it h l e s s th a n a 4 + f i x a t i o n ;

and th e

the

(j_)

( - ) c olunm, th o se

w i t h no f i x a t i o n .
I n t h i s s e r i e s o f 508 g u i n e a p i g s , 76 .0 /» o f t h e s e r a r e a c t e d w i t h
p e r t u s s i s a n t i g e n i n tu b e 4 ,

w h ile i n t u b e 6 t h e p e r c e n t a g e o f r e a c t i n g s e r a

was 3 8 . 4 , i n d i c a t i n g t h e s y s te m in tu b e 6 t o b e more s e n s i t i v e th a n t h a t
in t u b e 4,

c o n s i d e r i n g t h e s e r i e s a s a whole.

T h e r e w ere 422 a n i m a l s t e s t e d by t h e

" sta n d a rd " p r o c e d u r e f o r com plem ent

s e l e c t i o n and t h e r e s u l t s g i v e n in T a b le V II w ere c l a s s i f i e d on t h e f o l l o w i n g
b asis:

t h e colum n (d -i ) ( s t r o n g p o s i t i v e ) , i n d i c a t e s th e number o f g u i n e a

p i g s e r a w it h w h ich t h e r e was a 4 + f i x a t i o n
(.] +)

i n b o th tu b e s 1 an d 2 ; col mm.

(we ak p o s i t i v e ) , t h o s e w i t h w h ich t h e r e was a t l e a s t a 3+ i n t u b e 2

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a n d l e s s t h a n a 3+ i n t u b e 1 , colum n
moi’e t h a n a

i n t u b e 2 , c o l man ( - )

In t h i s s e r i e s

t h o s e w i t h l e s s t h a n a 5+ b u t
t h o s e v i i t h no f i x a t i o n .

o f 422 g u in e a n i g s , 77 »3> r e a c t e d v :ith p e r t u s s i s

a n t i g e n s ; l e a v i n g 2 2 . 71 o f t h e a n i m a l s e r a v h ic h c o u l d b e c o n s i d e r e d s a t i s ­
f a c t o r y f o r u s e a s co m n leiâ en t.

I n o t h e r v.’Cr d s t h e r e s u l t s g i v e n i n b oth

f a b l e s YI a n d V I I sh e.; t h a t , i n t h i s l a b o r a t o r y a t l e a s t ,

a very

liyli

p erce n ta g e

o f t h e n o rr aa l h e a lt h y

r.uinoa p i g s c a n n o t be u s e d t o s u p p l y

ccraplcm snt

f o r the te-he - i t h

pe-’^ t n s s i a ; n o r i s i t p o s s i b l e to

p r e d i c t fr o m t i m e t o t i m e hou many s a t i s f a c t o r y s e r a m i l l b e a v a i l a b l e .
In fa c t,

there

vas a p e r i o d o f ab ou t n i n e m onths b e t v .e e n 1 / 2 / 4 3 and

1 0 / 1 /4 3 ( g r o u p s 6 an d 7 , T a b le V I I ) ivhen i t mas n o t p o s s i b l e t o o i n . a i n a
"standard"

co'.ipleinent b e c a u s e i t

" p r e l i m i n a r y ' ' com ple me nt
th e guinea p ig s .

v;as a l s o i m p o s s i b l e t o o b t a in a s a t i s f a c t o r y

( s e e g ro u u s 4 to 12 i n T a b le VI) u i t h

vnich to t e s t

The p r o p o r t i o n o f r e a c t i n g s e r a seemed t o i n c r e a s e from

t h e e a r l y p a r t o f F eb ruary 1943 , r e a c h e d a p e a k i n t h e l a t t e r p a r t o f A p r i l
o r e a r l y i n Any, and th en d e c r e a s e d a g a i n , th e r e b ,. a l l o w i n g s e l e c t i o n o f a
s e r v i c e a b l e c o m p l a . i e n t s er u m i n e a r l y O c t o b e r .
A e l a t i v e s e n s i t i v i t y o f t h e " p r e l i i a i n a r y " a n d " s t a n d a r d " p . -o c e d u r e s .

ibnong t h e g ro u p s o f r e s u l t s g i v e n i n T a b l e s VI and V I I , grou p s 18 an d
13 r e s p e c t i v e l y a r e t h e r e s u l t s

o b t a i n e d u h e n the; 30 g u i n e a p i g s b l e d on

12 / 2 3 /4 6 v/ere t e s t e d by b o t h o f t h e com plem ent s e l e c t i o n p r o c e d u r e s a lr e a d y
d e s c r ib e d vvith t h e e x c e p t io n o f some s l i g h t m o d i f i c a t i o n s i n d i c a t e d in
T a b l e s V I I I a n d IX r e s p e c t i v e l y i n which t h e e s s e n t i a l f e a t u r e s a r e g iv e n .
'The tu b e s a re l a b e l e d w it h l e t t e r s

in o r d e r t o a v o id c o n f u s io n w it h tu b e n m ib ers

g i v e n i n o th e r t a b l e s .

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-3 0 -

v iii

F e a t u r e s o f t h e " p r e l i i . i i n a r y “ p r o c e d u r e u s e d i n t e s t s on

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( 1 and o- b e i n g i d e n t i c a l v.-ith 1 and 2 r e s p e c t i v e l y

o f fa b le I I ) .
G e n e r a l l y , t h e t e s t p e r f o r m e d as i n t u b e A, r e s u l t s

in a low er

g r a d e r e a c t i o n t h a n t h e t e s t p erfo rm ed a s i n t u b e B ( c o . : p a r e r e s u l t s
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ot' t 8 o t h e r 2 o g u in e a p i g s e r a r e a c t e d ’.-'ith l i . r e r t u s o i s u n d er th e
v e r y s t r i c t c o n d i t i o n s r e q u ir e d by t o i e t e s t , t h e r e b y i n d i c a t i n g i t s
- r e '.t e r s e n s i t i v i t y .

F u r t h e r : : o r e , 'ùhere i::, a :i ot lie i‘ p o i n t , i n v o l v i n g columns t and h ,
uhii;h s'iuau b.; ^Mentioned bec.-:i.u3 e a p p a r e n t ly i t h a s n o t come t o t iie
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in '''e stip a c o rs.

Jhe.se t

columns a r e s i . . a l a r i n

t h a t t h e y a r e e ac h an a n t i c Oi.ipleiaen.tary c o n t r o l o f t h e g u i n e a p i : serum
( t h e s e i s no a n t ig e n p r e s e n t ) ,

its e lf

u n der t h e c , a d i t i o n s of th e

" p r e l i s i i n a r y " p r o c e d u r e , 0 . 0 4 m.l. o f th e fO h e a te d g u in e a pi"; s o r a i.^as
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v.he re as u n d e r t h e e m a c ti n .: c o n d i t i o n s o f t h e " sta n d a rd " p r o c e d u r e , fo u r
s p e c im n s i;e r e fou n d t o b e soj.ieu'hat a n tico :.; lojientar],'' f o r th e p o o l , F - 2 3 .

In t n i s r e s p e c t , th e n , i t

i s n o t s a f e t o assume t h a t any g u in e a p ig serum

r n i c ' i h a s good com plem ent a c t i v i t y Can b e c o n s id e r e d a s a t i s f a c t o r y
reagent.

O i.v io u s ly , th o u :h, t h e a n t i c o . u p l e m e n t a r y f a c t o r s in any

g iv e n serum may have no e f f e c t on t h e i r homologous com p lem en t, th e y may
b e e f f e c t i v e i n r e d u c in g th e a c t i v i t y o f h e t e r o l o g o u s com p lem en t.
C e r t a i n l y , su ch s e r e s h o u l d n o t b e i n c l u d e d in th e compl.cmont p o o l .

From an a n a l y s i s o f f a b le X, i t fo llo m S 'f t h a t t h e r e a re t'vo r e a s o n s
f o r u s in g o n ly com plem ent s e l e c t e d by t h e " stan d ard " p r o c e d u r e , f o r
r o u tin e t e s t s
1.

in p e r t u s s i s :

I n t e r f e r i n g a n t i b o d i e s h ave b een e l im in a t e d from th e complement^
o r red u ced t o a n o n in t e r f e r i n p minimum.

2.

A n tic o m p le m e n ta r y f a c t o r s o f th e g u in e a p ig serum i t s e l f h ave
a l s o b een d e t e c t e d and e l im in a t e d f r o j i th e com p lem ent.

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-

34-

s e l e c t e d Coiaijlejnent on th e coi^M.ileident l i x a ui.on a j/a lo m.

.Jue t o uiio yu'tiC Gice o f a o v e r e j .r e t c s t in ; ; o f ou.lnou oi, ;s f o r corn..lehient 3C3.Lecx-ion i t iias o ecn f .o s s i b l e t o i n c r e a s e t_io so n sii.iv it;-/^

e ffic ie n c y

o.ncl ù ü p cn d eb iliL y, o f th e I'o u tin e coiep len ieat i i ; : a t i o n t e s t by a. la r y o roar y i n .
I n f a b l e XI i s

y iv e n a cohios.i'isori o f th e s a l i e n t i i f f o i ' o n c e s i n th e c o e io le -

nront fi.-o a tio n sy sto je b o fo i's anci a ft'o r i n s t i t n t i n a rle; y r e .c tic o o f coie . l e o.ent s e l e c t i o n .

s a l i e n t d i f f e r e n c e s i n th e coiuplorient f i x a t i o n s y ste m r e s u l t i n g from
com plem ent s e l e c t i o n
b e fo r e

j

A fte r

P r a c t ic e o f com plem ent s e l e c t i o n
1 , .in ti,g e n : T i t r a t i o n
r.iethocl
C o n c e n tr a tio n
t o l e r a t e d by
t h e sy ste m
F req u en cy o f t i ■ tr a tio n

..n tic o m p ]e h ie n ta iy
, CoDinlohient f i n i n g
n o se
1
p o te n c y
25 b l l l : i o n / w l . or
O.LV.[.— n.O o j .l lio n / ii’LL,
,.101‘e

2 . F i x a t io n p e i'io d

18 h o u rs a t 4 -8 ^ C.

5 h o u rs a t 4 -o ^ C .

3 . J e r v ic e a b i3 .it y of
s t a b i l i s e d co m p ler:en t

a b o u t 2 w eeks

a s lo n g a s 3 m onths

once f o r each l o t

d a ily

ks r e s u l t o f em ploying; a s e l e c t e d com p lém en t, i t vjas no lo n g e r n e c e s -

s a r y t o u i t r a t e a n t ig e n s e v e r y d a y .

The r o u t in e com plem ent f lo t a t io n t e s t

c o u ld b e p erfo rm ed \'v ith in one day u s in g th e 5 houT r e f r i g e r a t o r f i j t a t i o n
p e r io d ; and t h e com plem ent rem ained s e r v i c e a b l e a s lo n g a s 3 m o n th s, or
p r a c t i c a l l y a s lo n g a s i t

s t i l l r e t a i n e d a s a t i s f a c t o r y com plem ent t i t e r ,

e s p e c i a l l y n o te w o r th y ( i n T a b le ] 3 ) i s t h e m arked in c r e a s e i n th e amount
o f a n t ig e n t h a t c o u ld be em ployed i n t h e com plem ent f i x a t i o n s y ste m , a s

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-3 5 r e s u l t o f co m pl em e n t s e l e c t i o n , v . l t h o u t clanper o f c o m p l i c a t i o n s .

,7h i l e

t i le com plem ent f i x a t i o n s y s t e m w ould n e v e r t o l e r a t e jjiore t h a n a 2 . 0
D i l l i o n / f f i l , s u s p e n s i o n vihen a n u n s e le c t e d comiplcment had b e e n u s e d , i t
vjould t o l e r a t e a s u s p e n s i o n a s h i p h a s 2 5 . 0 b i 3, l i o n / x ; i l . o r more vnLth a
s e l e c t e d com}dement; a n d i t m u s t be k e p t i n laind t h a t t h e co m pl em en t i s
s e le c te d c h i e f l y fo r i t s l a c k of i n t e r f e r i n g a n t i b o d i e s .

A goo d exajnple

o f th e e f f e c t coniplei.ient s e l e c t i o n h a s on th e a n t i en c o n c e n t r a t i o n t o l e r a t c d b y th e s y s te m , i s b ro u g h t o u t b y th e f t . l l c n i n g cxp criu aen t i n w h ich
a s i n g l e l o t o f a n t i g e n was t e s t e d i n v a i'y in g am o u n ts f o r i t s

" a n ti-

c o m p l e m e n t a r y " a c t i v i t y on v a r y in g am o un ts o f f o u r d i f f e r e n t l o t s o f com­
p l e m e n t : t h e l a t t e r w ere c h o se n b e c a u s e t h e y v a r i e d i n a n t i b o d y c o n t e n t .

The " an t i c oniplem ent ary" e f f e c t o f a t y p i c a l a n t i g e n on d i f f e r e n t l o t s o f
complem ent ,
th e f o u r l o t s o f com ple me nt c n o s e n f o r t h i s o x p e r i / i : e n t , a r e c h a r a c ­
t e r i z e d i n T a b l e k l l a , w h ich g i v e s t h e co m p lem en t l o t n u m b e r, d a t e o f
b l e e d i n g , t h e number o f g u i n e a p i g s b le d , t h e number o f s e r a i n e a c h l o t
and t h e r e s u l t i n g volume.

A lso g iv e n a r e th e r e s p e c t i v e t i t e r s and ages

o f th e l o t s a t th e tim e o f Lhe e x p e r im e n t.

TABID X l l a
D e s c r i p t i o n o f fo u r e x p e r im e n ta l l o t s o f com p lem ent.
Comple­
m ent N o,

D ate o f
b le e d in g

N o. o f
A nim als

P -2 6
M-2 5
P- 2 3
d -5 0

1 -3 -4 7
4 -1 -4 7
1 2 -2 3 -4 6
/k -1-47

40
27
30
2 7 ___

M ote:

No. o f
sera in
pool
10
4
9
6

Volume

2 3 .5
1 9 .0
2 3 .0
2 5 .0

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mJ.,
iml.

Titer^;-

1 :1 4
1 :1 5
1 :1 0
1 :1 8

Agei'
o f Serum

3 .5
4 .5
1 3 .5
4 .5

w eek s
w eeks
w eek s
w eeks

a t tim e o f th e ex p e i-im e n t.

I n T a b l e A l l b a r e g i v e n t h e r e a c t i o n s o b t a i n e d when t h e s e r a i n c l u d e d

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-3 0 -

i n e a c h p o o l w e r e t e s t e d b y t h e " s t a n d a r d " corApleiiient s e l e c t i o n p r o c e d u r e ;
and t h e p o o ls a r e g iv en , from l e f t t o r i g h t i n o r d e r o f i n c r e a s i n g a n t i - .
bo d y c o n t e n t a s j u d g e d p u r e ly b y i n s p e c t i o n .

}[IIb
R e a c t i o n s o f " s t a n d a r d " com plem ent s e l e c t i o n p ro ced u re on t h e g u in e a
p ig s e r a com p osin g t h e f o u r e x p e r i m e n t a l p o o l s .

i -2 6
Tube
2
1

3

-

-

-

-

—
—

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r-25

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1

2

3

1

2

4

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:

ir I n ay,g l u t i n a t i o n t e s t s t h e s e w ere t h e o n l y two s e r a t h a t a g g lu t in a t e d a
s u s p e n s i o n o f H. p e r t u s s i s a n t i g e n .

'. n t h t h e f o u r com plem ent s e r a c h a r a c t e r is e d a s i n T a b l e s H l a and
R l l b , t h e e x p e r i m e n t was s e t u p a s i n d i c a t e d i n T a b l e l l ï c .

The a n t i g e n

em p loyed i n t i i i s e x p e r im e n t was t h e v e r y same l o t t h a t had b een u sed t o
t e s t t h e a n i m a l s com p osin g t h e fo u r e x p e r im e n ta l p o o l s ;

a n d one u n i t o f

i t was e q u a l t o 0 . 1 m l . o f a n 1 1 . b b i l l i o n p e r m l . s u s p e n s i o n .

The v a l u e s

o f 0 . 2 5 , 0 . 5 , 1 . 0 , 2 . 0 and 4 . 0 u n i t s s p e c i f i e d i n T a b l e R I I c c o r r e s p o n d e d
t o c o n c e n t r a t i o n s ( i n organism s p er n l . )

of 2.9,

5 . 9 , 1 1 . 0 , 2p . 6 , and

b illio n respectively.
I n t h is e x p e r im e n t, t h e r e a g e n t s were b r o u g h t t o g e t h e r i n t h e u s u a l
t o t a l v ol u m e o f 0 , 3 m l . , a n a a l l o w e d t n o u s u a l i n c u b a t i o n y e i ' i o u s o e f o r e
and a f t e r a u d it io n o f th e s e n s i t i s e d c e l l s .
t a b l e a r e i n t e r m s o f p e r c e n ta g e h e m o l y s i s ,

The r e s u l t s g iv e n i n th e
( - ) i n d i c a t i n g c o m p l e t e and

( 0 ) , no n e j i i o l y s i s .
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

.2

37-

-

TABLE X IIc
ï'he " a n tico n ip lem en ta ry " e f f e c t o f one l o t o f a n t ig e n on f o u r d i f f e r e n t
l o t s o f com p lem ent.

oonipl.ement
No.
1 -2 6

U nits of
A ntigen
Ü .0
0 .2 5
0 .5
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2 .0

4 .0
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2 .0

4 .0
0 .0

1 -2 3

0 .2 5
0 .5

1 .0
2 .0
4 .0

0 .0

3 -5 0

0 .2 5
0 .5
1 .0
2 .0

4 .0
N o te :

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u n its of Oonuilenient
1 .0
0.9
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70
70
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95
95
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gO

e . c u a l s 100, u, and (O) e q u a l s no h e m o ly s is .

fh e f i r s t s e r i e s i n each o f th e fo u r groups o f r e a c t i o n s i n f a b le
h llc

g iv e s t h e

u e r c u n t a q e uo; iio]ysis due Lo t h e s p e c i f i e d am ou nts o f t h e

r e s p e c t i v e comqlejuent seruja c. lon c; a n d s e r v e s a s a i^ o n ar o l on one o t h e r
f i v e s c r i e s o f t n e /"'roup,

n. coi'ûpax'ison o l a . l l i oui' con'oi'cl s e i ’i u S j

snov's

th e c u r v e s o f h e m o ly s is t o be i d e n t i c a l ev en th o u g h t h e com plem ent s e r a
had t o be u s e d a t d i f f e r e n t t i t e r s .
h i t h i n ea ch group t h e r e vjas an in c r e a s e i n i n h i b i t i o n o f com plem ent
V iith i n c r e a s e i n am ount o f a n t i g e n a d d ed .
f o r com plem ent j - 5 0 , l e s s f o r r - 2 3 ,

e n i s irinii.-io.Lon u a s r.iost marked
and l e a s t f o r 1 - 2 6 .

T here ^las no

p a r a l l e l i s m b etw een th e i n h i b i t i v e e f f e c t o f t h e a n t i g e n a n a trie r e s p e c t i v e

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

coupletiient. t i t e r , fo r as in d ic a t e d in Table X lx a , S-pO had th e a i? ;h e st
u i t e r , and r —a j une lo v ie st,

Cii ta e o ta o r _-arid, sin c e the antigen, v.’as

l e a s t i n h i b i t i v e f o r r - i b (_corapleaient sei'uni known t o c o n ta in a v e r y lev;
l e v e l o f a n tib o d y ;, and more in h ib i t iv e

:/ th e o th e r complement sera,

(iaiovvn t o c o n ta in .appreciable le v e ls of a n tib o d y ), i t seems v e ry c e r ta in
t h a t th e ' . •'

o f a n tiy e n t o l e r a t e d by t h e s y ste m i s

U'.ion t h e l e v e l o f t h e a n tib o d v c o n t e n t o f t h e c ;,

in v e rs e ly dependent
snrui.i,

'.nils IS

f u r t h e r i l l u s t r a t e d b y t h e s i u u i a r y o f tn c r e s u l t s o f t h i s eiip er i m e n t r i v e n
i n T ab le h i I d i n ahicii i s

shown t h e a n t i y e n c o n c e n t r a t i o n t o l e r a t e d by

each l o t o f complement.

I'hbLa k l l d
V a r ia t io n in a n t iy e n c o n c e n t r a t io n t o l e r a t e d b y v a r io u s l o t s o f
c o m p le i. ie n t.
i.^aicimum c o n c e n t i ' . a t i o n o f a n t i y e n
to lerated

Complement

O rder of
a n tib o d y
__ c o n t e n t __

1—26

le ast
1

2 3 . 6 b i l l i o n /m l.

1 -2 5

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much l e s s t h a n 2 . 9 b i l l i o n / m l .

most

3 -5 0

b b s t s i g n i f i c a n t i s th e v e r y cmtreme d i f f o r c n c o b etw een t h e amount o f
a n t ig e n t o l e r a t e d by .L-2 6 , a s e l e c t e d " p e r t u s s is " complci^ient, and t h a t t o l e r a t e d . by .3- 5 0 , a s e l e c t e n "..-asseri.an" co m p le m e n t; 0 . 2 5 u n i t s
- il.) o f ^

(2.9 b illio n /

'per t u s s i s a n t i y e n v;ors more i n h i b i t i v e f o r 3 - 5 0 t h a n wore 4 u n i t s

( 4 7 .2 b illio n /m l.)

of

v e r y same a n t i y e n f o r r - 2 6 ------- i n o t h e r words---------

more th a n a l 6 - f o l d d i f f e r e n c e .

Such a c t i v i t i e s d e f i n i t e l y ca n n o t be a t t r i o -

u t c d t o ch a n g es i n t h e a n t i y e n .
I f f e e t o f a n t i s e n c o n c e n t r a t io n on s e n s i t i v i t y o f th e c pmpl/^iie n ^ fi^^^^^

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-3 9 -

.,ith a. selocuea complement on liand, thei-ei'ore, i t i s p o ss ib le to
<:i!aploy la i'p e r amounts ol a.nti,_.i;.'n; and the effeeL ox' l a r g e r ajuouncs of
a n iip e n on th e s e n s ic iv ity of th e coiiylenent f ilia tio n t e s t i s i l l u s t r a '
te n in Table xJLII.
TABI,]! ^ü:II
e f f e c t o f a n t ip e n c o n c e n tra tio n on s e n s i t i v i t y o f the compxlement
fix a tio n t e s t .

LG31

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Complemenu
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1 .5
1 .5
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The r e s u l t s i n t h e c o n t r o l s y s t e m a r e g i v e n i n t e r m s o f p h e m o l y s i s :
( - ) e q u a l s 100m
X-

Complement i - 2 6 , o b t a i n e d one mon th p r e v i o u s was u s e d h e r e , a t
a t i t e r of 1 :14.

The t e s t s

s e t u p a s i n d i c a t e d i n T a b l e X II I w e r e c a r r ie d th ro u g h t h e

u s u a l in c u b a t io n , e r io d s^ e t c . , and a s r e s u l t o f t h i s e x i)e r im e n t i t i s
s e e n t h a t t h e s e n s i t i v i t y i n c r e a s e s w i t h a n i n c r e a s e i n amount o f a n t i g e n
t o in c l u d e a c o n c e n t r a t io n o f 3 .5 b i l l i o n / m l .

Beyond t h i s c o n c e n t r a t io n

t h e r e w as n o f u r t h e r i n c r e a s e i n s e n s i t i v i t y o f t h e t e s t ,

ide anwhile^ t h e

e f f e c t o f t h e a n t ig e n on t h e com plejuent a lo n e ( s e e c o n t r o l s y ste m ) rem ained p r a c t i c a l l y c o n s i a n t I'or a l l c o n c e n t r a t i o n s .
th e s y stem would t o l e r a t e

I n o t n e i Xiords^

th o u g h

c o n c e n t r a t i o n s ol a n t i g e n a s n i g n a s l o . 5

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

—Zj.ü—
b i l l i o n / i n l . , i t was n o t n e c e s s a r y t o u s e much more t h a n a n 0 . 5 b i l l i o n / j i i l .
s u s p e n s io n f o r ma^iimuiii s e n s i t i v i t y .
m l . ^ h o w e v e ly

C o n c e n tr a tio n s l e s s th a n 8 .5 b i l l i o n /

r e s u l t e d in a t e s t o f lower s e n s i t i v i t y .

S e n s i t i v i t y o l co m p le m e n t f i x a t i o n co m pa re d t o a r y l u t i n a t i o n i n t e s t s

on

anim al s e r a .

a n s t n c y i r i y t h e phenomenon o f a . y l r t i n a t i o n ,

sev e ral m anipulations

w ci'e t r i e d a n d i t w as fo u n d t h a t f o i ' y u i n e a pi;.; a n d o t h e r a n i m a l s e r a ^
h e a t e a o r u n l i e a t e d , a 10 b i l l i o n / r l .

s u s p e n s i o n o f II. p e r tu s s i s- p rod u ced

a more p e r m a n e n t r e a c t i o n t h a n a 20 b i l l i o n / n l .

suspension.

A p e r io d o f

shakin,:^ f o llo v ; e d b y r e f r ir ^ c r a to r in c u b a t io n was a l s o fo u n d n e c e s s a r y f o r
maximum r e a c t i v i t y o f a n t i g e n w i t h s e r u m , a l t h o u g h th e r e a d i n g s a f t e r r e ­
f r i g e r a t i o n were o fte n d i f f i c u l t to i n t e r p r e t .
p l o y e d was a s f o l l o w s :
O.OZi- 1.1. ( o r l e s s )

T h e r e f o r e , t h e t e c h n ic em­

0 . 1 n l . o f th e a n t i g e n s u s p e n s io n was a d d e d t o

o f serum i n uahn t e s t t u b e s ;

th e tu b e s were shaken a t

t h e r a t e o f 1 0 0 -1 5 0 o s c i l l a t i o n s p er m in u te o v e r a w i d e a r c f o r f i v e mi n ­
utes;

th e t e s t s

. .e re r e a d i m m e d i a t e l y , a g a i n a f t e r 24 houi'S a t 4 - 8 ° C , , a n d ,

i n some i n t a r i c e s , o n c e more a f t e r 72 a n d / o r 96 h o u r s a t 4 - 8 °C.

Some o f t h e d a t a a c c u m u l a t e d w i t h r e g a r d t o a g g l u t i n a t i o n t e s t s on
g u i n e a p i g s e r a h a v e a l r e a d y b e e n i n d i c a t e d i n T a b l e X l l b i n w h i c h i t was
shown t h a t o n ly tw o , o f a l l th e g u in e a p ig s e r a r e p r e s e n t e d t h e r e , w ere
c a p a b l e o f a g g l u t i n a t i n g a s u s p e n s i o n o f H. p e r t u s s i s .

This i n i t s e l f i s

e v i d e n c e e n o u g h a g a i n s t r e l i a . n c o u p o n sucxi s. l e s t i c i ' co m p le m e n t s e l e c t i o n ;
but i t

s c a r s e l y b e g in s t o show th e t r u e s t a t u s o f th e s i t u a t i o n .

I n a s e r i e s o f 1 7 2 g u in e a p ig s e r a t e s t e d b o th by a g g l u t i n a t i o n and
coificlem en t f i x a t i o n ,
h.

t h e r e w ere o n l y 6 wiiich a g g lu t in a t e d a s u s p e n s io n o f

p e r t u s s i s a n t i g e n a t one s t a g e o r ano utter 01 t t ie u e c t in ic u s e -1, w h i l e

1 4 4 r e a c t e d i n th e " stan d ard " p r o c e d u r e f o r com plem ent s e l e c t i o n .
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

Of

—4 1 ”

c o n s id e r a b le i n t e r e s t i s t h e f a c t t n a t t h e c o n t e n t s o f th e tu b e s o f th e
com plem ent f i x a t i o n t e s t s ^

c o u ld bo rea d f o r o .^ ;r lu tin o tio n a t th e end o f

the. 5 h o u r f u c a u io n p e r i o d .

The s e n s i t i v i t y o f a p p l u o i n a t i o n un d er t h e s e

c o n d it io n s v<as fou n d r o oe a s h ig h a s i t vjas i n s e p a r a t e l y p e r f c r r a e d t e s t s
e m p lo y in g t h e 5 rw inutes sh a k in g p e r io d ; b u t s l i p h t l j r l o s s th a n t h e f u l l
t e C linic d e s c r ib e d a b o v e .

j.ii t h e s e and man.r o t h e r t e s t s t o be r e p o r t e d

b e lo w , a t no r i m e was t h e r e e v id e n c e o f a,.

l u t i n a t i o n , w ith o u t e v i a i ri ce

o f com plem ent f i x a t i o n j a l l s e r a t h a t a p ; l u t i n a t c x l s u s p e n s i o n s o f d . p e r ­
t u s s i s a l s o f i x e d com p lem en t.

S in c e a h ig h p e r c e n ta g e o f "nori,ial" g u in e a p ig s had b een fo u n d t o
h a v e a n t i b o d i e s c a p a b l e o f r e a c t i n g w ith p e r t u s s i s a n t i ; o n s , e s p e c i a l l y
i n t h e r i g i d l y c o n t r o l l e d co m pl em en t f i x a t i o n t e s t d e s c r i b e d f o r com p le­
ment s e l e c t i o n ,

i t seemed a l m o s t m a n d a t o r y t h a t s e r a o f o t h e r s p e c i e s o f

l a b o r a t o r y / a n i m a l s be b e s t e d .
T e s t s on la b o r a t o r y a n im a ls o t h e r t h a n i-aiinea p i p s .

Unless oth-aivise s t a t e d ,

t h e r e s u l t s o f com plem ent f i x a t i o n g iv e n a r e

t h o s e o f t h e " ro u tin e " t e s t ( s e e T a b l e I I I ) .

This t e s t i s l e s s s e n s i t i v e

uhan t h e " standard" com plem ent s e l e c t i o n p ro c e d u r e ( T a b l e I I ) u s e d i n t e s t s
on g u in e a p ig s e r a , b e c a u s e t h e r e s p e c t i v e tu b e s o f uhe r o u t in e t e s t c o n t a i n 0 , 5 u n i t s o f com plem ent more th a n t h o s e o f t h e l a t t e r ,

m ic e :

T h ese a n ii'.u ls w ere b le d un der a n a e s t h e s i a b ' c a r r iia c p u n c tu r e ,

and uhe s e r a t e s t e d i n d i v i d u a l l y t o a v o id any p o s s i b i l i t y o f g a in in g th e
f a l s e im p r e s s io n s v h a t som etim es res^lLt from t e s t i n g p o o le d s e r a .

Of 62

a p p a r e n t ly norm al m ice c e s t e d , a l l w ere n e g a t iv e b o th by th e r o u t in e comij le m e n t f i x a t i o n t e s t e.iid by a g g l u t i n a oioti,
o f 235

irnm m ized w it h h . p e r t u s s i s .

i u s t s .^ere o.xso i-ia,cie. on s e i a

The r e s u l t s g iv e n in T a b le 41V

show t h a t o f 9 b imiiiunixed mi ce t e s t e d , by coijpleii.ent .ii.._auion a l o n e ,
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onl.y

/; v'Jüi'K n;;,^G,wive; cj,

u. i',; Ouiiur

onl;^ I j

n h n l t V~- v;ci'n no: n b iv o lannnee:

.,e ru ne / i t l v c b

c o a . -Ice.ent

aj„:-l-n-bliintloa,

n e r a o i l o auj n a a o d l j

" n o a . a l " aiieen v.'eru n é s bob, Ir ' af ;; ;l u' b in -

0 l'iorij o.no. b;/ oo'on ûlin r o u t i n e coa]jiuiuont i'iao.-bion t e s t ooki th e " s t o n d a r d "
c o r .a le ,œ n t soJ.ec-btnn j:i'oceb r.re.
:.Liat '.brhlü ;;.bout h a l f
te sts,

The r e s r l t s

iv e n in. T a b le IW, i n d i c a t e

J c f 'bbc sh eep oero, noi'o nopo-bi've i n c'.p; , l a t i o i o . t i o n

o n ly b ,;oro n.: ...b iv o i n to a m u t i n e c e a ,.l(;..a n t .f i ..a t io n test, and

none u m .er th e vui-y s t r i c t c o n d it io n s o i t:x; "stand,o.rd" c c a p lv r .c n t s e l e c tion te s t.
ii n b b it s :

The i'e s n lt ,s o f t e s t s on th e s e r a o f 19 "nor]onl" r a b b it s a r e

r iv o n i n

To.ble XVIa i n n h i c h i t

i s shovm t h a t -.iUij.e 12 t o 15 s e r a n e r e

n e g a t iv e

b;

7 n ere n e y a a i v e ,in t h e r o u a i n e cojaplcjoent

a : ; '; l v . 'b i n a t i o n , o n l y

isolation m o t.
Cl

37 l'a il) i t . s irn n n n ic n d a i t i i b . i n i l i i e n s a e ( T a b l e h V Ib ) a i l s e r a

n e r e to rn d t o r e a c t a i u l i i . .

.'c r tu ss i s i n t h e r o u t i n e co n y jao acn t f i l i a t i o n

t e s t , w h i le o n ly a b o u t I / 3 o f thcoi pave d e .f i n it o r e a c t io n s i n a y g i n t i n a tio n te s ts .
h orse:

o n ly one "nom a l" h o r s e sem uii, a p o o l o f tw o h o r s e b lo o d

s];ecia ien s was a v a i l a b l e f o r t n s t r r u ; ; .

,'no i esL.J.t i n th e r c u r i n e coiriple-

n e n t f i l i a t i o n t e s t w as s t r o n y '.aiouah f o r a " d o u b tfu l" m p.ort^ and i n th e
" stan d a rd " coiauleinent s e l e c t i o n p rooedure^ f o r a " i:o si.tlv o " r e p o r t . TiuLs
serv.ia a l s o a p ; y l u t i n a t e d a s u s p e n s io n o f ii. p e l ' t u s s i s .

,i5 1-esi.tLt 0..1 th e t e s t s on a l l th e a n iin a ls i t i s e v id e n t th a t n o t
o n ly do I'u irie a .lijp s e r a h a v e a n t ib o u lo s c.i.paole c i ro a cu rn y i u oil
t u s s i s r .n tiy o n , b u t th e s e r a o f o t h e r la b o r a t o r y anin.ials eo a s '.v e il,

an

d e t e c t such .a n t ib o d ie s , h o w ev er, une a y y l i t i n a t i o n t e s t i s t o t a l .evid sr ice o f t h i s iias b een Siiovai i n t n e a'oove ^ a o le s o f
.

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: e s u l t s o f coT.n-.'lor.icnt fi::o .tio n and nyijû.rtinaL icn to :3:,3 on xiice
ir.a!ainized a;;c.i:r.3t f . o o r ti i s s i s .
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th e end o f th e 5 h o u r j.'i:caticn |,e i'io d .
To3 Ls n o t tu;.amined f o r a p -p lu tiria tio n .

TALLd :fV
Pu3\ i l t s o f cOj.T:lo:Jont l'ix a .tio n and a r p 'l u t i n a t l o n tenL s on s e r a o f
13 "noni a l '' sh eep .

d ep o rt

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I 24 hours
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shclcing
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5
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6
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The r e s u l t s p iv e n a re b ased on th e s t i p u l a t i o n s in T able IV f o r r e p o r ts
on th e r o u ti n e complei'ient f i x a t i o n t e s t ,
-Hr

The r e s u l t s p lv e n a re c l a s s i f i e d a s i n d i c a t e d f o r th e t;.b u la t io n o f
T ab le V I I .
The r e s u l t s p iv e n f o r th e 5 ho u rs a t 4 - 4 . r e p r e s e n t she a p p lu t in a t i o n rea d in g 's i n th e tu ile s o i ts o eonp.leiiienL, _Lii.^viiwion t e s t c.t tiie
end o f th e f i x a t i o n p e r io d ,

ac :

A'f ' tico iiip le; i s n t a r j .

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-4 4 -

. e s u i t e o f coi;i.;loLient l'io^ation and a g g l u t i n a t i o n t o s t 5 on s e r a o f
19 "noi'i.u;,l” r a u b i t s ,

n g g lu tin a tio n
R ep o rt

Co.’upler.ierit f i x a t i o n
te st
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±

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7--

T hree o f t h e s e s e r a r e a c t e d to
tu b e o f th e t e s t .

5 h o u rs
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5 ;:d.n.
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slir-.h t o :cten t i n tlio more s e n s i t i v e

T.iBIjÜ :CVIb
R e s u lt s o f com plem ent f i x a t i o n and a s ; - lu t in a t io n t e s t s on
37 r a b b it s irfjaunized n i t h H. i n f l u e n z a e .

;era 01

R r g ls .t i n a t i c n
.e o o r t

Goninlement f i
te st

Of t h e s e 36 t e s t s ^ a l l b u t 11 gave a 4 + i n b o th t u b e s .
The vjcaher r e a c t i n g s e r a , n o n e v e r , gav e a t l e a s t a 3 a u n d er th e l e s s s e n s i t i v e con­
d i t i o n s i n tube 1 o f th e com plem ent f i x a t i o n t o s t .
^ I n t h e s e 23 t e s t s trie a g p lu tin u m y/as I'ath or J n d e f i n i t e and e a s i l y
d is p e r s e d on a g i t a t i o n .

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-4 5 -

■j-'iie l'oisij. uS

01

l e s t s on t t o sf-'ro, oi lr.t)orn.toi ';7 pniiinls corrobornts

q u a l i t a t i v e l y , ;^t l e a s t —t i e r e y o i'ts o f o th e r a u t h c i's .

lo r io t et a l

—

( d ) ,

and. i'ooney e t a l (s6 j found, "norieal" h or 5 0 sers, to lave (H. y e r t u s s l s j
a n t i b o d i e s ; h'oi'det e t a l ( b ) , h van s e t a l ( I f ; ) , ann t i l l e r

( 6 0 ) found

such a n t ib o d ie s in yuinea piq s e r a ; end C hievitn e t a l (11), F erry e t a l
(2 1 ), l i e n s t (1 5 ), -wans (1 9 ), Ivans e t a l ( i d ) , .dueneicens (3 9 ), d r is te n s e n

(ht),

bony e t a l (5 4 ), F i l l e r 1,60), 0I n s te a d e t a l (?u) Found r a b b it s e r a

to r e a c t v iith F . .-ar tu s s i s a n t iy , .n s .

lev e v e r, since Liic t e s t s employed

b;- ti'tese i n v e s t i g a t o r s '.'ere n o t n e c e s s a i'ily o f very hiyh s e n s i t i v i t y , i t
i s iio ssib le t n a t t h e i r r e s u l t s u e rc c u a n t i t a t i v e l y d e f i c i e n t , and th ey
perhaps vicre n o t a b le t o sec t iie complete p i c t u r e n i t h a l l i t s im p lic a ­
tio n s .

These ii.;u lic a tio n s , t h e r e f o r e , via rra n t a b r i e f d i s c u s s i o n w hich

m ust n e c e s s a r il y follow a lo n y two p a th s :
ism o r o r g a n is m s , i f

one d i r e c t e d tc.-.ard. th e organ ­

a n y , involved i n th e p r o d u c tio n of th e " a n tib o d ie s "

i n th e su p ip o sed ly "normal" anijfricils, and tiio seco n d tow ard th e c o m p lic a ­
t i o n s ( p o s s i b l e and. a c t u a l ) r e s u l t i n g from t h e u s e o f su ch e x p e r iii.e n ta l

animals i n s t u d i e s viitii H. p e r t u s s i s , i'epr,r(.iloss o f rue o i'iv in o l th e
a n tib o d ie s .

T here a r e s e v e r a l r e p o r t s i n th e l i t e r a t u r e i n w h ich th e o r y a n ism ,

I r . bronchisp-i'ti c a , has b een d ir o c tl^ ' or in d a r e c t l y incij.no.na.ted to r

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L ions j.n souclic-s vLi. ni it« i-'ar m s s i s »

o r ' c;,)i.LSoi j.Li

'.'.ri.Loio.ls^ inc.i.utLÎiio î 'o.Lùjllsj ;"ulnea j:l' s , ;.nd f'oaos;

out n o t in ro.us o.nd ro.ce.
;:;anisn; i n

^Lowi': u (!?9 } lis.s iouricl

orolth

{oü)

';u ln ea p l::s nnô r a b b i t s ,

a l oc nnc o: non'ioncc o i t h t h i s o r -

/..iiea ( 7/.) s t a t e .: t h a t b r . bronchiseT.)-

t ic ü . îcaj ü(: i.iresenû oni 'niinea o -.ts t n a t liavo never snovjn evidence of i n f e c t i o r i .;ith i t .

S in e e .r - ir o o k s e : i l l e r

"lonr-jnronn" interreIa'Lic>ashijj b.rtoecn
p erh ap s t iie r e b y ir i p l f i n g i novb.ed

( ? 9 ) h ave inade r n fh r e n c e t o tbi;

. bronchioei tic o . and h.

-ertu ssis,

o f vhe n o i'l o f f e r r y e t a l ( 2 ;,) vhro

found b r . b r o n c a is e p tic a n i d e l y d is t r ib u t e d , ojiiony ra b b it,s and. p u in ea p i g s ,
and sh o v ed ( 2 1 ) ( 2 2 ) c l o s e i n t e ^ r e l a t io n s h i p betvjeen t h e s e o r g a n is m s .
r e s u lt cf th e ir cork , i t

seems t h a t

.1. ,

As

r . br o n c a is e o tic a i s i;ioi-e l i k e l y t o

produce p e r t u s s i s a n t i b o - l l e s , th a n IÎ. p e r t u s s i s i s t o p ro d u ce oronohisept i c a a n tib o d ie s ,

k ille r

( 6 k ) h a s d i r e c t l y i n c r i i l n a t e d h r . b r o n c h is e p t i c a

f o r th e i n t e r f e r i n g a n t ib o d ie s v h ic h he found ( " o c c e s i o n a l ly " ) p r e s e n t i n

th e c-onioleioent sei'iri.

t h i s r e p o r t o f b .f i le r adds c o n sid era b le v e i g h t t o

th e s ta te m e n t o f b n iith (oO ) v r i t t e n in 1 9 1 3 :

"The iinport-ance o f th e g u in e a

p ip a s a la b o r a t o r y r e a g e n t t o to;:in s and l i v i n r b a c t e r i a i s very g r e a t

and t h e t.Li.'.e h a s come t o a l l a t t e n t i o n t o th e n e c e s s i t y i o r r i d —

to d a y

d in g th e s p e c i e s of t h o s e i n f e c t i o n s v h ic h te n d t o i n t e r f e r e v i t h i n v e s L ip a t io n s

."

Con s i d e r i n g t h e p ' - i ' t u s s i s p rob lem as a v h o l e , i s i t

not liv e ly th a t

o th e r or'-:an;l.snis^ h n o vn oi' y e t t o be ioiovai, iiiiylit l i ; : e v j l s e n a v e e n e i ' t e a an
u n d e t e c t e d i n f l u e n c e on imn;.'- of t h e investigations reported in t h e literatu re ?

I n a r e p o r t by J p r u n t (S 3 ) i t i s x m p lx ed tr.au t n e p r e s e n c e ox

a P c i l l u s le o is c p t ic u r t i ( 8 2 ) besides b r . b r o n c h is c p t ic a in "normal" r a b b i t s
iic.y in'L cr.i'o re v .ith b.. d e r Lus s ils o i.p c i'im e n b s .
Vnc.-i 11UR h o m o v lo b in o C h ilu s ca n i s
h . p e r tu s s is ,

u d aii-’c. ( k / ,

u.Uj c io n c lo n e d

c.s an oi-panism r e l a t e d i n so:io nay t o

b ld e r i n r f h e n d r ic k 1 2 0 ) d e s c r ib e d a p a r a p e r t u s s i s o rg a n ism

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-

47

"

o f liiULian o i ' I ' it ij ï'ea a ced aafi-;c-;n icall;y and In o t a e r :vays t o d . ' j e r t u s s i s j
anci , .illfc ii (u l^ t’. l s o lo u n d i>ais or'.'c.nisni a;::Onr: iiuiiicU'i c a s c s %
idaanoscd a,s
"w hooping c o w .:i" .

liz u lc a (/^O) i s o l a t e d . 12 s t r a i n s o f or,%c.nisnis from

w h oop in g con ch p a t i e n t s w h ich h e v;as a b le t o c l a s s i f y i n fiv e , g r o n p s .
i'lono o f

oi:ci:i w ere r e l a t o o t o . .. i.n flu a e v .a e , n or i n any d e g r e e o f c e r r a in -

t y ; t o n . [ .' o r t u s s is .

^n. th e o; s i s o f t h e s e rei'iorts^ i t

seem s c e r t a i n

t h a t u n t i l a l l th e or,ga.nlsms i'o la t e d t o h . p e r t u s s i s a r e a c t u a l l y u/iown
and t h e i r i n t e r r e l a t i o n s h i p s o d i a u s t i v e l y s t u d ie d i n r i g i d l y c o n t r o l l e d
i n v e s t i g a t i o n s , i t i s n o t s a f e t o i a c r h i i i n a t e , o r t o h o ld any p a r t i c u l a r
s p e c i e s r e s p o n s i b le f o r i n t e r f e r e n c e s i n s t u d i e s v jith h . p e r t u s s i s ,
in c r im in a .t io n i s

duch

e s p e c i a l l y n o t w ai'ran ted on th e b a s i s o f w ork, r e p o r te d

i n th e l i t e i ' a t u r c , i n w h ich the. animals used had n ot been r i g i d l y p r e ­
te ste d .

h e y a r d lo s s of tine ca.usa.tive a g en t, i t i s obvious, from th e data pre­

sen te u i n t h i s r e p o r t , t h a t i n emi'loyinr la b o r a t o r y anim als, t h e i r blood
or serum in enpei'iiiiental work w ith .1. u e r t u s s i s , one may r i s k a c o m p lic a ­
t i o n which may have a marked e f f e c t on th e f i n a l r e s u l t s and consenuently

le a d t o erroneous c o n c lu sio n s.

I t would be s u p e rflu o u s , as w e l l as a l -

m ost im p o s s ib le t o d i s c u s s :my . s i g n i f i c a n t number o f i n v e s t i g a t i o n s w ith
I'egard t o t h e Aionner i n w h ich t h e f a c t o r o f "normal" cniii'jal a n t ib o d ie s
may h a v e i n f lu e n c e d t h e r e s u l t s b e c a u s e , in t h e . f i r s t p l a c e , i t h a s u n -

do ubtedly been p o s s ib le an soiae cs.ses I'or rnvest:igatoi's uo se u ceimain
s ta n d a r d s , on a b a s is o f p r e v io u s '.u'.perionco, w hereby t h e i n f l u e n c e o f

t h i s f a c t o r could be reduced to a n o n - i n t e r f e r i n g eànieiuiu ev en w ith o u t
a c tu a l iuio.iledpp o f ii.s e x is te n c e .

S e c o n d ly , t h e c o m p le te rnfo:c,nation

ro c u ire d f o r sucli a d is c u s s io n i s o f te n la c k in g i n the I x u o i a t u i e .
Cn th e o th e r hand t h e r e a r e undoubtedly c e r t a i n in s ta n c e s i n which

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

—L^'ù—

L-ii0 '’norriifio." anj.iiia.1 a n tib o d ic s iiavQ in ilu e n c e d tlie course of v a rio u s
investifÇa uions r.iuca i n bue s aire way as une a.ntibodles iri / uinea oiy se ra
h ave i n f lu e n c e d m e b e h a v io r o f tiie com plem ent f i x a t i o n s y ste m .

The d e­

c r e e t o w h ich t h i s h a s waken p la c e w ould o f c o u r s e d i f f e r w ith d i f f e r e n t
s t o c k s o f an im als^ d ejjen d in y upon t h e a n tib o d y l e v e l o f th e rroup a s w e l l

as oj.' wiie jj-io.j.vidua.1 a n im a ls ,

I n t h i s la b o r a t o r y , f o r in s ta n c e , youna

noi’i-:al nu.ce iiave s e e n found to be 100.j f r e e o f d e te c t a b l e ( II. p e r t u s s i s )
antiu o ciio s.

T h is uny n o t n e c e s s a r i l y ue tr u e f o r sto ck s a v a i l a b l e to

o th e r w o r k e r s .

I t w o u ld , t h e r e f o r e , be w e ll to mention b r i e f l y the types

of r r a n ip u la tio n s th ro u y h .fiich the. in flu e n c e o f sucl: "normal'^ a n tib o d ie s
may u n w i t t i n g l y be t ’nrovvn i n t o the in v e sti.f* a t io n s «
1.

Use o f "noi'i'inl" animal b lo o d and s e r a f o r prepn.ration o f the
c u lt u r e jiiedia t o be u sed f o r th e s t u a y o f

c u l t u r a l c h a ra c­

t e r i s t i c s o f the t e s t organisms, and upon which the a n tip en s
fo i' su b se q u e n t wort: a re to be grown.
2.

U se o f "noi'mal" a n iu ia ls (r a b b it.s and g u in e a ui,"s e s p e c i a l l y )
t o d e te r m in e t h e p o te n c y o f a n t ig e n s and t h e i r p r o d u c ts (tœ zL n s)
by means o f h i o r t a l i t y and lu o r b id it y ( i n c l u d i n g sld .n ) t e s t s .

p.

Use o f "normal" c n ü m .ls f o r p r e p a r a t io n o f a n t i s e r a t o be u se d
e s p e c i a l l y i n animial ( u s u a l l y .wousc) p r o t e c t i o n t o s u s or i n
s t u d i o s o f a n t i, o n ic in to rre J .c.t io n s h i.p s among s t r a i n s and
p h a se s of h . p e r t u s s i s , or b etw een h.

l e r t u s s i s and o th e r

o r g a n is m s .

d in c e th e e r e s e n c e o f b lo o d i s n e c e s s a r y f o r t h e p ro p a g a tio n o f ^
p e r t u s s i s , c,.\n th e a n t ib o d ie s p r e s e n t in th e blooci u s e d f o r p r e p a r a t io n

of ti 10 mo .:ia, have so::.e aniiuonce on the o.nti p'.nic s oru.CoUio ox t n is o r —
::anism?

Toomey m Tokacs (Ü?) and ,.'u e t a l ( 9 2 ) h a v e e^xpericnced marlied

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-Z;-9-

clele borioiiG e f f e c t i-e horse hloor: on
trail,s.Lors on g.icj.i’ ■■■'0,a.a.

.

;crtn y o is ciiltiironi a fte i' r e . eated

f.rrt on viuO'Cij es i n niK; ;,ooiia tic jo.vo an e f f e c t

on f . -'Oi-'tussis iins . een noioivn Lç :,orùet
Ï '!: oilfi lioiuici

no.

1 01

nreoonce ox i t s

h e n -ou (6) i n 190? i n some

f i e y ih/iuia t h a t h. ne reus s i s docs

n'on i n the

a n m n u i . (n<croio jic-nio;. a t h6 . ) in liciuici Oiodiiaii; b u t,

as iL .TOWS, i t d'.c..o.e5 a.fluuinoouet and. i'alJ.s to tiic cottoi.i of th e
.OU.0Ù o n tih o o ie s :h

. lo x^'^oiLin I'O;

of '"nut0-0. . lo Gin.htoc " toon
time to time?

flash .

fnccor c o n ri'iu u tin j to niic yciencioenon

o ., ::on :-o ortoo

- variou.s : uthoi'3 from

vr y.'otmms .ronn i n tie; oivoonce oo' a n tib o d ie s could con-

C'sivabl"' ausorb thu.ie aritiwodies from the an hi. un, and a f.flu c in a te spon­
ta n e o u sly when SUS:'aided, i^'- s a l i n e .

,.hat a ls o cun ta.- sa id about the use

of such a n tip en s a i t h adsorbed a n tib o d y i n o th e r aork?
those and o th er

The answers to

ouesu ion s c a i 't a i n l y cannot be found viiuaout i‘ia;idly con­

t r o l l e d oap eriao nt:. -io n .
In em plo^inp "aoz'mal" a.n in al.s to s t u d y th e to :d .c p r o p e r t ie s o f ^
p e r t u s s i s o r i t s pi'o-i.mct.,, co u ld n o t t h e a n t i t 'o i i c s p r e s e n t in th e a n im a l

i n f l u e n c e th e co u rse o f l i e
th e l i t e r a t u r e a i ''

u

: r ,I t s ?

The c lis c o i'd a n t c o n c lu s io n s i n

•— ^ect to t h i s p h a se o f s tu d y m ig h t e a s i l y have b een

due t o d if f e r e n c e s i.ri the l e v e l o.f u n d e t e c t e d "rra tu ri.].'' ...h.v.:nr.p/ o.f one

v ,ario u 3 s to c k s o f a n im a is .
I n th e use o f 'b 'lo m a l'’ anlm .als J o r pi’ouuct.Lon o l s po ci_Lic ,..ntise.Lc.,

;

■ -/.'hut '.-.r.tsi'it can on e ejipect tn e i'osultxnp .‘Liuaune sera, oo i,-e j,.>ocx.;.j.c
Tor t;;.e product in. -. c te d , r f bie am.i... ..fs ai.r'.h..ip
Z'clhted an tib o d ies i n teo.ir

stem?

...ueut i t jiot ao pios.sri.oLo

' .j'oiv'Cb i l l i a c t ' .L .1" ,_1er oo hiicronse une 'v.i. uor

1;

te d

a.ïitlbodios t'u;b are ulreacp' ..u'ostuif?

T e is s ie r e t a l

batT'.nym s e t a l

jjmmmolo.y..i.ci.Hy

o .l

-ue

tni: ^.a m.LhoJ..o ...c ,.ii.J_y l e —

I'ov .u is ..once, ,,van.;- ( 1 ? ),
n o b e r ts e t a l (Y o ),

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

-> Ü o x lv o r t lio r n e e t a l ( V ü ) , e t c .
a a tlb o d ie o ,

claïu aed t h a t t h e y vjerr- 2t i e

to

i'.i'oi ace

( i n some in s ta n c o C j a n t i b a c t e r i a l a n t i b o d i e s ) unon i n j e c t i o n

01 p e r t u s s i s to :n .n j i n one f o n c o r a n o th er^ i n t o e x p e r im e n t a l a n i i m l s ,
v fh ile i t

seem s from ttie u o rk o f io i 'd c t e t a]_ (? )^ l a p i n ( p i ) and o t h e r s

t n a t p e rtiis sx s to:m n h as l i t t l e oi' n.C' an ti^ c i ic p ro ç.c rtiijo .
th a t h lo s o o r f e t
nue;CIS i s

the f a c t

( i p ) consih^ac p e r t u s s is bo::in not very a n t i, e n ic in

i' s i ' i t . c a n u

.:./c:.,use a p o a i'in i^ly m e a n \,i e n ic iL y cn'

t u s s i s to:JLn h a s o een demons t r a ie b
iie a lt h p j end u n i n i t i a t z b : ) .

onla- i n la.;o.-!.'s.T,o]\

ov~

aniL.ials^ ("noi'm al",

from t h i s else.' f c l l o a s t'no p ' O s e i t i l i t p t h a t

o r p a n isn is u h ic h may be t o t a l l j s u n r e la te d , a n t i p o n i c e . l l y ïria^/" a p p ea r t o be
r e l a t e d tliro u y h t h e medium o f u n d e t e c m d " n o n ie l" a n tib o d j.e s a lr e a d y p r e s -

e n t i n th e a n n sa ls u s e d _ o r p r e p a i'a tio n o f tiio " s p e c i f i c ” a n ti s e i'a .

This

p o in t ca n b e s t be e m p h a sise d by c c n s id e i'in i th e e x p e r i e n c e s o f bi.ii.th (30)
I 'i t h i n f e c t i o n s in la b o r a t o r y an im als^ and nio s t a t e m e n t t i i a t ".hi a cpu aint i n c e ,.i th t n e s e i n f e c t i o n s i.s e s s e n t i a l ir> £.Yoin:lnn s e r i o n s m is in ie rp i'o l a t i o n s —i n th e iii.m n io a tio n of anim als .ior n. ;p iu iin in s and o ih e r c r i t ib o a i e s ."

Judpin™ from t h e r e s u l t s p r e s e n te d i n t h i s report
t h e s tu d y o f p u in o a p ip s e r a e s p e c i a l l y ^
o f b m ith i s

i t is

vdidi ropard t o

.e v id e n t th a t t h i s sta te j.:o n t

f a r i.iore i n c l u s i v e tlu u i th e w ords i i p l y .

to per.LorO'. n s a t i s f a c t o r y

j

Cort;\in].y_, i n c r .ie r

com jilo...ont f.'Lxation t e s t i n a stuoy o f o a .p e r i-

m e n ta l m a .t e r ia l, i t i s n e c e s s a i y t o h ave on hand .a com plem ent serum w h ich
is

frvio o f a n t i o o d i o s t h a t ai'e ca^.'able o f r e a c t i n g w it h th e t e s t antiyen.

fLcroin a t l e a s t l i e s a p la u s a b le e iq ^ la n a tio n fo i' t h e r e s u l t s o b ta in ed ^
d u r in y t h e c o u r s e o f p r e lim in a r y w ork; i n s t u d y in g th e n a tu r e o f th e a n t i G o m p .le i;ie n ta r y

1.
lo ts

of

a c tio n o f

l l

,

;.)er t u s s i s

a n 1.1 y e n s .

Tiie v a r i a t i o n i'h aiiticom p lern eiiü aI'y a .c u iv it y m t h Li.i. i, ± r-in t
com plem ent due^ a s i n d i c a t e d i n T a b le :.IIc^ t o d i f f e r e n c e s

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

-5 1 -

iii tiie aiiuLoody c ont e u t ox' ta c ccjaixLeixeïit serx'.,
2.

jJie anbicojn.pleîiien'oarj' a c t i v i t y o f a n tij/e n f o r c o n s t a n t

aniounts o f a /'iv e a Cvj.wler.ient rem ain ed c o n s t a n t o v e r a vviào i-an^re
o i onL.x';en dilunioiiG b e c a u s e c o n s ta n t ojoounts of a serui;i co n ta in

a c o n s t a n t l e v e l o f a n t ib o d y .
3.

'i'ho in c r e a s e i n the a.ntico!;iole;v:enta.iy a c t i v i t y o f a n ti r e n s

t t n r.acx'xaso i n vno xmount o

c. ix^.leixent scm /. xiseu^ v;as due r,o

i n c r e a s e an one a.;xornt o f antii^cdy availahl.e i n the J.aryer a'ticunts

of sei'Uix.
The anticox'joleiXLentar;/^ a c t i v i t y o f .'.n tir e n s in c r e a s e d .; it h
th e a y e o f th e coxT oleuent serujii b e c a u s e o f th e d c c i'e a s e i n c c n o l e n e n t a c b i v i t y v a iich n e c e s s i t a t e d u se o f la i-y e i' am ounts o f com plem ent
serum tiioreby i n c r e a s i n g th e aiuount o f a n t io o d y a v a i la b l e t o the
n n u ify n .

A com plem ent seru ia, vtiich a v o id s su ch c o m p lic a t io n s as t h o s e , i s
(E v id e n tly a ssu re d by the co:uple:.aent s e l e c t i o n

e r o c e d u r e s described, h e r e .

Upon sujjci'- ' i c i a l a n a l y s i s of some o f th e d a ta r iv e n (T a b le s VI and

V II) i t viou ld seem that the " stan d ard " p r o c e d u r e h a s l i t t l e advantage
o v e r t h e "preliminary" p r o ced u re i n com plem ent s e l e c t i o n s in c e i t u a s
shovm t h a t 1 2 . 7,J o f th e y u in e a p ip s e r a t e s t e d by th e form er c o u ld be
c o n s id e r e d s a t i s f a c t o r y f o r u s e as compilèrent, w h ile on the o a sa s ox tu b e

4 o f t h e latter th e r e w ere
fo i' th o cobiplexient s u p p ly .

2 4

j w h ich c o u ld be c o n s id e r e d s a t i s i a c b o r y

I n f a c t , th e " sta n d a rd " p ro ced u re seem s t o be

much l e s s s e n s i t i v e th a n tu b e 6 o f the "p 'r e li.r a n a r y '' p ro ced u re i n i A lien
o n ly 1 1 .6): to 18.0,.: of tho total were satisfactory; and in such a compari s o n , t h e " sta n d a rd " p ro ccu u ro would seem oo have no va,]-ue.
Ilow ever

is it safe to assume that the an im a.ls represented in Table

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

-5 2 \^I and V i l ai'e conv--

ul6)

j,n tn e

s o u r c e , b iu d and i ^d- »
utij'v.aOk'Jii ;:,nd

u rn n ^ d ia

j as L ;j<-cause che./ vjere o b t a in e d from th e same
sarae colon;/?

I'hero a re un l o o t o n l y i.iany

-"Cioln ''U i'ia o le s anvolved i n

oi tn e ana ria ls r.iiit-' <5an

h a n d lin g and housing'

!.'ec"o t i i e i r s e r o l o g 'ic a l o i c t u r e , e s p e c i a l l y

{r.

i i tile r e s p o n s i b l e ■• v-nt dm & o a c te r iia ia
n u n i c a b i l i t ; / o f '«.'hie..

t h e

o r o n c h i s e p t i c a ; , t h e cor.i—

t s f i o t c c .m ily c o n t r o l l e d i n t h e b r e e d in g p e n s and

s to c k c a -io s .

E v id e n ce o f n' io u s g r o u p s viitfiin

is '’h e üiax'kea v a r i a t i o n i n I ' e s u i t s onion,a th e v a r ­
ch s « fi« s .

a r e s e v e r a l grou p s I a ^

b,

g u in e a p i g s e r a ,

are

I n f a b le V I , f o r i n s t a n c e , v;iiile all e r e

S, 10, 1 2 ; i n v;hich t h e r e were no n o n - r e a c t i n g
o th e r s

p e rc e n ta g e o f them.

1 5 , I d ; i n w tiich t h e r e w as a g o o d

i n Table V II t h e r e i s c o n s i d e r a b l e v a r i a ­

t i o n i n th e percent'-

o f n e g a t i v e g u in e a p i g s sjiiong th o v a r io u s g r o u p s .

I t u o u lü b e natui'aX - o

s u s p e c t v a r i a t i o n s i n r e a c t i o n aiiionp: t h e g;roups

t e s t e u by one . iv e t

i-octtd 'u re, t o b e due t o v a r i a t i o n i n t lie p r o c e d u r e ;

an d d u e t o l a c k o f

fin ite

c a n n o t s a f e l y bo d c s l / . d ,
u e r e I’a t i i e r r i . i e ur ^

e v id e n c e t o th e c o n t r a r y ,

su ch a p o s s i b i l i t y

Cn th e o th e r hand, th e c o n d itio n s o f th e t e s t s

ctLd -'lot a l l o i j niuch

c h a n c e i o r v a r i a u io n , e s p e c i a l l y

s i n c e n o o n e but 'Uio t v r it '- t ' nas had a n y p a r t w h a t s o e v e r i n iie r f o r m in g
tlierii, t i i e r e b v , c;lso '—ZLihl'iei.'5in,g' souits u c;ch m .ca l x a c Lors »

xhei s f o r e , u n t i l

t il e c o n 'd it io n s i n i.''-' *

brc-cxing' cuid. s t o c k eagres c a n be i i ' gi d i l y c o n t r o l l e c i ,

t h e o n l y w ay in uhic. t

th« tv jo p r o c e d u r e s

ca n be com pared i s

o y p e r f o r m in g

b o th o f 'tnetTL on ohc -louiie f-rin iia l s e r a a s vjas d on e u i h i tn e ,^roup ox _iü
n u i ne a p i g s b le d 1/-,-a 3 / 4 6 .

F ro m t h e rcsu l'k '-i

, / i v e n in T a b l e s VI and V I I i t

t h e s e v e r y id g id cu . i i t i o n s
nj - s e r a

w o u ld

be

w o u ld s e e n p . i a r ,

under

o f p r e t e s t i n g , a v e r y lo w p e r c e n t a g e o f g u in e a

v c L n d ^ n r v ic o a b l e a s co m p lem en t w ir h a c o n s e g u c n b lo w

R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-5 3 supi-dy Ox blie r e a g e n t .
ti-Lie;

but i t

xn t a i s l a b o r a t o r y , t h i s h a s un t o r tu n a L ely b een

w o u ld n o t n e c e s s a r i l y agpl:y t o o t h e r sto cJ cs o i gua.nea p i g s

w h ere t h e s i t u a t i o n L a / w e l l b e d i f f e r e n t ,
uc u t t e r l a l l a c y

t o r e l y up on l e s s

c n t h e o b h er haaid, i t w o u ld

s t r i n g e n t ju eth o d s o f p r e t e s t i n g j u s t

b e c a u s e s u c h .;.etiXoci& a ll o w

o n e a ;_,i'tjauei' 3u p ]jl\ o f co ju o letu en t,

le s s

, y r c t e s t i n : L i o t h o u r s c:..p au le o f d e t e c t i n g

o l vjhetiicji' 0 .1:' now t iie

LuOiii, t n e j.n ti-:rierx rig a n t r o o d l e s
t h e i r p r e s e n c e w i l l s w x l l a ll o w
t h e b a la n c e , o f t h e s y s t e m ,

heg'ard—

.y o ï d i j . oe p r e s c i i t ; and y.-hat i s

Lioro,

tliem t h e opi ' o r t u n i t y t o i n t e r f e r e v ilth

f h i s h as a lr e a d y b een i l l u s t r a t e d

by t h e w ork

w i t h 1 - 1 3 , a ‘b jro lx L u n a rp " cour^lom ont, s e l< ; c t e d b y a p r o c e d u ie e v e n more
s t r in g e n t th a n i s

c o n s i d e r e d n e c e s s a i y b y h o lm e r ( 4 5 ) h a r r i s ( 3 b ) and

th e o th e r s fo r s e le c t io n
p o o le d f o r t h i s

o f t h e " ./a ssern ia n " co m p le m e n t,

complement s t i l l

c o n t a in e d a n t ib o d y i n h ig h en ou gh co n ­

c e n t r a t io n t o be d e t e c t e d by th e verp r i g id
h llb ),

and t o

dome o f t h e s e r a

" sta n d a r d " p r o c e d u r e (T a b le

c a u s e ii.ark ed e f f e c t on t h e am ount o f a n t ig e n t o l e r a t e d b y

t h e s p s t e a l ( T a b le s

a I I c

I 'll r th erm o r e , i f
t o t a l l y ig n o r e d ,

and -TC Id).

t h e a n t ib o d y c o n t e n t o f t h e g u in e a p ig s e r u m i s

and t h e t e s t u n i t o f a n t i g e n i s

determined b y t h e " a n t i -

co.niplementar^/^ d o s e m e th o d , " one c a /i c;xj3f;c t m arked v a r i a t i o n i n the, a n t i g e n
c o n c e n t r a t i o n t o l e r a t e d b y t h e t e s t 3,gsteiii from d a y t o d a y ,( d e p e n d in g on
t h e a n t ib o d y c o n t e n t o f t h e : u i n e a p i g s e r u m ) , a s w e l l a s r is j c t n e p o s—
s ib ility

o f h a v in g a s y s t e m w h ic h w i l l n o t t o l e r a t e th e q u a n t i t y o f a n t i -

g e n n e c e s s a r y f o r r.gui:Lmum s e n s i t i v i t y ,

.k g p la n o .tio n f o r t h i s i s

B y r e s o r t i n g t o t h e " .in tic o m p le m e n ta r y t i t r a t i o n "
a u t h o r s , t h e v a l u e o b t a in e d i n t i t r a t i o n s

is

s im p le .

method proposed b y other

t h e l a r g e s t am ount o f a n t i g e n ,

w h ic h , i n t h e p r e s e n c e o f coimplciiierio sei'Uin a l o n e , i s

j u s t bar'..;lg Ccijjaole

o f reta rd in g coiTijjloment a c t i v i t y to an a p p r e c i a b l e e x c e n c .

in e p i c o o n e n t s

o f such a m etiiod u s u a l l y recom m end, a s a test d o s e , an am ount enual t o l / 3

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

-5 4 -

or

1

//^ t h e anticom ploB ientary 4 o s e .

d in c e th e evidence alread.v jn-eaentcd

i n d i c a t e s such ''a n t ic o r ip lo m e n t ary " a c t i v i t y t o be due to a n tibo dies i n
th e complement sei'uiH; such a Lust aose i s i n r e a l i t y l / ^ to I / 4 o f tiie
es

0

;a'.iOLi,n Ü-

suCii C..I1

c± c.aii-'i^Gii

o^.

ca.ole

i^cv.c uany ivj. on uhese ariI/j_bocJj,os to

L i.Vj oO Iji'ijiy iMOout an a.pin'ocicLijls j..iavbion oi ahe cjimj 3_o—

aient i.'hich i s rJ_so y r c s e n t i n th e , iv en auiount o f ,ruinea
lia v u r a lly ^

i i th e comple:_.ent serum con.,;\ins

serira.

hl. h ccncentratiun e i a it.iijo iy

Lhe s o - c a l l e j. "anticom . le io e n ta iy " o.cLiviLv ir, (ajiv'esporidinyly u i-h :
o a l ; si.a.ll

amounts o.:' a n t i ; 'en can

e t o le r a t c i .

and

un the other aandj i f

th e complement serum has a Ion ccncen'Lra'uion of anLiboüpj coriv:sy.;o.ndinply
l a r n o r aj-iounts o f a n v ip e n ca.n oe t o le r v .te d .

i n th.is l a t o r a t o i p , cha.nces

f o r oho o c cu ren c e o f th e l a t t e r c o n d it i o n ha.vc ;_cen very rare for a s in d i c a t e d i n Table t l i t had n e v e r been p o s s i b l e , before adoption o f th e
comolei.ient s e l e c t i o n pi'occdui'c^ t o employ 'uiuiyons fo r coaplement f i x a t i o n
t e s t s , i n c o n c e n t r a t i o n s above

2

b i l l i o n / m J .. (e q u iv a le n t to

200

m illio n

or- 'anisms per tube of tlic 'uest ; .
e v id e n c e in
fo u n d i t

th e l i t e r a t u r e

ir r d ic a t e s th o .t o t h e r i n v e s t i p c t o r s

e c u a l l y i m i i o s s i b l c t o u s e t h e h ir h e r . c o n c e .n tr a tio n s t o l e r a t e d b y

a syotcTfi i n u h ic h a s e l e c t e d com 'plei.icnt i s employed,
c u lt

h ave

a t i s r a t a e r daff]_-

to c.nalyC'0 i.iany 01 t h e i r r e p o r t s i n tei'm s o f amounts co iiiy a ra b le to

chose e.iUi'-'l oyocL b;/ Ti.io u r j_ue r , u'e cause .'aOtjù j.i ive 5 t l a, u.Lon^

d.Ov ....

i/P

t o 1 / 4 o f th o a n t ic o m p le m o n t a r y u o se " (.vjh ich , o f c o u r s e , y iv o s no i n d i c a -

t:i.on o f number's o f or.p'£.nisri3 ;» b u t a fevJ o.mai.ayles can be r iv e n .

C a ie v itz

c- 1Lever ( 1 1 ) iv ere a b le t o u s e 4 0 0 . . l i l l i o n o r r a n is m s f o r ea ch tu b e o f t i i o i r
te st;

and tho same w as t r u e i n uho u ork 01

t h e same p r o c e d u r e .

(6 b;

100

m illio n ,

have s p e c if i e d

1

acooscn

. : loyoi' ( Al j \'.uio used,

1

^ a to n (72 J v;as a u le t o use -cOG jid.ll_on., Olali e: uuus^.pi

oryanisias f o r each tu b e o f tn e t e s u s .

btnei' a.u u n ors

t h e c o n c e n t r a 't io n l.u t h a v e n o t p iv e n s u f f i cien u c.a u a ,rom

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

“

54

~

or 1 / 4 tiio a n tico m p lo iu e n ta r y d o s e .

J in c e th e e v id e n c e a lr e a d y in -esen tod

i n d i c a t o r su ch "anticoriijlef.ien tary" a c t i v i t y t o bo due to a n t ib o d ie s i n
th e coHiplemcnt sormi^ su ch a t(;st i^ose i s in r e a l i t y 1 /3 t o I / 4 o i th e
la r .'/o s t amount o i a n t ig e n ca ija b le o i rea c tin ':

.vith tjie s e a n t ib o d ie s t o

su ch an e n t e n t a s .0 lu'iny ^ibout an a p a r e c ia b lo i:L:.ation o i th e co irp lo :..ent n'hieh i s rJ.so ...I'csent i n th e , iv e n auiount o f ,auinoa _i,"; seru]:i.
h a u u r a ll^ h di' the. coap lci-ien t servuu cun .a in s

.:i. ,:i c e n c e .n tr a tio n oL' e.n: ibod-

uiio s o - c a l l e a " an ticom ,.leoen tr..r2 " c .c t i v i t v i s c u ri'esn o n d in y ly ui./h ; and
o n ly 3;. a l l ai'i.oy.nts o i :.n ti./o n can

e to lo iaeoeu..

on t;u> otJier n a n d , i f

uhe com )l(5.uona soiun. has a ].o.v coric-. n tr a e io n o f a.niy%acci;;^ coia-es so n d in y ly
l a r p a r ynnounis o i a n ti; on can ce t o l e i ' c . t a d .

t n i s l a l o r a n . n ' v , ch an ces

f o r Lae o c c u r e n c e o f t h e l a t t e r c o n d it io n have f.cu.n v e r y iv.rc: f o r a s i n a ic a t n d in i'ab ln %'I i t liad n e v e r boon I 'o s s ib le , b e fo r e a d o p tio n o f th e
coaiploiuent s e l e c t i o n ^.n-ocedure^ t o eryyloy .-.nriycyis f o r couplcinent f i l i a t i o n
tests^

i n c o n c e n ti'a tio n s af'ove 2 b i l l i o n / c l .

( e a u iv a le n t to :.00 r n l l i o n

or a n is c .s p er tuLe o i th e t e s t y .
E v id e n c e i n th e l i t e r a t u r e in d ic r .t e s a h a t o th e r i n v e s t i y a t o r s have
found i t o c u a ll y i/y.j..ossiblo t o u s e th e h ig h e r c o n c e n ti'a tio n s t o le i'a t e d by
a systecL in n h ic h a s e l e c t e d corcplei.icnt i s cm yloyad .

I t i s r a tlio r d i f f i ­

c u l t t o a n a l/n e many o f t h e i r r e y o r t s in tei'ms o f ci.iounts comwarable t o
t h o s e e m oloyod by th e a r i t e i y
to l / h

be ca u se

:.;Ost

iiiV'..;sti ya ïu o n s eiyyloy

‘'1 /5

o f th e a n tic o m p le ia o n ta iy aose" ^.vchicn, 01 course^ y r /o s no a n d ic a —

t i o n o f numbers o i o ry a n isn is;. b u t a fe n cn cn iolos can bo y iv e n .

G h iev itz.

b I n v e r (1 1 ] u e r e a b le t o u s e 4OO . .l i l l i o n or(;anism s f o r ea ch cube o f t n e i r
t e s t ; and th e same y a s t r u e i n t h e so rb o f J a co b sen b L^oyer (h .l] who u sed
th<7; ocuro o r o c e d u r c .

i

a t on

(?ij

m s a. o le ro u se uOG iid.ll^.Oj y Gl.-.n c: i^unsw.yi

( 6 b; 100 n d l l i o n , o i’y a n i siiis f o r each rube o f une t e s us,
have s o o c i f i e d

tu n e r c-.ulnors

th e c o n c e n t r a uion out have n o t p iv en s u f f i c i e n t d ata from

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-5 5 -

;^iica Lo calcuAaüe biie iiu:iihc^r o f oi'._;ari;:.SLis usoci oyr f u f o :
ot-.'i'ii.K.iii'.

.1.,;y Uoou cl ]_u

j u s o o n o lo n :

D a ii-h trj-

. oil0;-'0nsj.on^ .oooiiiJ.d ( l o ) n 2 o i l i i o i i / i i L .

fv iin s 2 . i i i t l a n d

( 1 ? ) an :JüO r i i l l i o n / r l .

o n .io e n s lo n .

I f hcu been sbonn (Tnoln 2 I I e j uhnt ,vlt:n n biojical a n t i ;on snrh as
th e one ei.ivlo/ed;, th e conciuiLration of a t l e a s t o .J b illlo n /r r il. l o r b;0
- i i t i . o n CO’ " :ilsiio

" r i.ia-o oi' ene l o s t j la.o aecesooT;/' loi' ^ii'octest sens 1—

a i v i t y o f th o s .:s tio .i.

p e r tube o f tee t e s t
, cper 111./----

c o n c e n a r o .tlo n oi' f .Y

e illio n /ja .

(o r

jiiillio n

Vo u iillio n ;ioi'e tiian the aioount used b;/ C h io v itz V

) re su lte d

In a l e s s s e n s i t i v e s,ystcu^ ano i f th e ni'itci"' s

e s t i m a t i o n o f th e le.iou n ts oiiip loyed ey ou h er '..'orkors j.s c o r r e c t^ i t
i-atiioi' c e i 't a in

t iia t veip; fcn^ i.f a n y , o.f th%i. ./ere o.:.le

I u; not'ip; of i.ntipen necessai'p f o r iiss.:L,:u_u sonsi oivfiy/ .

seci.is

,o e n p lo y the

tuns in; : b;;- the

n r iu c -r 's n ::p e r ie n c e , t s r e f o r e ^ i s i s s'ei'y 11 sol;, t n a t they^ tooj nod b een
.ieolin ;_ s i.t il .'in t io o d ie s p r e s e n t in
h ave d e f i e d

tiie oori leni^ n t sei'u ..; .o n tib c s .ie s iV iieh

' e l e c t i o n b,;' i.ho no tho se a v a i l a b l e i.a the t i n e ,

i f tn is is

'.nie^ i t i.s a l s o o v ia e n t isio.t .such a n t il'o s lio s c o u ld h.ave ixuin lo .r y e ly r e s 'o n s ib le f o r t h e .f a ls e r e s c t i o n s r e p o r t e d i n tiio l i t e r a t u r e :

p 'a ls e noc-a-

t i a a s L.ue to Ion Loi iiunce of she system f o r su.Lflunni. :'s,.'.nv,.nn.^;s o.i
- n i l on ; ana f a l s e

o . j i t i v s s su e

so p a r t i a l ;

f ix a t io n o f c o iils v ie n t by

t:nn . n t ,i .odiios c o n to .in e d i n t a o com plem ent so-runi.

JU j

c o u r s e o f oho .loihi le a d in p t o tine - r e s e n t I'e p o r t, a t u a s

n ls o found t h a t r e a c t i n g su b stan ces i n ''uinea p if. sera i n t e r f e r e d greaoly
with complement f i x a t i o n tests for s y p h i l i s and gonorrhea, n e c e s s i t a t i n g
s e l e c t i o n o f a " s p e c i f i c ” coiapd-ernent x o r each a n t ig e n used.

-f'f jo... or ._.nce

of t h i s cannot i:o o v e r e s t i i i . t c d , s in c e .it i s v ery p o s s i o l e th a t j.i..!.lures
in t e s t s

f o r o t h e r d i s e a s e s : ay have i.u m '.h.v' t o .la iJ r r e ac e l i . - . n i u e j.roZ:i

the: ccap .lom en t s u p p ly , t'uiose s e r a .;h ic h nave " a n tib o d ie s "

capa i e o.. iec.C'a.ng

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- 57 -

Sm3/IARY AND CONCLUSIONS

I n much p r e lim in a r y tjork l e a d i n g t o t h e p r e s e n t r e p o r t , t h e r e w ere
e n c o u n te r e d marked i n c o n s i s t e n c i e s in th e b e h a v io r o f H. p e r t u s s i s a n t i ­
g e n s , w h ich p r o h ib it e d e s t a b lis h m e n t o f a s ta n d a r d iz e d com plem ent f i x a ­
t i o n p r o c e d u r e w it h any d e g r e e o f f i n a l i t y .

S im ila r d i f f i c u l t i e s have

a l s o b e e n a common o c c u r r e n c e in t h e s t u d i e s o f o t h e r i n v e s t i g a t o r s , and
t h i s h a s r e s u l t e d i n many c o n f l i c t i n g r e p o r t s .
s tu d y ,

The r e s u l t s o f t h e p r e s e n t

h o w ev er, ca n n o t f a i l t o p la y a m ajor r o l e in s o lv in g some o f th e

p ro b lem s in v o lv e d b e c a u s e o f th e f o l l o w i n g .
1.

R e s u l t s o f p r e lim in a r y work in d ic a t e d t h a t i t was n e c e s s a r y t o

e s t a b l i s h a p r o c e d u r e w hereb y a com plem ent serum known t o be (H. p e r t u s s i s )
a n tib o d y f r e e c o u ld b e o b t a in e d .
2.

Such a p r o ced u re w as d e v i s e d , and by means o f t h i s p ro ced u re i t

was fou n d t h a t t h e a n tic o m p le m e n ta r y a c t i v i t y o f a n t ig e n s was r a t h e r due
t o a n t i b o d i e s p r e s e n t in t h e com plem ent serum .

I t w as a l s o - e v i d e n t t h a t

a v e r y h ig h p e r c e n t a g e o f t h e g u in e a p ig s t e s t e d c a r r ie d such a n t ib o d ie s .
3.

Though e v id e n c e in th e l i t e r a t u r e seem s t o p o in t t o B r.

b r o n c h is e p t ic a a s th e a g e n t c h i e f l y r e s p o n s i b l e f o r t h e s e a n t ib o d ie s ,
o t h e r p r e t e s t i n g m eth o d s,

su ch a s c u lt u r e f o r t h e s e o rg a n ism s from t h e a i r

p a s sa g e s , a g g lu tin a tio n t e s t s ,

e t c . , a r e e n t i r e l y in a d e q u a te f o r s e l e c t i o n

o f com p lem en t.
A.

By u s e o f a r i g i d l y s e l e c t e d com p lem ent, i t was p o s s i b l e t o em-

u lo y c o n c e n t r a t i o n s o f a n t ig e n f o r g r e a t e r th a n any r e p o r te d in th e l i t e r a ­
tu r e .

A ls o ,

c o n c e n t r a t io n s f a r in e x c e s s o f t h a t n e c e s s a r y j.or maximum

s e n s i t i v i t v o f t h e t e s t had no d e l e t e r i o u s e f f e c t on th e com plem ent.

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

- 58 5.

In. com plem ent f i x a t i o n t e s t s on s e r a o f o t h e r a n im a ls ,

found t h a t t h e "norim l"' r a b b i t s ,

i t was

s h e e p , and horses i n t h i s la b o r a t o r y have

{H. p e r t u s s i s ) a n t i b o d i e s , w h ile t h e n orm al young m ice do n o t .

These r e ­

s u l t s , h o w e v e r , do n o t n e c e s s a r i l y a p p ly q u a n t i t a t i v e l y t o a n im a ls a v a i l ­
a b le t o o t h e r w o r k e r s , and i t

i s a d v is a b le t h a t a l l s t o c k s be p r e t e s t e d

in order t o a v o id the c o n c e iv a b le c o m p lic a t io n s w h ich may r e s u l t from use
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6.

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immune aniiTials in p e r t u s s i s s t u d i e s .

i s a l s o recoimiiended that t h e g u i n e a p i g s e r a t o b e used f o r

c o m o lam e n t i n t e s t s w i t h o t h e r a n t i g e n s b e s i m i l a r l y s e l e c t e d .

Reproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.

- 59
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