ELECT ROPHORETE‘C AND EMMUNODIFFUS'ION STUDIES
OF NORMAL AND IMMUNE SERUM FROM
GERMFREE AND CONVENTIONAL CHICKEN?»

Thesis for The Degree of M. S.
MECHEGAN STATE UNEVERSITY
Gary F. Dardas
I964

114E513

LIBRARY

Michigan State
University

 

37::LJLCTROP1-iififi5’31C :63) I'LEE'JUFOEIIFFUSION STUDIES
OF 1:0an #31) Emma SEPUhi mom
G'I‘T‘ .m-m CCEV‘E‘HTIOHAL CHI GREENS

by )
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Gary Ff‘Dardua

Submitted to
Richigan State Univeraity
in parti&l fulfillment of the requirements
for the degree or

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EASTER GP 90131

Dapartment of Eicrcbiology and Public Health

1964

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The author extends sincere thsgks to Dr. C.K.
Smith for his inspiration ana guidance throughout
this study. His prompt Satisfaction of needs and
genuine interest in this research.has been greatly
appreciated.

The counsel and suggestions of Dr. R.B. Durdos
and Er. T.J. Dordas were also appreciated.

To my parenta go a special thanks for their
continued inspiration, their fine example and de-
votion to their family.

To my wife for her patient understanding and
gentle encouragement goes my sincerest gratitude and

aamiration.

11

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vii

ETRQDUCTIC;

In sgite of their well established antibody
forming potentials, certain peculiarities in.the avian
immune mechanism hove discouraged the use of the
chicken in basic immunological research. The nature
and explanation of these idiocynoracioo are points of
great controversy among workers in this area. A much
debated issue involves the uncertain composition of
soluble antigen-antibody complexes formed in different
salt concentrations. Several attempts have been made
to establish the chemical and biological nature of
the voriouo constituents but as yet no completely
satisfactory explanation has been presented.

fintibody heterogenity has rooa;o-J been il-
luminaued by the acquioition of new knowloago con-
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primary and sooondary responses. From studios of a
variety of mammalian and nonmommolion vertebrates, a
general proyerty of the antibody forming mechanism
has been recognized. It appears that the first anti-
body type to be produced in reaponso to stimulation
nith a suitable antigen is of high molecular weight
(usually 198). This antibody usually, but not neces-

orily, migrates in electrophoresis as a beta2 or a

gommoluglobulin. Following prolonged immunization.

1

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body activity under some conditions. These antibodies,
Called imTano globuli1o , have bean intono .voly studied

in some 3161i a 23 re om1rés their phyoicol 1n£ im-

manoo11omioal

clarify some oopocto of tho primary antibody ros.onoe
of 40-doy-oli cormfroo and con ontionol chicks. This
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puriflcztion of one pri11rv roogzn1: anti-novine serum

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human gamma-globulin (HGG)-s ensitiz ed chickens was
lower 9999 tllut of their res active controls. Hora»
over, the enti~P GG res case had 9 shorter induofiion
period and apparently imposed some inhibition.upon
the reayonue to B39. Eben EGG and hemoglobin were
administered simult9ne wly, no antibod' 993 produced
to the humoglobin but the resyonao to HGG was d1-
minished. Gold and Benedict (1962) stud led the pri-
m9ry and seconflary responses to para-amiuobenzoio
acid (IEBA) and bovine g9mma-globulin (EGG) in adult
chickens. A primary induction period of 72 hours

w9 3 sufficient to provide de+eot blo antibo‘ ;y union
reached a maximum titer in.5 to 7 days. fifter peak
titers were attained. the antiaBGG antibody was rgpidly
eliminated in t the anti-P Bi am ibg dy persisted for 70
days. The authora suggested tb9t a slow rele9se of
anti-hapten a.ntioo&y couli account for the unu9u91
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119110919999 01’ the prim:r'J res-901199 to 1331 1-93 studied
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all phaoes of the antibody r9zponne, ie., shortened
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longer persi9ten¢9 of antibody.

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fr otiwn conwirtca cf moi eculeu aiti $111dut tlon

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3333 3333-3 33 to 3333333133 with 2~“3 ant 2 rasisw

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tanfi 3133tro:33r3.1o £11331333 “firfi 33333133. 33333n

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t 33‘3333 E—EB 333 3130 33d 2 reesist 311$ 33333. Ultraa
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inti 3333 fr3213 P““”"-13:333 3313.2113 £~V3 (131. ct}?

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63 €333, Iolfe 333 333333 (1951) 333..3 31 33

t

“*11333‘3 n 33 £33 3 af 3313 an the precifiitin r3—
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CA 01—AC:.":1 31.211.11.11“ 1. .3110 £53.. J $-01 fin? (‘11....1214333.

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14

The? painted cut tfifit the effect of high salt ccncen-
trrticm W713 exm‘tfire’e 8.3033 thv‘.‘ 17-. €011.in CC: 01. :‘:7'1;3. .31 of
antigen :33 ratiboiy cud at“ not a nenspecifio 833ting
:ffect. Gaufirvn uné fiymgry (195?) confirmed the
Bancifi citv of thn high salt effect on the preciritin
reactinu vith nvie' n {3313 (rum. In zxrotrer sin y, Gooé~
mun, folfa uni caliber: (1954) nctad thst difftra3t
03ti0n3 Vt‘rie d in fizeir ability to cause precipitation
of zdn Jr~w-H3333333 333313333 at the afime 10310 3trength.
Ffiutscn, Nichol and Cara (L #9) observed t34n
593 of the 33eci£ic precipitate farmsd with ahickon
33313053 and 333 cansiateu of an alghmaglobulin. They
found that thia conconvnt inc;og.t diril3 1.3 3:11-
zatian tn» ceuii be raxcvei my 4;: 333103 1.: the
Specific pr 0131+3t3 An.incrnasa in the hetag-glcbulin
fraction use} 3’: “WA for tim first “ti;:;:-'t "1th fine “Pi-P ',
31 333333. fiznovitz 333 Tolfe (1393) rape tzd the
experi;3ent3 0f fiautach at 31. and cezu L3 nw:t “_3112333
tbs regultfi. The? stutei that if the alphauglobultn
was present it was oifiher m"*~~n 0r insoluble in 3331-

333 330333. ognavitz, Sings“ um olfe (13:4 could

suggaated the greseuce of two 33313333 tygcs en the
L3813 of tro 31:23:333 3313312 on“ -33 yeka in elec-
troghoresiao

wo- .. ., ‘41 ‘g‘ 0 {33:7 ‘\ . ,‘ 1. '. "
veuhozi3n :2“ .0433 (1;;01 nets; t3a 9113c: of

15

dilution on the quantity of precigitgte forxkd with

the primsry rfisyonse BS , Lani-Rmx Lgbuono They found
& d¢= cr ease in the 5:0 r1t of precipit1+e farmed when the
antiserum was diluted and prep sad that flutibody multi-

glioity :ccaunted for thir effect. They also observed

PA

greutcr LT?Ci§it”tion when non-dgcomglementod enti-
23 “im “:5'u32d. Since the amdunt of complement in
these Serums was too lowt to accc“fit for this, the

posribla L vnlv,flnnt cf a non-specific ccmponent

3.1.

uhic h 00- ~p. L tate£ with tha complex was considered.
Genzoni;n and ”c1lfe (1353) studied the effect of ageing
antiserum or the precipit* re¢ctinn. They conclufied
that & co~xr€"'”it ivv frctor was indeed present in
scram £33m normul and Jug-Sensitized chickens. The
idfintity of a canyonfint 0f canfilflrent Iifzd the co«
precizitrfi f ct or :33 di so 1 imrd. hey found that
aguirr scram at 4 C or at ~20 0 resulted in: (a) an
incrc;sea interf Ciel antibody titer Wham titrated in

*5 Salt, (b) cecceu ei tctul nntiboiy H precipitated

I

in 83 salt and (c) incrc::c wt 1 "r>clpit"t94 R in
1% salt. They pointcfi cut that inactivation of anti-
serum by versaLe was :reffl able to heating since the
lattnr incrczzci co-grecipitation 1n 8% salt.

Oriana, Rose and Clapy (1952) demcnstruted the
very small complement uptake by the chicken anti-BSA

system. They found that comglenent uptake wzs

16
unrelated to the antiboiy content of an natiscrum 91d
that the chickcn cyatem wouli not fix guinea pig cog-
glemeut. Cf interegt mas the fLOt thg' yrcci1itvtion
was inoreqawd in tug pruaanie of 1101*‘xl suxum. Enhanced
prccigitaiion ass not Cutr hated to & cezpcncnt of
comphmant or to £1.13 co~1rv..ci;'t tic-11 .-ucroglwulin
of E~A;u04uu' Cengoziga uni Czaning (1353). Her 1:3 it
ettriautct ta 3 agecific u;“ be ;y 81301 in was 2.230 rhea
by a heteroiogoas gntisaz-antibnc" 935303 and re

..- """ (1“.‘7—9-‘I »,.'\ 01"." ‘~\\ I
DJ aura-2.1 {£53in1. 1;.‘;11.;44.2..1011-~.n, fit {31. ( _;O‘J) (at..'.~.1).‘(1‘

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- 4.... .. , 2.1 :1 ‘ . . x \ ,... .g.’ -..*‘1.‘ “r .
LJUCHihl kid “a 311c42egdogeu10 40'1-;tfl of a ort-pu

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13.53.91, .6401; Cu; ... v n 94.1. .....Ld Ln...!..'wl. Loni“; .:. Ml bah t'-".;-‘ "

, \ 5., a F..="'L" 1:9 4.x” . v-.. a: - 4‘. J- - .~—~
pruxC¢4491y aJJ o1 hue ugcci110 k£%CigivnuCo It W¢e

- - - — ~ ‘~ 1 3 . 5"". 1' a -~ ‘-.~- -- 4 ‘ :7. , ~. ' ' :1:

not y‘cu‘aul‘u 4.4; "~21”.le Col..1ylu.cv f3;*;..-.,... :11 0.15 Ed Emlt

~ ”3, . .. . .-. .5... . .‘ , .4. 1 1 ,‘3 , 0 .

an' free“ scrum bat $13 ¢ucorformucq cacn 1re$1 serum
- ~ .2 "1...: .. .~ 5'." ,'-.. ".._ ,, ‘,,...o.~.‘“ .

was reactaa W¢fiu “4L1an La 4; 84;». Htuuu¢ QJL-garum

v‘kl'ich was react-m i“; '23.; gutpmue 0.5.“ 43‘1"; 1.5.5.5; 2.01. in-
corpoxaee 00—}. ILEEMGUlQCtIOghCYESiS revealgd the
urcsance of 2 éiffcrent ;a*ih-uphutluouj co mglexes.
Ul*“'cnuur¢1ub l duuly$ifi of tbs soluhla camblex Fran
parution revealed the following comgnn its: 43 (B39 ),

73 (antihon gzmz' D..oaulxn); 03 (a olable com lax);

13¢ (soluble ccmglex) and 215 (m acroglob1d in. Coep).

17

Regardlasa of the age : tha antiserum or the region

of ths re: -ction 1n t;e precipitin curve, Uo-“ we a 1n-
cursor Ht dinto the antigen-antibaiy complex in 1.5 H
salt but only with 359$ ecrum in 0.15 E. This
arterial was cansidersd to be analogous to the rhaup
mLtoid factor (a) or proyardin 1n.maamalian serum. It
wan notewcrthy that attempts to prevant acuprecipitation
cf tIL‘e m20r0510bulin by ramov 1 0:153 anfi greater

com ponents Ie;ult€ :d in.tta 105 a or 50 to 75% of the
antibo my nctLVity of the antiserum. ficngozian, Eakino—
dmn ani - rter (195 2) disoavered oo-prcoipitating
mucrcglobulina aimilur t0 00-? 1a serum from turkeys,
ghe cznts , duch 25, rcta, mi oe uni guinc:a pign. Further-
more, thev provided some evidence for specificity of
the reaction b$tWfifln 80-? and its homologous gunman
fllflbfillin.

Orlvns. 205 a and L;n met (1961) aug"csted :ha
involvnr ent of multiple nntiboay types in the anti-ESL
untibo y r65 Izse of the chickén. ?hey damcnstratad
the preaenoo cf tro fintibo :1183 which differeél 1n
apeci ioity. rolecular weight and in the type of com-
plex forged by fiheir reaction with antigen. They
noted the inaurgnnusblun of.“ £11. untLo..-;..LI"& 31.123.122.ch9
at? fih.‘ Cl “Lion h:: d an electror horatic mobility and

a diffusion consta 1t shie-1ex Lea: ea that of serum

1:3

- '.. . 71'- ‘. f I' "h! v‘ I. I ‘2— .r
s-:.ltm:n.~.rs.. 222.5225. 2132-021 c:ii..l.£ucn 5.22.2.3. 522221...-22‘ *0 192...”; 22.2.34,

til-eye 0.322032 -.- {i that £1 1122322; 322231510213 (7:3) 32.23 unilecant

~.“-‘< ." -'." ' 2". 0' . ‘1 *u. ‘2‘ . C.--. ‘--“_- 'l‘ -‘
3.3.2. 11.; CO-‘yl‘muu. ‘ 3.122112% 2. 3 T”. 2.6.. Ightlgdrfi‘vifll 2.---1 ‘2 ., oom-
,.‘ . .“m. 2.. -,.A.‘.. . - 2., ...,q .12.. 4,. .... .9 .
2202. erzo dgy22LaQLa were 11“~*”tuu 22 ex; RLn tn

2“’295192: (a) avian ant:ise.xm
0031,?32d an mniefierwianfi yrcrortinn cf ncnprreoigiu
$251325 ”30322172273; I:f‘.i‘=:f*...az‘;2'?;, 7.7? 221"‘§.i‘::(‘2‘.1('“, (b) :31";
0.15 fi'figfll a rarxicn mi tnesa fittfi.c 2.123 v2r3 inccr~
vorut2d i320 tna lzttice farmed b? he 51721553 antiv
b95195 235 (c) afi‘itian l yreci itfl ion 132 82 £361

3.. -,. .. L. . .2. , .9 ..- .,.
259 awe to 226 fulfinuicn 02 cm L as of nanrarnciy-

( «-2. J. 3 .. I 2. ‘o 2.12.. 2' . '1 ”I , + .-2
it . 5136:) 30...; kchtJQJ’ t'lflahnLCRU-fl; '0“ e\-1: [y bell-3:1. 90 Pl L ”€01 lit Itipbg
r a. h" 4-.3- J ’2 .- ’i '~J .. at u. _._ .“ 7 '1' . I ‘5 ' ‘-
ooh 1.233 mange 3r39292t2525n Vufl ufia titre? b0 u
..2 .,22.,.o, . 2.., ..., as: a .. .
$221524,“ 3 0:215 6:2,;t '35 C}? '5‘. 12222;: 13.20”: 9.1.011 C... 2.1. {2031*

2...?

8:20‘22 0 C312; :~~ of the 35122. Crlana (1963) etujiefi

“it
the 03m20v*tion of 132%: ano;rsci§itates of chicken Pnti-
13021-1233 22:20. 1;";32‘216'232‘3 :.:.3';ti.'jfi'::;.-, 22:13 2.:z2rtcl*..:_;?.c-c1 {22: 2'3; the 1:2-

2 - .. ' ..r. 2 -‘ v «- in ? 2. _‘f . . 4‘ n. .2
arm sad 52622‘ ,1...22n 052 .322 14 52 salt is in“ 622 ta

a

a ltin; out or to Co~f but must at trinu ed to anti»
bofly multiylicity or an inter otzannctvv xantihcfiy

’

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45 N201 uni attrib uted them to 2ntibuéy zualtiplicity

ES snare: 353 d by Ozlnna (1953)

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figfilutinnticn of latex particles b

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19

3.1211351511 22.1; 02259212221 by 222.293.1222 9‘s". Li. (1957}.
fin-222‘ in 12-52) cawzdarcé 51-22 1.02251221111gy 2.1.2.1. 5.213
activity might 13.402122: 22319 presence cf 2.22 5Vian

CC“ 2.111.113 331. 1.0 “1.1.0 1.2:. z.u;.a—J.1.-:.;.2‘1 l“1‘.ex.2£._a.‘¢uid {5.205913 Be

.. 1- '9 ..., ..2. .. . 1 q.-.-- a. L 4‘ «H. _. _.1
bufibvvbi'thif Q' LLO‘usU"u?Q.L' Ltumt it L‘StUP' 4.32.5, 51-12.42Pb921u;ti98

532.552.1112. 11: 2.1.1cl. en mm; 511:. at; (1.0.2.;ch1 13:01.: 1 ”123-22.

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'36- 2".-"~.r.. -‘ 3. 2‘ 9' ‘r '-. . ‘f'"'.fi'\ r. .- v d
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530:2. 2.11:3. 2:32;: 14:19. {19-214) using --'i::511u.ss twin; 50211.21?
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vantinnsl ahfnkc. $33" mhnei'33 thwt the 33033..303 a:

fl°"”1 03113 3.33 1'332312 31333333 333 1333 firafiuant

:.ew:31.3; 3511 wo¢n1flfifii in ‘r*f“a. 31in A3 333 in

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ific in fi9rfi£rca LLé C9uvqrtLOKLl ch: ickr. It rue 3‘32

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be the oxlgLL.LL LL LL91 gicley 223M m: inau actor

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the LLrLfLLe chieka were not exyoacd ta inLLLtiefiel

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5louulia uLJ affared. :hey Cdfigéjtfia tuéfi in ufifiition
to yrnuuctian of “natural g-mma H- 1.1obzlin", t? 250 2nimul.a
rare ex;: 092% +3 diat r5 2qt132n3, mnfiigena in their
W2ter 9H5 befifiiug 231 youLiL. 13 0c 3L1; virus an $30-

tions. L oatman (1959) Lain 2d out that tbs prime ple

difference in tbs serum.prptein patterns of 3~month~old

R)

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... 0‘: no . ’ j, ..4‘ a _ v . ‘ ‘ L l". ‘ 4‘ . » ‘ 3.1 ,.' F‘.‘ ‘ ~ . ;. I ‘ J '«. . a .‘P’.
$Aux4.¢ Q-Lhkgyad 0‘33 1.4.114 3;“; 1.47; Olgulth—efind-H glut-LAM.» .'-'~ bait»-..- "

1' " . ’ ' ,‘ ' “! D '* ‘ -‘ "I ' ‘th‘c - ' ' . ‘ 5' ..."
«...... 33:1... in“) U; ¢n4muus 3.43 {LLMM .L-3 Von-.433 3:63 10 ‘04:de 4.5» ‘4..."

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3.2.2:. £3.25 .1152 3.0 21013.0“-

H:

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3...ng a”. Lu&33‘~'ums.l'fll A..~L.L~.Q 513'»... 57:23.". 3.

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| ' 0‘- : ...~ 4| ,—:k g ‘1.-s.—.~r ”- .3 :u. .' n..._ .‘ ‘ A‘ g, ‘I. .. .3 ... Q A
«was; $3.17 I. tug-Q L0,} 32:: Cu. ...... .-.....-.‘~:.5 .. 2.33.0...Al .44.»..3 .. ...; .3... ..L..-..-<..

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--... +1 ..., ‘15:! .5 Q -_~a t w". 1—: ‘ pan? ,‘ firm}. «b I .. 4"? tro‘ a
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if L14. G’s. ...L W”. 1.1. GL5..L\.:.L§ ...“; k} 4.; . 2.3.9 t... .25. :14. ‘ 4:. x. o... . .- 0,331...

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{3. Lu..- LIUL 3:]- J» haul-‘1}. $05.; 1. smut) ...-.1 Q35- 1:.” . ”at: 9 ml Lab v Lumpy-o. A -"-.an‘&:~'
’ I; o ..u. #1-.!£ . - ..‘I LN" _ - .0. Q ~ .. .2 .I s... ’ + 1 ‘ 7 .1 l“ t
‘ S ‘ . f . . - *-’ , ,, .. . 9' ."‘_" g".- ‘ 9!. "‘ “i ‘. ,-‘ \‘
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- . .. - ‘, , . '3 ‘2" - ..
res 3:131:39:- 1'! .1 :‘A O 0:232.“ 1J3...
“‘ 4 l .. . . n‘ '5" .I - \..; . uk- .. ‘ .50 ' ,'. .’ ' . ' ' 6 d -. 3‘
"as. ...]. (1:139) :14 6“ :3 we} 3.1:: yr»: “(mute Lu. n:-.ts.>..:*z..l.

fl... , ,_ . . . , a, .w . ,1 ..., ‘ 913'. 5,, _
:ultfi-Jaulee in 14:?) 3:21.: cf gar: ”Erna 0.-..c.-... ..a ONLI’VGC‘.

’2'
a.

‘tixe E.;~._’.'.‘353«'..I‘if21{3(} of 259 510.5 {3"‘1555‘5114’3 f‘hi ch rem apa-

pare tly aveeifio in: rat 333 r3bhit erythrocyt33 at
thIt tr: 33 d y or lira. 3. 3130 notca that in 33-

day-old 0333"“L5cn5l 35-355, mnti-bgcterial 5r*‘ut*3553

3 mew-333. in 1.33 33353 .353 inst 4r 3 3"!)"‘""”"“" rev-3 w:-
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C1535 33:3 not uetcct3d in 333351 33353 tram germtrea
. " ‘ ‘ - - - ‘ o .Q < L O '0‘
051C33 reg'rtlcrn cf 35;, 35t anti-L.C“*‘3“*"3i' anti-
D It. Mav’fl‘- ....“

b33133 3u332“35 5t 153 to 170 3 33. It 333 33b3aquent-

1y 52.10321 that 200-3135317311 51:: 3'33 52:33:34“ in the diet

 

33333 333 £33 tn 33th 5:3333 but 3£ter 3339015315 , the
5uuuuota5acity of thase 3ntig3n: 313 consiéer: Ely re»
53333. $33 5-1‘"ed .5 l5tinin racgonme 3383.13 t.3
5533£r33 25:3;13 03313 *33331 33 be 53393 tad for in
t3333 of 3331535 d3fici3ncy. The £30t thgt the hyyo~

5 555550551155513 in 33331r33 3155-53 333 353 to 33-

.1,!- , 5.: 5, "...a. _. ...n . ,. 1- 5 . .9
5131553 35535155103 535 13% «33 to n insne l'tQ 33:5—

reaponfi to bacterial 53t15333 3‘103 3330 intsntionfllly

.9120
L1.
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5.:-
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533 appareatly 33053533333 3333333303 0f 3nti~b3cterial

agglutining, hefierohemagglutinias and 3ntibofiies to

' n. ‘ .'\ '- 3‘- -' H! ‘#'r ‘ h . I? ‘ "‘ v
3133(11533353 35535:3333 tnn53 53 vu~~031 t?3 nan»

O

1man0gLn10 oi‘g.. 0:: gems LLtihuL- as. ngaver, tha in-
clugian of Ln: janio proteina in their diat and water
and the .ru.-Lcm cf enviro.r nzontxl antiQLns tenia to

.4..- ‘ .. . 2.11, ,4 L L. .... 'a‘ A ,. . ._ .._... ’.“
317413 iii-ii“- 5» ’3’” o r-L‘ T 1.3-.,,:i4‘. .1971" 19.313. ‘1’. .3. 30 41.16:: (41-1339)

H1

Lroviied an explanLtion for the apgearnnca cf auti-
blood group B L;,L1+in1n3 in $23 33.1m cf 39 mfrae

‘6 L29:?.:;. Iii-ism; Jib: 13in (2..:9 .1.....::~11:3.3 in L1. 1.17.1113:
frag c.:VsLL.0L 1 #313 La at who mt tne ECLh day of
life but nst from gcr:frea :Li:Lla ufltil 60 £335 of

Qa’s; .n. Um .. -_ “ ‘a .- . ~— ,5
en,y3uL: gurszie cLicLs L910 chLaed

 

to rm. 111, atrain 0:5, anti—B substance antibodies 3p»
peerefl fihortly thereafter. Reyniera at 31. (1960)
"r“: mted £22111: ur‘1‘“ fa? ether anti undies v hich
Lra prose ont in serum frcm gar rrrea chickm we.
Lritp their "iLLLnologioal aéolescenoe" germs

free e. iLLLna more eupnhla sf antibefiy groi1ction 1n
res pens: ta suitLble etixuln an :ostman and Olson
(19 63) at: 1ed precipitin yrofiuction in 90 to 110.flay-

u - , -' g ‘ . 5...! 1!, 5.: .- r. ‘1 5“ _.. wv ‘1 ‘
34.1.... 9'” 5“wa J 9.3.3.1»?333 ‘1.Ias$avv:1 ‘r: 1.1. U 1.4..‘T‘3n £6; :‘1..;5-.:4-G *r‘ Lri~

. ‘1 - ‘- ‘ 0" -"'- 1‘. n-
1n<..:11utir:n .1... 1111.31: 1' ~ 3 . .. ‘3... L L 4J, 3 1:3 l :1;

.a ‘ 'vh-l\ I’d ' ' ' c v- 9 £ ‘- .?q a .‘- .L\ L .

l canLLigan of t.;:r1mzry “gtLoQ.j rasr*n.e a: ofl883

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(b) co v"“t33321 ani..la 3rru;3ced 21th s.ntiboéy

by the fiflrnfrfic chicis and (O) tLe Lntibofiy
centeat of scn‘m from g3r~a rm; chioka fieclined aloaer
€333 twat from canvantianIE. the reaaan far these
airfarehces snened to lie in tax unicria 312323 1::-
yLoiu-mgo;33hag3 5333;; cf the warrfraa $313.13.

9“ - .-l ;- n i, "I.
”ha “lactrsfiuérexw

 

9".“-

gwh',r ;hc: 313 Ln"*‘vch u diffsreatifil nifirfi~
tlcn 3f cnzr"u collniirl particlrn tfirourh a snitxble
medium uninr tlo inflating L=I :3'1 cleatriCfil pfitzntinl.
It therefrre 33 a LCII‘.3 for eopcr.ting 3 mixtura of

. .4“. -“ a ‘ t L ‘ ’ ‘ J F‘ ‘ ‘ ‘3‘.
clou3lv rclgv3d ca: ..3032 an +16 b 313 cf t3cir not

in *- o .n. . a“... r I ‘ J.‘ 4. O i A 4 .
03¢ pa 3;; 313333 3 3:3: Len. In 3:33.123. an:
_ ‘ In 'I ~ . , ‘1’ .‘9 r n-s’.’ I .‘ " ' ‘~‘
332cc:;m, 3:231: Cfififillud . 333,36; 3 s~nfl13 of 13

A

mxteri l 39 b3 33313331 to an elactrie £1313. ‘Un‘cr
tL£.J 123133336, thc ? 210:3 constfith3ta are dist.i
butpi in 11393315 zoLns in¢h can he lficvtM by 331%-
able at~inin; techniguga.
afran (19 0) raviaue .he theoretic l banis far

high volt 30 3333? eh,c€ro*30“n'11 031 fiineusned acme
factorn which affect the 33,3d of‘migrfition and the

wxgrau of bex‘rwuia”. fie B3w:*'*3fl 3n :t Ln§ electri-
cal cenditicna o the ”a;rort‘31'n;“Lum t=sere data mined

1

by re atizm mi 33 betr~fin e“rr an uni volture whose

values were daycndent upon the rssistznce of the 999199
in acoor99nce with Chm's 199. since the resistance was
in1t1911y fixed, in arder to increase the rate of ion
migration the current had to be elevatea by an 99199t-
ment of the 9331199 voltage. umith (1950) pointed out
th9t either volt1ge or 99. :19r9 g9 should be at b11199d
thraughout 9n electrophoretio 9939r9tion so as to gro—
vida 9 more nearly'unifarm electrical enviromment. he
inflic9tcd thfl h9m a can9t1nt volt99a 999 93-1199. the
99999959 199999999 during tne run 999 to a dimunition
in 3.19-9t 999 091999 by heat 5999919199. 299 999993
of heat which 919 prcdnced ( I “v 199:9 ) innreased
with the current and 099999 919 tillati on 9f b1ffar
from the 919111. $99 rate at pretein migr9tion 999 1n~
9909999 99999 the influwrhe of 9 const 9t volt99e. On
the other hand, whan oon9t9nt 099:9nt was applied, the
'99 M, 9 99 dropped 9-9.9 a result 9! the fiimmition of 1-9919.-
tzztce (1.911.199 ’93}: 91+. 1.9993119 a 19999-1113. the rate of ion
m15r991<9 9's 9crn'unif9rn thraug31out the r19 nnu
aoIV9nt distillation 999 decr99999. El9ctro-9arctio
buffer flow 09119911 1.21 9;);‘191'9112: fiifirfitiafl of Ezibatztzncca
even figmgfh they were 11110211111599. This effect 9:118. zirc-
sumably 99;u999 b9 19999119 chwrn 13 ( uaually CDC H )
which 191~rt91 oggoaite 0919999 to 719019111 901V99t

901999199 999199 their cathpdio flow (Smith, 1960).

ercticLlly all of he churnctariatioa or electro~

ghorutio Bfipflrgtififl are influer cad by v*riaua proper $135

tLe bag iur L"LLem em; lcyed. Lturell and Laurcll
(1335) LthqufiiLed Lufiinr conii‘ Lions ( b:.rLiton La at
px 8.C.'u=0.3?5-6.1 3 for tLa eLo ctro;ho refiio analysia<xf
8&3 11112.
':&L“ ~t Iidfl. d cunuliions, some electxo5 hereuis
of: xcruul hLLLn 5L rm ~ Eeyuratfis five aim tine» canyon-

{3.2.

is: Lluuain: L19921, £3193 L?’ beta Lad gunmawglobulin.
LLrLLL (1334) CbJL vau u siailfl r pro otein olacrluuuiun
of Lergl Larum.fraa guinea Liga. floamenn (1333 and
13:53.1) 11:526. .25. LWLLWALGL ii. hibru’i. .Lc- 1681;211:5116 with a;
bu:bi*w buiicr at ;L $.6 ta caaggra the Lara. protein

:1
i .i" '7:- “ ‘r ,- ' 0,. . ‘u- q; f-n
£4141 c ...}. :31 cu; L...ulu..;;.4. La! 3.3:; 21 8 g (It:

. n I5 I u: ‘ '1 .
2: LLu’vf..b a.""'2 .;C‘1"~LI.L‘ ‘5 LA.
P .. ’ t L ‘ ' . . ' . _.“‘ ~.-' -'-" - a .. ~ ’
4» w 4.4 1} LA- 4. £3.36 u. LLLQAX lg. .5..fo t}; dis-0‘3 -3. HI 1‘3 SC. .5. *4“. 1:. U3]-

V. , I . °. 4.“... . . . .
\ " ‘ " g '- 5"“. ..‘ .t . . 1 ‘ 7“,“ . 1, 1‘.) ‘ v‘
1"“..c3’1- ‘g‘v'.‘-clr.l 33;; ~ 04;-do f" v} s—nda.‘ J'tflaovu “Jul Uighiu-{l &-$*CJ-‘VI)U ‘z‘ma in

#014 . ,u'el at“... .v‘ ....3— \_-L ‘ . f ~ a.“ . if .- ‘4‘ ’1 . > 3n" - . ...”... ~
‘4ifl £~ ..6--‘-'—$-3'-";.A-.’ Q'Aa;n r 3.. 0 Vi an. ‘v $32100“; iCE-‘tfl ULJ. laifii‘g:

V‘- w . . 5‘ I. _ ,L' ._ . , I. ‘ . s - ¢ _ 5-: b
{Eli-.351)” ”13*! g§..:.a;.-$~.~‘;:.:.Q;;L:LL?'£ 319-5” ' 4.55 14633139 Oboiil Vud 11. Sup.-
‘ 5

(3.18.? r‘.‘ x1 -.- vs» . w Nat. King" ) “u p1 n- V» 3' :6 rv. J" "13"" ' t"! r “‘1
"1 J? qL-‘l_g“ :6 |_ g -_:“' “ J “L “bill \I {Old }.' 9‘" ".v ‘1" W0 CV15- ;e‘iuli Motlé‘

-. ~ r. ‘ ~ 9., JO...“ ' . q t!- 10: _ g "‘S .- .. - ."' .9 .
?L‘."..Lf’.f....4.8 01. Luv? 552?”! £1,557 o 9.21 2......3'53g this [AJKLLII'CG £3711-

.m“ 1‘ 3 N 1"“ I; a_ .. {'3 i "n ~. a .I “ '1 " "I 1' -" . .‘u" .‘ '
~L|_.'.-L:: hark 1.3.6; l‘ .‘u I} 1‘; "m '34. Ere-:3 ...JLC. €,a4£§.aua"‘;.$£vUTMJJIQ

f”.

3 .A . -y.‘ ": ... .... ‘. ‘ A “ -. ’ l v ' \m ‘I' .~. -. ,5 ...... .. .. ‘
a coa5 31w~1 L LLL Leme 5::LeL. Nngt£WmB from L-

g g -.o..- a.» ,2 L o. I. L. . .3 |_. E... z‘ A sl- - ....“ \_ 1a- . , _ ..
year-c <11d “,2LJrse LL3 CCRJLntLeaLl guinea ngs Lemons

" "'4' "q"! q a " L! n r4 :- o~ V I -\_I_o “ .‘. .lf' - ‘m " n
55 {'1‘ Lab. €;..-L~~z.£-3..,t."v @471? 3:5.) 1:61.). v.83 Qt.“ u." U.LUZ.. 94V!) u¢1.£q'.'. :B’LSQQ .

~. .,': L... . ~97 ,., , : .,. ,,. . ..- .
1.! Li L,‘..’.. i (21.: 1.1L? ‘5 ig— .1: L.x~~.x~:aK}A;..-Gl01.1%an

cf
L4
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6
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frtctions were abccnt in t o : erAm of 5?:2ftLe snimals
thick were fad a seemingly adagunfie AiAt. Ya waver,
uLen the Aiet w -3 CA.LU A, its hi5he r nrotein antan
w29 rmflm AA 111th: aggezranca of these froctionso
vreALnAL--r Stu riiesv Hr bbits in is 39A no app rent

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ifs» execs in the ser'xa fro . tLrse t;o 5r0A5'gs. Lu

so gucut i avastifation, hcwev var, rAVLLIAI hJ”C*2L13-

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51: LQLAreL*A in garLlre-J rabbi ltu char cteri:t :0 of otmer

a.

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agecAeu. Lezwfi pLuLAlA pat tLrns obtzin .u frcm 5orAAruc

an} Cantxtlbn‘ “”U&d«1t3 diff.é xei on? y in Lair rev

333 Live 5 mug-5: lcbulln lAvcls.

*Q"- h ‘a ‘- ’ «.u .
AL.MA¢U. ,Lan: LrssAa cf- 15A” 9’ t n {“00'SGLV9
- ,- 0‘ . -~ Q l. 1‘ . +" 'r . - “ -\»‘ '1‘ II
stL,-s:...._1 MAL-.5 in ALA: ;.'4”.‘::.'3ic§-'ZLLIIT4LCL1 A. rrcLer.”

:Leraaie ALA free diffusion. Therefore
3:9 use 0f n LAAt.L13 grcjercd :fi*‘r~ru* 93031693 an
«ntifinfl’c analysis of .Lc test : t"ri'l. occrilngly
KirsLLfeld (1363) pointed out that alectmcwficrcticzlly
idaLtic~l can {39333 coul b3 IQALIVLA by their im-
5;AocLuAic l diffgrcmeea LLA thAt entifenicull3 re-
13t9 9d 003:0 m1 t3 cau.li be recaled L3 iffcrvucea 1n
electroghoretic "chilit3. fie illustrutaA the resolv~
in5 Ikcnvr cf iA'nLovle repl arctic hflu3y313 (15.) by

its LLility to 56; r tc 3L LL? eat 0 A;oret1~mll y ‘na/or

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m... i ..J O C d E ..2. ...... Q a a . .1 ...-“ _ . ........u l w... a. ...
t S u r J 4 ~ 1 r. a. f ...... +... c L. c l ..,. r .
A.“ ...... a... 9. +9 . .. s . a. a... .1 mm .M a.” .. H. . .. .3. r.
m..." f q .. p... .l .l 9.. ,6 G l ...u .2 A b . 1 ..2. .. . Q 8 .f .v.”
G C l S C .1 .. ...... ...“ .. u .. .. r...» 1,:
".... ....u i ...... ....» .4... l. ..v 3 C P. v w u .... t. ...u L L .- m
1.... ..u 6 .. _r .. .r .... 8 -. .. i .«u 3 .u v, H L .... v n.-.
w... 0 u...“ . 3 0 I n a ._ w ... . S w... 3 R u f ....» ...J
O 1...... i . .3 .1. "J a - u 3 ....u ...: .H n. w. .M in. 1 .
J. .u t 1. «J -..; ....) ,1 .29.. .1 C . 1 P \I “a a... H. .J A...
o a» x; 1. My nu :1 ”u ”U u a; «a w «a w“ a; .2 a“ 1; rv~
t. ..u 6 ...» n ,. u a i .u C ...... .. x . C t. .3
“In ..L My) 1c...“ — A“ o 1...“ w.“ .30 ... u i ....“ \p‘ ”h. :11. «3.4 . J A...“ '
... e .. . J l ... 3 . ... ...; .... t C A 1 .5 o
r l ......1.. Du {In . ..u ( e ... e P... a .9 3k to ..ot. ( 13‘ c l
..1 r (\ i r ...... .. .. .3. ...... .... i .... a .2; .....U
\ o n- .... ‘5 . I . .O\ .\
T. fl . _. .... : 4w C .. u .J c o . 4 K
G ... I ,. . .. .. .. ..... . l . 4 .. u i l ... my .3
C . C . 1... . .. 3 .w v a...“ ...... a. ....w 9
l... b (I
G

c
p rtic“l r Cfiggflugnt r920 v
6
IO V~- ..
,\
et-

a .

I y at ...-u. . ¢ .3. ....u m... H

b ..u ..., u ....v 5 n" O . a J. . J 0 R .a

my an. r... 41 i V... ...v x” _.... war .4 4 ..L. rJ
.... ...... _. n .... a .... 1 m .. ) ... .
a.“ 3 up «...... ,1 1.9 “b ... C .. u . H u. . 3 l S .3 x)
V 1 .... .3 r. T. ... flu . ,~ ....m «I. nu. .... .... a L
r l . t x) n M i 9 .... r 3. .1. Q 0 w t ....
n» O _. . C. b .....L 1...” fig ....“ “....“ an :— 3...» r 3. ... ”a. “,3... H1,
3 E . C I... «1 C. 3 1.» .1 D. ,. .\ U. f x . 8 u r... l

p rti ul ta dratrad £2313 as a manna of diffaremtiatinv
salute 33160 163 of dif f.re at 26123 alar dimer -tions.
as a result of these at2dicn, a antics of crass-lir;«:ed

Q

M a '2wma2auz wag m22da commarcially availublao

5.:

£313 0'
Lor23; and Pleiin (1362) exglaitad the molaculmr

«m 1-‘1 L ‘ 4 - -~- «A ,-. q. a .. q a .- u -. .i .5, ... . . ‘\ ‘2 - '-
GACALfildn plugfift} Cl “6:3“.181 2nd uflVQIOde an 3132103

cbrafigficgrsph.o prac g2; aura o 116' ml fi ltr.fiatian.

crme 3r2ctisnl. Flafiin (133?) fraction 32& havine
fllnama by 581 3513333103 in nfifinfivx GmT). GanZ) : 3d
Prué?‘.}.2. 31:2,;“30 7:233:31: 330:2}: ’2'2'43 £21“? 3. 1.3!“??? 5‘75

33133 Ema mahcrabie by 3:33: electr03horacin into

nlhuxfin r19? “”1 paor avg inna. Gel filtrwtion in

C}
4
M
f

:2

0-103 251 GwPGG yielded tw an“ tfrta 3303013 weuka

r33 eat .evelv 2truc2 2trif3§al unmlyeia cf the v:r20us

fr‘fitifiws :3uf21 330% tie 0-592 331 ravetle' {3” £31-
. - .. . 2., 2.. . '. 2. ,. L. fl 1","? ..- . 2' ., ,2 2- n, “a

13312;, cm;“u2anan-: 12222 1, 122.;wmfi 7~; :32»:.:. 7,;

s ‘ ' -“ ~ 1.. '- 1-, 5‘37.” '9 v P. a, ~1 _

‘ 4 2: '33, at .. . 2323/1 '1' E111 27.10:" (1 .»-) 322.2143 33223;:

:34" +1 .... ,;&-.+--na '2 :.t1‘:"}. of, 37"17‘1’21'1 COIHZT Vlacte ivy: yjtwt22=incd

... u-Aai- Hid-rd 9. ..Ln..'.

.2 V ‘V . . * q 9 _ (.1 (A \ .... ~ ' ...“ 1-." . _ n
by {”1 Iiatfsviflfi 13 33313331 G¢?v$. U? fun mac 02

.3 .1.»

*~’ 127*;27-72-3'33222' +2222. 2w~~2~~t~2 cmfiw‘tuazéfia cf

mkoabw. v ~-. .52..» .-~..'..&.

-.,1_ PH. 2 :2 .1 2 .-.M,‘ ‘ t
tflc 3 rue :9»;2. 2-6 f2rct £233; -.2 2.1;332 332 be 2
a 2

31

lCJOsFttazi in the: 129002“er 3’30 32:, t2 9 tE ..‘LP'i 00:12:11: 100. ml-

buain, Lfinoglgbin, trxx:fzw111n and a 3.5” 51y003r; “in.

.!

D;:I‘£i:-13 (1:55) fr 102101‘tezi 2‘0 1:131 and {.ziziwffiigl’} Mai-
mfirum in .«Ln'icx 5-300 an: obi. med three prshcin
ye;km. The first tyeuk osnsistefi of grofieéne Wuowe
50190115: weight excaefiad £30,330 and inaiuned an

al; 31”: 1001113, 153 as two high fielecular weight flflti-
bofiiea. The aeconi “23te1n peak 0? ntxlnwz 75 con~
utitnanm a, as: 7.6 mitiz unii-fggfi antiboay activitv.

Elafiin (13’2) etugie d tx:o 11* fl :euce of ca"tzxn

uffor caaiitLOds 0n fivrum vrouuhu.fr¢cticnatimn fly Eel
filtratisn. Buffer yfi 11d net affect the sagurgtian

ea 10:; as it was camp31131e w5tn grotcin 3"01111r.
Ru11u18 05 law zenla fifirfinqu, hqvever, 3110 cu groteino
D“Luohd interrot ions to 000‘" 1hich r<m miiad in tug

fGrE?;'1 an of rwwtonuo1; atubie 0:37:9195.

Ifi'*“h”1ff”v on

*‘I‘flvv’u'w ”a www.-

’ I». r‘ y; ~5 4~.'\ .--- -..- l: . ‘- ‘5‘ g, +‘.. ,:" _ ~ N.‘ ‘ IQ. .... ( lull
.qo-¢ 1&0 newly ua'210;nd fieuuCJm a: b:QCue¢:c.
r of I v _- . O‘ O ' t x p .
”w; “:1 L1. 1 ' . h ‘;"f‘~r.4, u J-qg‘s ‘1 A?“ hf 01(1 “n 0‘)...
D 0. ‘y , ‘ n ‘ -.. .. V." . 4' '1‘ ‘ J 'f
3.3“: Clea-£1; t‘ ~‘L 2492‘.» c)? ”~J;fl:-ft4rm 3153 (...; 1%.}. I‘X 31‘633

:4.
.. ,ZCI‘.

«-#€ (1?" {sum {311; alum}? (12453) 1m.e:*>»aen:zer1tl.‘$'

.14..

ff}

5-" “‘ l ’1 ..l' .:.\
I)"; h~;.{:l"‘ ...;1ti {up}; CLEEIO

- f .‘ t . 'T.‘ ' 3 : .".‘ ”\"I +“ . '
develejnd firfl.Li av. tar the a11afl.:onal
h ‘ . ‘ Q ‘- f.~'v' ,4 ’ . g .' I" +
in :31. ihz:_ “IGCJQC us acre 513111r bub
\ ' ' fit. ‘9 - .; A . . u. t‘b
219 yrcviaion mnaa far r@.c. A» c

"it, " (‘3. q _ ‘5 V I.“ . .Ii" _. .“flb ‘ :.J. J“ j . ~;. ,1. ,- '1“ . .
3a “(1 km auv1uaa a gxwhultrb+V6 grucavure for

single éifxiw i an In gel. E18
same of tha mast b sic newects o; innunoiiffusion such
2onitiva carr&1.tion bet ~1precigitute

inteflgitv um} sntibafiy cane ar.tr.tion, (b) tha effgot

and (c) tau lufluenca "3 cross re cting antigens upon
;. 3.33nu3 antigen-antibalg 5;3tefis.

'n .,.+n.ud-~t ~ & a ' V, ,Q'Cb‘ -‘
(‘- git-0‘ “0“10w‘~c:~l tra’i -6 492110;: H 0* t-sc ‘u‘l‘Q 2" Lin ...-c3 L:

«:4

éouble fiif-aaimn wvs "20v; 'dcd by JV We ”16?? (195%).

i viriety cf an; orting med; . have been em-
pl ycfi 1n -mnuui-.1Ju¢~u technigucs, but 95x:
been 128$ mos t often.

Grab r (1955) obeerveé th.t tag «*er-ge anfi

. 3 gr. .- 55 ‘ i d“ ’ 'p C‘ $ 4 . J- ... n ‘h I- O‘- . a '
min.“ 1 gate 6138a an we.1 Us ude .0. 1 pore area of
n. , ‘ -. . ".9 9 - Q ‘ a . .4, I . ‘ V -- ’1: 1.- -& I

.n agar gel sin yffisflTtlfifial to tne uni eucsflurJtion

C
€31 t area; regulated the rate of molecular ciffusion.
Conaden xnfl rm (lg/J) 133$ md .093 the axe Of celluloae
acetgta raz~-Jre ha a a2"::eztifig Kecium fer immune-

dif&‘.‘u ”-1 1-0

0
‘1
O
'r"
'53
A
3...:
...-I
V
C‘
:3
d
‘5
:1
d"
O
c 5
f...
}-J
F:
1‘!
‘1
L.
*J

- .- - . n - Jo -._ 4 12, .. ‘1 .5. ‘1 '~'~ -' 4 ‘_ _"‘" ... ~ . ...;
E.3l‘0;-'CrthTLS G L :4. A v‘; :£‘31 ..'- iv}. . .21»; \JCj'A...’ MCI“... I. U g

.- ‘A y I‘- ..J‘q Q a. .I I ’ ck ‘ a. ... ’ I”... . "H. , p_ _ . - 1....
u 1. £113 cu-ger"t.aa .. tnfl 1e-ctlon .Jzurxbce. .hcu
. ~ - ' v ... ...... ' . ‘. ' _.. -.--.-.--
react L33 «3.3 in node r.te inu;;uuca. the at: JJLr
' - .. ~\‘ ' xr . A. I'tn‘ ~.'-l» . ' ' -‘ I‘ .1: "".-- , 3 ‘
IC'CtJflt al.f-ne& ward“; 1L3 xztcrfmce w“. diluteu 1t~

= I, . 1 5* ‘5 I. . ‘A . - J ’1" . \ J: v ' r', J I” :13 . ' ‘5‘ -"
fidif :13 1% 0"}:034‘31 )téq- “5:1. C‘JJ.JC4.£?o.¢:‘z.zgl 1:13;: C). ..e 9.8. but)?

s.;*.*.";:ri.‘:t.. 0’s:".f-.2:.tr2tir.rm :‘i’ 1":9 two renctugita wore

a t 1393, r grcoiait2ta "in tarnsfi which hecgza at.-

ill; 1 Cf n:;¢ ITS“CItLCZS 0f -xtigaz Lgd Latitody
bsia 23cr33r2fci at :2 .22 2 r 13. 313g» iLa2uz-
Irwfiigit2tfis fh3:e& Ezlacfliva burriera for aeralegic~
211; rclztzfi :clwc1l‘z, 1: Ln: gab;1ul o to 0123:22222te
bafiw e5 the cs"x.;.2£2 of an mailman 21x2235 1 d; "m
at “1. (1:29} {0134 tL-t tL9 ti2e ra.u£2‘c& -03 .26”
0121: ti fcvz'ti-n '-s 9“-221*2L2«L to re.cfi at Con-
crut2 iQL, ziffmeifin distuxce uni fch-o;; Lffucting
diflz;;:2 r.td.

u2cltsrlun: L35: ob2grvca thht u prccl2ith

133v soul: In chu3.02exlzgd by its 312%, dcagizy. width,
egt‘2ziar, £t2231‘23 2; {9L2C2cy .9 £22 1102.6 Eb
~rwzonufi u byuivm Zcr ClLLSiflJLtLQfl 03 tr; 2:1 Liouw
Hairs brt: on 13Le3 in a c3222r2'123 r2oiyit2ta Lnaly-
21:. 32.3.ivn2 22:3 0122:: Lu :3 type I. II, 2:: r

 

.’ V J D . .. 'I. ’ .. ‘ y 'r . . -. . ? J ‘- - ' I 't
15 ’1 “A?! '12..-.“ 11‘. ‘2‘32 is“: l; .. 22.2.‘2‘2 {if gt-LQ‘ZLRA 54.}. 2.3.156 1.122503“
2..“? ‘1 'W 1 " (a .3 I (~" . 4‘.
luvcc'}-& will“ It :1 »-.'$ .~u~sl$ {ALLAH ‘:-' -Lelaau.
.. 3“ ”It. ‘2“. 1.0.. ' .
at 222.22 2' .....“.:*.22.:2<:.~ twig
.. ,‘4 .‘> ~ g ".I- ‘~ "" ‘vf'y I." In“. - *‘r . ~9-
2222202299 are un22ae .Lsmg 022 r serum 2 chulins
. - - .... ., . . 1H , ' . __
n tLeir 2allitJ to combine s-vci2zc2llg mitn h mo-

10903.18 “I LtL-f c-LL.

fa mntibuiy purifiGLthm.
groceflur.l fifiyro: cLes to LLti‘;

Ill {ZQI'dEi‘z 1, 1:14.

1350191.

3-,

,J!

This 3roperty hhe served 23 a bnais

1‘8 421'9— t‘ 2'0

n (Sehon,1963)t

34

(l) non-3 33 1:10 LLLHGIJ .3333 i3y3lv3 fz;ction3tl3n
Of lt7tifi,.u3u C231 '33:. 2} b'-Si.:" 31’ fin-Jr'uxefi 21‘s V5101: :{J‘G

-‘L. x. . - . ,,-.~. 3. _- ., , .. . 3
(343;.er 0;, £31.91,“ wd 2:, 0:: all - 533-0134;»...UU-3}!wiiubtllinu. an‘:

9

H.

(A) 3:36 i 10 Leahoda which h;ve 38 L361: baaia the 1m.

333333331031 specificity of 33tlganw ..Lmhvj inter-
30tio33. Surly atteapts were made to isoluta eracifio
3;ti33;g by the use of antigen 5'30 0-333 £3"33-t135
33313. Svcascon (154 ) 34333333 t‘"nL to or 13r-
033l psrticle3 333 3331 them to inolwt3 ageoific anti-
" 5;.“ at 31. (1335) xiroru d 33t1g33 on 333113

r
“v\ n . - " r.‘ q ‘0‘; .
re 3313 to ruchvur 33311 3333393 cf 3311b13"

...fi.. ~, .. no hwy... . .p 5.. .. (- . 3.»
33:3: negt 313333133133 03 £33 C333133. 33t3331333

'-
(>3
‘ \
9. J
n
y .1
1...!
A
'31
1
K”!

-:’_:) 1:002: i.‘.~-\:V.{.-.'.1tie.-:5! 0.2-? :n 3173-.” 9.103
made by Lanni at 31. (1356) th¢t 3? 11 but significrnt
33 3333 of cvzl3 sin b3o:.:e £t3037>y adsorbed to the
33113 3! £1533 yraeigitim t3 33. ficeczéihrly, they
3.333 3:111:1‘.‘..-';-.-:.¢i..1.:grim-6!. 51.13313 her-($3 t3 Z":::i.f.-“Ie 3111;1-
0v 1J3nin untibufl” with 3 t“rity I 90 to 95.'3.

F33 31303 cell strorxtn th0 been erploycd by
a number of investxu.t3r3 as 33 133 s3bge summortin”
331333.f3; gatiweu or h3gt33 caupling. 3333ataiacr
et 31. (193;) 0339131 divzctized 3:3 tens to red 3311

t

., 3,. . ‘ , 4' ._ ., v, L. ‘2 . 3 . ,. .: xv :
“tPOhmtd 333 33.3 t3333 33343g3t03 for 33333.13 33

1, 2.3» «t a A, . 2.. t -J- .
bo‘v 33303,.103. D3333cigt333 of ant3~hr;.3n anti-

. r . , q , ..‘.
be 31.33 31 33 £33 3 3: 31333 c333-.x 3.3 acca3illhnea in

dilute acetic acid. Eisen Lné TL Lab (1349) uLLd a
aiail.r Lathofi anfi obtgined an antihafiy prn_; vr tian
whicn Lgs 90% pure. ‘Lth at filo (1360) L: d iLmuno-
Largi'nn ta ififll ta antLbuflies Spec Lifi 0 far a 2.4-

3;. 9' :1- J! .__ ._l-.:.A.‘|.ei
CHILL]. 3.10 n L6“. 131, mum bf. I‘L Lanny.

L ganr 1 netnod for Lu;- if :.in3 saecifio untin

p

protLin antibagiea LLL Le cribai by L vfirocrver and
Iraaaaann (1333). Thia Lahhoa wag bxuwd ucon an
antigen railiiCation 3m: 11thzt itn Lbil ity t0 precipi-
t t0 Litn the dut1HJLV LLB nct i: .LLired LnJ.could be re-
muVed frcm tug rczceion mixtura fallowing ccmplax
diaaociation. intiaaiy wielis of aggroximn'w 4y 333

.'cre oht 1301 by this grocedure.

choific iamunoaorbenta were preparefi by Camp-

bell at 51. (1351) far the 1301 Lion of a variety of
anti-protfiin antibodies. These Lu.hors re 190d cellu»

lose to farm L uoLL1r Lo ocL330und -:hicn coulfi be

".

didactiaai and couplai to wratein antigens. Specific

antibodies Lara Lbsorbed fram oar"“ Lrataim mixtures:

the ififiufiflficibfiflt LLa Lashed frsa of nansnccifio re-

‘
§

Lat nta Lad attibc L‘ws “tre e "t ufrom the complex

k".

at Q3 3020
Ion exowwu a raging n ve been amt: loyed La sup-
porting meuia fer "Wtigéfl coup .linLo lslinfier (1953)

Lia-0:17: s:;.._;.~i._1onsate;i or o: rt) '3‘”). trari I'L- 3:1:er and comic!“ ted

I
‘ '~-' 4' “ ’9' V ' fix? 1" ,- “' ‘ ‘ 'Q I ,~ " , ' ‘ \w, 'a ‘
than .u cox.esqu»ng uaLqury] r? -ch CMlOPJQQQ.

r45
:3
73
I

p

I.
’4
L.
{‘3
(.3
db
0

..' .; .... m I _ fl
..11t1,.Qu,23 mum c mzlafl t0 it“s-Q ruff:

suticeq snarled leyQQiQ-Lc?y11”“ ”G euniam tea
hLve been used to iaQILQQ Q v:?iety cf fixti~yrsfiein
L .K '9 9.. ,. wun “A ..
«n3‘1fliL5/{O‘Ad‘o as (5.39.05- “a at ::l’ 1;; (3 CL’ ”3‘03 ct R~l.' lfé';:t}.

”th at L1. (1_ Cl) uQLd tLiQ rQQin CO"plQi mith hum n

‘

, .5 ‘ ,...,,.,.' ,,:..: , a” .4 _L. f
v ‘. g1:.r;.l.n ‘8‘) 1001‘ 1.3!} 3 {éli;uFr-\:&Lt {’DL9OOV&-._01‘J-tt:-IIJII1 {323.13}..-

M
t.-
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3'3
5.1
'J

. ._ ._ Q . ,, ... _ r, I .- 5-;1 _9 ‘_._.3"1.', -0 . . 1“}..- ._‘4,.g ,
110-111-322. [“51 62. a.-.” king 1335.13.11L-u1 Li ijj; Ht “1m
- . o In ', '9: N - 3‘ : 9-" f.- r ‘ - r. K I 4- c" If '-
EI‘UCChfi-ulu 1V? “”3": t *ct4L.-..Ljr= c153 1154916L$ ’02 9‘3 24‘.) \lfi. Of
‘ ‘4 .: l , ' 9' - _~.-: . “-q ‘ ‘ ,- -‘ 4. ~09
£359$boicf 1.3 1 E: {3; of mama: CLO-Lanuln 33.11.45; :n 6.3 f.) 1: cy Of
., , J. ‘ w"? a“ . » . _ ,. .1 .
9 r0 .m.uLl: vi. LQi EPQCQQQrQ wQQ Q ”limab19 to L
.- M . : _.‘ ... . ..: .. *9 ...: . Q , ,. , ..
LL 3 it tLVB Qt ruin. 1L3 01 LLQLLQ LmOJfitJ of Lati~
'3!
b 1" ‘h‘o gum-$19.1 1’11 1 b1“? ‘- ~n‘ . "v i‘n (P ‘+ I ‘..o)
D a"! u- ”31...,1t -.. . Vt: fB\.:..utz-L-quo ‘91.", ..I. ‘v 0') 1.3.53: Cs...
.. * .e.- ..-.- ‘wma . 9 ..
.1::“‘:;=le' Q: for ¢..L.:n,3.....~-;:.~;.' 3,11,: 1110-1“: :10 f*"“ --.:=.QJ Q, us;—

- - r- d" ‘9‘ f- ‘.'\ — (Q 'I - m ' 1:"
fr;cfie.sgfltud LtL; CQer. Rubbit LL1130 lea ta bOV1ufl
QC gm uihziin LLQ QVQLQQhun were 1Q0.Qtak, puri1ied and
._ . .94.,." t. 4,., “.9... .,9 9. ,4‘.
it“. 4.. ...J.» ..C'L‘J by t21.3.3 :_ CC $43,143.53 “Hits: 5.1...-13.l "4? 381.4412...

”'9 - . w“ 3:. - an 9 .-.. “.-‘:.
bgenefi e: LL. (1:24 Cv\JLtu ,‘ r eoLmyle f: seed

0
I
.-

4.: 9 9 . ‘W""'* .d
Qrfi - anfi to pclyah L QLiZQ L LQ»3_L1-Q;s13 agrena Lr

.. h .. 2.1.9 .9... 9" “I 9 3. .,4..: . .r
r 9ve:\ C:‘T~C”;S;ulzL253 L133L123 L31 ~~1~LS~J~Jfi~t~M

viii ...)Udiefl 5‘03; t‘A‘g 591.19.}: Of 1' :9;‘f;‘-‘-'~p{h,l 1:15.41“;
. .; . .- ." .4‘ a". . A . .I. J‘ ,( - ‘9‘. n ; , 4' 1.. ,1. “ “fl '9 '."‘.- .

Vidu la. Lawcrula;uLQ ahLiaegg CymtAdd LO yL$jmw1n0-

an , .‘ 0 § an 1 '9 , I _ '4‘ {..- - .~- v $.. 1"' 3‘}.- ' .,

gag-'Qtfzcue had no 9.551,," $14.5: C {:01 m! f r ’5--ch tasters.

5 . \ _v: . .3 .‘ . .3 ‘u 1 J ._ ‘fl ‘ g: u..— +~ ,4: . . .0 ..2 " ‘.-“ f- , ".I Jo~
:LtibQQ; cnzla flaw Q; Lhzwafim‘zlr QLQQLQthQQ *0: 938

’no‘
‘3
é
$3
:5
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. . q .. _9 c, . .9. ....-- .. H .-;
realn bound SfltLt“fi $3 L:r L_ aye; L

...“.-.N- , ..N u p. . 2 a.» u-usipsa‘.
_, N 2 , . b ‘- ‘
1i”. “.AegnlhlJzfi .511)», 3‘1.‘ Li,“ ‘2'):5

.r 0.. «a. .n..t . '. ‘4 ’5 ' ' a, .1 ’ '- I A). 1’
1 0923c:C1;l 2 ch c2 unite 15;} 2:2 3 czi~2w an:
m V w ’7 v ~ 1 w. .' v- v- a" 4 .2 " ‘ I . .
acr¥2u gm a souzcs cf $33: to ,zavlue tie “22u.la uzc

t ‘5 : .z'i ‘ "‘e‘w . . o- d”. A ‘ .- .- an, 94.2.. ' I "w
g this gpwfj. k-ra merw bwlectud 1:43 a axrgla clutom

Fr vfi- 9 _ h . ...-. + _
3cnn;rce Or a Gfindfifivififlfil
v": -nc.‘ "I0 ‘0 A ‘- ...: 1"
flub$333393tg 2rswy: a; Biiui 43:: 31rdrgeu uefimau~

grsximttel? £3 chickena eaar-wi ad each areal: of con-

3)

vantional birés. T3. marker of Lfirfifirga G? M0 par
3rou; V*ried from 6 to 12.

Group I consiamt d at l J-duy-old germfree and
convantian l chicg‘ v.hioh w re‘used a3 a aourco of
normal ngwn.
ran a IIaV cons istafi of 43~dz y-old 3*rw’rpa
rad convunti ouul cuéghs 2hich wcra 3130 “med

aor*ca of nfivwfil a..%..

Graups VIaI? consistufl of 43~0yy~olfi gmrmfree

1:23:33 cenxrmntienal chicks: which researiv‘zm azn‘tigijeimic :Etimo
ation and v:._-a blel far grimxry rec;aa antieerum
at the ‘13:: dizzy pcatvinocmlgtion.

Gram; I csLQi'fiefi cf gfl-dwy-old garrfree :mfl.

.~ .~ mw ~ ‘9 tr. ‘1’,qu 1' - , q ”I . v -
cozwmntieul enemas ..2-.4021 were aged {.1— m mace

37

33,194,;

 

Gfir21?90 chi ens were tudafar‘.1fied diet
pregurcd accori *3 t 03:3.23 (‘ *"Dlimhea). wnis
diet, havever. was deficient in certain @fiSefliiul rc-
quirenenta and wag mufiifiad by Smith (Ragubliahefi) by
increaain: tLe amount of thiam;na and Vitaain 92' This
Buy leanCeu uiet greatly improveu tuo nutritional
at $43 u: in» germfrue auifiuLd tharaby prov'dind maze
agitJLia chicfigna for tale 534d]. 235 congeaitLon of
Chichgu dict Lv-ujzx ia.éivna in.1able 1. Conventional
oniuhcua ware fed a nutritionally adequfite. atundnrd

paultry ration.

gy - wt Etna

t

 

 

Fl xible plasuofm1801.ztors similar to +hoae
described by Trcxlcr and Eovnolds (13 57) find Trexler
(1959) ware engloyed. A mcdifioation at this design
was muse Which substituted 8 fiberglaaa door for the
one describafi. The dime.3 ions of 11-23 1301:4t0r chamber
were either 24 x 24 x 26 in. or 24 x 24 x 36 in. de-
lou21u2 ujon tr :3 sine of the cage'ueed to accomolate
the gargfrve chicks. Either plaetio or perforated
9+eel c~"*‘ vere uficd u0“9fi ifiV‘uwon t*e sis 9 and éea
gram of crowiing of the animnla. $ha chicks were

hut. " 319.13. in atiigd Jra KEYS-ft 021131210 3'11 '51 13134-3 cfE‘e

A A- “m

—'I. VQVF‘.‘?'.GTLQ 131:3.8‘13108 CO... Media. Pennsylvania

v.
*3
i

 

I 7“ ... ‘ ~ ' .m 1"» W ‘ ‘ ‘1 4 ..6 0 Fit!" t -
2 H.: l. Tn;rnc1¢323 93 C113333 d.et IF? 5P1
5 q .....- 3.? 4" _3-- r“ .v '- ...... -'\'1" tip?“ :a'
13...”; EMAQIR. {.13 its.) $464.13 UL-.-¢m/ ”5),;

4:3. (an
.“(.I

)1
=4- 4.)..-)

Corn 3231
Hey he; a £4: ‘
'1
a

6
~‘1 51L 9 .f 3941 2.79
“.118» . 131?]. 3:019 1.133111% .31.!
41.32.017.62 ' 415:3?
‘ELt hcrugs 4.55
1 Cl (1031363) b,é7

Vit“.ia a {0030.) 200,6Q3 3/5 2.27
.- . .H. 1.13
uroléne 61 41-55
-$cnrbic ac aid 6.54
Hiecf13vin 5.88
C lcigm 1.3 toiscn”. Coll
ELL Cf) v.13? 1.2:". 1-1139 0.14
155:}.- TC) :3 {<2 ...5.’ 6 £5.31 002C};
lfiflflltfll 5.54
Wivtin .3332
?0110 1011 , 0:1
5:“...1. ...! 1‘16! .‘iul 3.13
fit-1.413..“ P a, Coil
1:;x—z;.-.r-.::.;. 1) 305.0.) 430,233 u/g 2.5.3::

A

nafiel lfll, 9nd Vere latcr tPEangrrfié ta fi 361 153
03323 for “itigl rrnring. Ferforntgd steel c 393

vcre uawd to axint in th; Vfiixhlp unti. t3“ CX“9?1-

g." .. :1 i. . . g -‘ . .‘ "\. _‘u .5 ‘
'.'- .‘p-‘i _ «#11. *“fl'”. ) 2"»“~'
l. hf .... . ..Lz“; " '. .L .-. :. ~.'_'a._-' 'f {i '....-._- . 3..)

.-

L
I

. pup—1. _ a, 1“? .I J .; q . .-. . .;\__ ‘ ”v _ 5...: 0‘ ‘r‘ q 4 i
“5"» 3 fr: ‘-~‘- . '95-} a “h E~V1~ttuhfl 3. r’v‘ . 90131:..3 .311 Of If = 09..
. W.“ -.

 

 

if

8036 “ scr'afi 38 6A3 guxziciéo in $218 -tudy. ~30-

I Q ...fi

Gwflbl'. “n alJ.T§ f:.r"l Lafifuuth, 335 33633 to t

9“!

13.3
' . I'; .. ' ..‘A . a: . . I ‘ ‘n... ‘ I A . "_ a: v.-. S! " " 5‘ . “ ..I ‘: I.\- 3‘ I
£1LI¢§4¢¢€3£1 15 I‘Ji. a 4.;15. L 0'%31A0‘334.L1‘E..UJ3IJ¢ 1 {.31 0.‘n’u} £52,.“ OJ.)- OViCCd

“uh," I 3 o.— J u- . 4" 4- $- ' 6' 0' ~. ifi;..l a l.
£331.--qu 52311C--9J 0.. .5333 u¢3314¢¢tfinbo

.. '. ‘-~:. : I: '5 ~54 ‘¢ 2 -‘ ‘
3i". .1 o v “2131‘ W's. 3 333.1. 1.1.5.3153“ .LLL C 1J- bb'r fidmIVtJ. tIMJB
r .. ."3 . .'. ..«. _ ‘ it...- ‘1. - I -. ' ’ r a -' -" r-" ' - - 5“ ' ’ .3 r f. 0 ’;~""
ch.33nai 3n Julwrb- -.;L’ IlHUAs by huuUU1uVLh; 3dr
”

:3 min at lxl \ and lb lba pres I‘VV.

 

#:3131333 its V3. €3‘~“4r,-. Diet au‘ supglicm to be

 

usci in the is olmtore were s39r111““ in "t inlaVa

o .

sisal 0:1 HiIu=r$ 14 in. in 1Vm3th an? 12 in. in diameter.
Cylinfirica thtfl V8 :9 coverad Vi tr~ air f'ltav Ladia
PE~115J uné allowefi air and atezm.trhnaf-r C3233;

“fl oavered

H

;.&
iv

:1.»
$9
*3
l

auVOQZVVLng. Thu Open and of t} L? or
. 6
with 0.091 ifl. transgc n' m'l.=;r £11 . secured to tha

r7
“um.uit1 Leotch brand t3pe numbara 471 and 853‘. Diet

3 3322333303 ¢¥+uk°3£ ii5l"df V§?itg x303. Corfio.
r; “5.! 1."

.‘= a f. o M' a

Lmericnn ;“eri ‘36: C AntPV. Erie, ?9““"Y1Y”nia

Oar. -’;‘..£’-LI-Q;llilfif Z‘i‘nvrgjj‘ 4.232... a L"'.“D"‘.. 3.201” ’30-1‘5530: 51110-
3.1. Du out Ac*=wuur Vnd Co. 130.: 131~“;303’ 331'
Limnesut3 ¥3A335 ”1:1 1490 00130: Anne 99113! $133.

I

‘3 cxu: A km

0...

41

was cantgined 1n 3luminium puns 11 1/4 x 7 1/2 x 1 1/2
in. Keefiles, 3331:1333 and bedding were cantzined 1n
perforated paper bags and placed within tie 33331
oylinfier.

The cylinders 383$ sterilixea in txe autoclave
by: (a) evacuating the chamber to 28 in. of vacuum.
(b) LL.-ttzng steum to a chamber pressure of 10 lbs,
(0) a: in avwcantilg tha chamber to 28 in. Of vacuum
333 (d) aanitting sta.u to a chaub3r 33933333 of 15
lbs. Sterilization was completed after 15 win. at

21 C.

in iren. Salina calut“ 533 containing an ro-
priate amounts of bovine nerum albumin (933) were
33.4311“3fl my filtrmt fan tizrcugh n ?eit3 filter 33d
su3gorted in a 33inn33 filter. Th3 filtrate was inn
Jacted through a section of rubber tubing into a sterile
10 ml 51333 am3ule. The ampule 333 he3t 333136 and
mzrnyel with ycrrc3tlo acid bafnra it a 3333 30 into
thb 1301flt0r.

ipolfit,3§. Internal surfaces of the leviators,

 

cages 3nd 3 :tio floor 33 ta were sterilized by apt t;y-
ing with tho 2% per3oetio acid salution. 3 30-min.
333 aislenca period wan 3110333 follawing'thia treatman

to 30331t maximum ucfi an a: tne uieinfeotnnt. The air

inaié tha iLclLtar chamber was than replacefi with.eir
which hLd bLen filter steriliged by pLaaLge through 4
1*yars of air filter $11111. Filtere were sterilizLd

by dry heat at 15% C for 4 hours.

k"

Lanafer‘gg_3ufiglies 11:1.223 Isolstq; CLLLber
S1L1re~1.k £11 ska cant 11155 sterile 11 tar were
cheoiac<1 for a vncunn meal before pass? 56 into the
isolator. L flaxible plLfiiLé 1161? a $13 sprzne1 11th
per cetin 1011 1nd t 311 to 1L1 ribarglaea deer of the
isalator. ‘31 “111 1uzf;£1uf tha L1tar T1133: were
then eLrLyed 111 plaeua iLto tLa 11131: sLL-vL. The
stainless ateel cylinder'whieh‘contLinei 1311111 fiiet.
bvdiing and afiher suppliea 113 atigchmd ta the egg; 131
of the aleeve. Following leading and attachm_nt of the
cuygly cylinaer, the sleav1 chgnber was 3111361 tLrough
Lorie on either Lids and 010211 with nLOLrene atopyerao
1 30 min. residence Leriod 133 1110161 to permit
atarilizution of tha iselmtor 11¢ LL. :Ecu tLLL film
hLd $1.952“, thL plLstic inner cover of the $1221; $28
door was removed and tL. LLter £11113 were taken into
111 iaolntor. The mylLr cavar of tha supply cylinder
was Emmanerd and 113 cantents were tanan ed. Litter,
»mgty water flasks 111 fee 1 “3:111 were 313211 into

*
I '5, '1
a. ~-‘ Mk1 § .

the Luurl- cwlinfler 131 the finer door 1:1 r1
.1’ J

57“

4.3

The sleeve uni cylin3cr were removed, the outer door
8 8303333 with tfi§a nd 3:3 3“*'Ved befu: 3 its ports

were closed.

£§_€fi mlitg $3.

 

a
9

ch lccke mrv. free: fcczitl -:mrlea 3 are col—

H
rt
5:

in cle.n,a marile t33t tub 33. Fluid thic-
5130011333 r3;inm 333 1333313333 mith thaae 3333133
333 izmc zb:wt 3 at 24, Sfi 333 37 C for at least threa
33333. ,we rs were :refi.”ou :33 etwined accordir g tn
the method of Gram for airect micronoogio 32:3132'3 on.
35; oholls troy newly hatched 051033 were incubated in
a £133 of fluid thioglyeoll3ta 3t 35 C to establish
their fr333cm from bacteria. 333 gerafr33 status of

311 small aLL“ ls acre ovnfirrrfl 3y t3333 methods.

.‘wlb 4.3:». 5 .55»;
* m :7 v V > '_“ "lfl.u- W

‘5‘» L.“'".f" 3H» --5 ”an «res». 3 n r.»- "fin , {M -’.“'3 '3 ‘3- . -
.2c333 H :rtjx . 3‘1h33: c , 3333 no

 

lean. unn‘xdcd 3:33 verc 1h-3 tedf Gr 19 1/2

C)

anys in a J 3333; incdbrtcr. They were than Q“QVG -d
-.'I.‘.rl:ll‘. (2132:1133 ta £331.33"; living 3:31;... 335.3. catic 2.21:1:
- 5 fi 4. heal-1‘ .- . ‘
30133133 333 3333 ta hecegtdflin;;a tha 33311 Eurf.C@30

‘1

"LV .4 ‘fi-Q-‘If‘ F9." .. .l d , ' a. ,V-x
3313 3343.10n 3.3 pr p.23<1 in the £33 33.ngmm3nncr:

\JJ

.- y

I 9
33 3613 {13333

If;

m . _rv' «t ,
31 of a 333 solut3 3 cf war ca 13

M

230 m1 at distillad 3333r and 31333. 3h3 solution

3+
C)

van 333“*€03 $3 33 5.0 by tha naaition of 3§yraximmtaly

p
3?

‘53 :13. cf 33% Emmi. 12:..0032151 3......3 “2:305: t2: :2 Tim). mm.
car‘rfiisx of 9.1;? 2..-“1.21 £1.12 3:: 11%;"; :1“. "2:23;: iii/1...: tail to
{333 m1 2:11:21. {listilled mater.

Iixtmrmd surfaces of the 6.53453 W333 clawed mm
aacontaminated by guceeusiva waaid nga n &a slhuline
gatergent anl the diaiflfectrnt. Th 556 G3 . wera imp
magixtaly place-d an a etarile Git-311319533 steel plate
in the koletcr floor where they were arrayed and emu
cloaed for a 55 min. M1139? ca pericfl. Following flan
Cimt.;r..rain..tiun, £11533? '1; me try“... .-5‘1‘1‘21 into the itselator
ch1mber xtere they were érlcfi and Get far hatching.
Tia tempczratme of 16.3.32 isolntsr *1 mint-sited at {3:}:-
g;roxim¢.ztcly '53 C uni}. 2 waclm 5:31:92? 11' 15122144; at which
ti? a they were awe ed to a COlQfiJ m;intenance room at

t... 1'
‘- ) ‘4' 0

\..«$- .-
-.L"v.: ”i :13
W

fannitu'tid bovine 8935.15}! :..3.1 ‘1"... in (2335.) was whiz".
52.3 the: 1.1....cirIl 3.11.1 51.921 in 1.2113 stuuy. It r. a 0?:-
tginaa frag Cglbiocham,fl lct 533515, and prop red from
(303111 frwo‘r .1023 V, garage C.

ghee? mud L1::c¢ cullJ AHIE collectei in A1—
Levers solu‘mn frum a, heal My aiult £4.16 mum—.11 «I‘d

also salve-:1 as an rmtiggen.

A . ...

a. CglLioehem. L03 ixaaleeu Califfirnifi'

SerumJlequtiog

Blood was collected in clean, fir? 25 x 156 mm
glass tee tubes. Habbit or chicken serum wee obtained
from 10 to 15 E1 of blood/tube a ter syneresis for 2
hnurs at 23 C and “hatrs at 4 C. The serum was

.p

clarified b" centrifugation (2.590 R.C.E.) and frozen

at -10 C until its analywiSo

~ntimnrwn Hrs mgigg
.rimgry rampage antiserum mpg progncué in
4D-dwyunlg gernfrce 33a canvcntion l chickens by a

3L v30 injncilcn of 332 or ahaeg r35 blogd calla. inti-
E$& sensitiZwtion was 1 *9”*lia.nd by uh 191$: vsnsue
injecticn cf 40 mg/ kilogram of bofiy weight in 0.5
ml of }”“31LLUFLCAL saline. Dzicnsna v. :re L.i.Ll *:d
with sheep red bloofi calla by an intravenaun
injection 0! O. 3 ml of a 3; gnu 11vension of calls in
saline. 3.t scrum w: a obta i.ed by cardioccntesia at
tho 6th ézy poet inoculation. Gran? seruma were

fiGOlGd ccco..;ing to antibouy titer as revesled by
an interracial )rcaig it n Mu.ljb‘3 er hemggblutinp
EtiOfl.

9olyvalent rabbit flhiiwchionfia uniiucrum has

produced by a eariea of insectians at an alum pre—

cipitate of normal earns from.4D-d¢y~olfl convantioncl

chiekans. Each of 4 adult fintch Belted rgbbits mere
given 6 intr3333cml r 1333331033 3t 13 any ittc:V3lm

.. .. .3... 4. ... , J
fol 10303 b 3 einb 3 intruxeri one 1 isuccticn of

"CI
. \

serum. The rni $13 acre 2131 DV 0 ”fi‘oc~"*rh*b 6 dmya
vftnr the terminrl intrryertiranl innauer an.

$39 133.2113 333 9:333:33 by di 13:13; 5 L1 3!
whole chicken serum 31 h 16 ml of flistillewi .f.t3r 3nd
1% 31 of {a 135 colut ign of? l (304) .1? F-G. Thig
mixture was dJumLHQ to yH 8.5 with 53 ugjfl'LHLOH re-
multed in the fornatisn or a 39333 w-ita prgcipitnte.
The precipit:: te 33:9 33 3d twice 31th physiolochal
331133 cont inind 1:10, 033 rcrthiolafie 3nd 333 r3333-
333333 to 25 ml in this solution. The 1333133 333
stared It 4 C for no lcn;cr than 14 3: v3 bcfcre its

1338.

7);. .unifi'fi imhv.”~‘.q‘-‘v‘ C%At¢\-v§ “A“
' '4 ~. F ...-o -$v d- A} '
"-fi“..-‘-. “.3....5 :Ww u..- ”cw-«nut» can‘t-go: “:39."

‘

:' " " 1"? ‘WQ ‘f‘""" "Pt. “3.: '.- f1‘41“":‘7~ ; ‘.
""L' “J I§«"~*;"““Je {11” 1-4.5... i t....' ¢,"(..(._

i-ni
0

per.
‘

_’- & - ..- n - ..vp .-' 'w ' ' r ‘ "" I"). ‘- . . . -. ... H ‘ .
$33. a L tue yaLCiVG renurLl3tinutzen 333033393 33

kl.

“-i

'7
described by *Wb t 333 Layer (lfifil). Fresh aheop red

bleed C‘lla rm re 3 w. 1 tkrve 1333 13 3hosrkmte b1f~

. J
(“W V '. Pi .- . . -,
sdxin" (I33 at d 7.2. a 3.53 333333313. wr.

$5!

fare

.3“.r33 by tde £..1?£‘3 cf 1“ Ml of ext} 001 13 t0 40

{:2

3. 3f buffered 5 line. T nnio :cid treatment 33s to-

cc"*113“Ld 15* 3131"; 633:1 volurm of the 2.53

suspension and 5 1:20.050 51155155 ct tw 515 5015 and
incabxtiug for 10 515. 5t 37.8. The tanned 05115 were
055Lr1£5¢55, washed $5105 555 r555531555d in #0 51 of
bufferei 5 11515. 5511555 ccuL153 555 x5535 1 1'55 by
555155 1 51 of 5 2.55 t:.LLcd call 5553553155 to 5 mix-
ture of l 51 of the 555L515 5515L155 5nd 4 ml of 13?
at ya 6.4. TEL; concentration 5f 555 550L515 salution
v r155 fram C.‘ to 1.3 55 533/51. 3 +55 10 515. at

r5 5 t5npergt5re, the saluticn 5 5 05:1Lrif5ged 55d tne
05115 5555 5~5h55 255 55555355155 in bufferea saline
Lt 511 7.2 to th 015515-1 551 1 vrla“5. 5 cantrol

5.5 .19 of 55115 555 55555555 in 5.515115r 55nner but

.3.

)
3.11

It?

without 5rct5in 25555555t. i5 M1155 w 5ich were t5 be
55v5y555 by this pr5c5£555 5555 $5159 5haerbed with
5L5ep red blue; 55115.

’Ltiaxzwm 5:15ti 5: were 1'55'511 in‘bmffered
551155 to 5 £15 1 Vu1555 5f 5.5 51. To the5e dilutions
555 55555 c.55 51 cf L5 t “55:1555 contefi r55 5511
5555555155, 351% L15 coatr‘l ttb,5 55‘5 iaoluded 15
5155 55513515 55 £5 11555: (5) 51:55: 55115 in 5-1155,
(b) 555555 cells 555 55t1515 55, (c) thnefl. 035t5d

cells 555 551155 551 (d) txunefi, 505555 05115 555

.115". 11.: 4.5 ., ._
~51515105 yngeras Lara 555d

1, 1*.-

:.-# ‘c. :‘1 fl' ,»‘,‘.'. .3 .‘0‘!t ‘2 ‘ \ . 1-._ ‘ I I
51L5r an 055551u5t 55555551~n .5t room 5 *:*r.t::5.

T .,. .4. JO. ‘ ‘§. . m ‘
farm £13 Jr 2.3;?“ e: 3

 

 

A fiirect Lhurr31utiimtisn ansayv v.13 er319yed in

. ' p‘ w; -\ R Van»; 0-, .
1.3 tit .tlon of £31 M?rj res awn: hauiSCtum to sLee;

ing 3.5 ml fiiluticnn of heat i“nctivata&5 fiticc crum.
zemagglutinutian pgtterna warm rand after gn over-

night incub tion at ream ten.w rtxreo

1,- Qr-s s. a 1‘ w? ‘T'zn Aug-g. J JI- 1‘ It 4-1. ”5-17.75
éAvva-s" \J' F "L'n .J... L—~\— «than. fi- w- J
.... .. 4": ‘w—w —‘ ... 1-.

 

 

Ethl vcluzes 0f primary resyenao anti-Bi;
eatiaurum Lfid 0.23 L’LQlCUUtO W.nol ((-m; were mixed
and in mph +53 at r: 3n t;:jaratdre f5 24 huth. Follow-
ins tnia fire :tmcri 9' than uture was 61? lyzei fig inst
reyeatQJ ch ages of ?B5 at 3H 7.2 to Inmove the E-ZJZS.

Th astibofiy can eat cf haze nmtlsurnms were titrated by

9‘. n a. ‘- w .m- Au. - ¢ 4-4!
wasIvc acmcgmlutInfiyian.

 

 

 

 

v- 1.3- .9 f“ 3.51.1 \ ..2.“ - '1”: i a“; “129%.
_L “{- v-» v} 0.5. - 3.». ,ant‘fl ’. n“ ‘ ....C‘ ‘33 J- 4T0 J CHIC}; ; 1:. oi‘idl- L
' “In .3“. ~— m A ___ m
‘ 3" I‘ .. ' ‘. _”‘..- l' V ', 9- p 1. ‘ ‘c

B m h} n “alcgous primrry resgcnce 031033- unti-

serum ? are wired in the Optimal groportions region of
the eq"1v;lence z :13. fine ml of undiluted antigcrum
mga uizei aith.1 ml of Ext in a finsl £:1t cs m.tr: -
ticn of $5 to pracigituta the mat gen— hutiuoxv comglex.
The BS; 004"}?cntr'fi10fl erle-J frfm 1 zfxa/xl ta 0.25 r~/

degm M‘iuu upon the »mtibudJ titer 0f tn antiserum.

’1)

4b..

fiber 2.31.31.11.15 . ”he (:4 ism 12-13 1:10's.;.bvt1+c.‘i far :5 hmrra
; E? C 3; far 34 he ouze at 4 C. The resultin5 pre-
“it .e n cantgifgged and 1.2he; xi1h lfifi velumes

L: WC... 0

ask?

iii-1..

“91: iz-tisn

by the :6: tisn of 11¢ to 210

.-~ ‘ . n G - a, 1' I‘. ’ -‘ I H. P 4', "- q . ' A 1
., f» u\_ '5. ‘ ‘ .. . o q f.- ‘7 a y
rflvulst (.33.. 1 »‘ LL;‘G..A LA 91.33.. V“: p #51

Ram .u ”.~ -. a .
“,bl, Tuig £111'x0 wrs ¢uc

‘ 4’ . - ‘ I” r: -, . . 1- '9. ~ _- 1 -
“a: at 4 b 1or u h0133 1 HM frag-

V""y'§'|
.. ladd-

.‘ _‘
c1 tfi tLan, mr,
a,

ll
tf‘fi U

Q

fluid v-3 01“”

greci

'111‘511‘3113 u t 3 7

r t1er vice

git ta 139 .ocmnzlihued

tvEf-s-i a3 t_ic (9:11v161208

r.

L1 or O.L,J¢.
3 far 6 houra

3

_S’
I

t9

-\

agit1tifin. liter

t

. ‘1“
a". ‘3

16:1

own a:

:9“

fiery-1.“

“guhle 001110 .rw

firep-

3ti$ao
1.1 1‘ 11:16.1 T133151
.fl...mmw.,,quw.
0113:1r111? 511 c ifTurion 11 ifs 1011 1rafifrcd
‘ccerfiixy t0 +16 procaiuréfi Cf Smith (11$?) :11 .111 C01
and hu11nvnv (3353). T3” ingvhdfiewf of the bafferad

. 1 '1 _ -.. . _, - 5
. . . ,1 ' fin“... .
-3 r an; 1 .“dfi are + 111 wnl bi
-"‘ "'7’! "3‘ "3 " - A
g. -,. ,1 L. . LU}; atj‘ti Karl or
.1 J" _.. (5 ~ I -r _ax'p gr fir»;
113‘; {Ls-9:113 LU}. ”1-5 0-! Ut-I-k ~ 5-H?“

 

Inbrefiient

Ion 5 r 39. g

3.13 3’3 Ev/fltgt!%
a. n

0.15 3.3:rKNQ
:2 4

Win
0“”

90

C0 3lidiu3 d L& r.t3ri€9 Ira

us. - r- {VC-OIMQval

1

Oouauu¢naau§~0oauh - —\ - 1w 1m- ....m

‘1". '1‘ 2f.
"J‘vO'fl’A at

1?,
V O 4‘91.)

7.511

Lv.’
r“

l

{1}“

1510:153 fleibgt 5' 111.

o;

0". - ...

Jvnn E. Comyus Winn of +re buffernd aer gel
(3 413;.1'3 £03? Odell“ {'33}

.g

'0 J 501 “if-fl 1.55.!le Plii'" (.3, ODE-”Lt.

t1 r-{I

 

 

 

T w v— w». vru~\.¢ b‘ "l" ~11- t mm» W. . ‘- ,_
f TIT:};,‘ ‘( 3 1’: ""'?.N.“ ‘15
I 1L: 3.“; {... in L3 s.) .315. Q
“Ma” ~ w ‘_“—v , - “ _‘Z
Q ." . >- I "‘K .- .2 I _ A A > I
;:VJ19J051031 a line ¢§.uml

h d
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To: $31 of tE‘s h

0
c“?

3611 ic were poureJ ov~r -he surfJog

l x 4 in. glar~m L nbern c 113 anfi

b
(I:
kid

or 1:1 T’iCifll 013'.”-
mllowed to OJUl. Lffier aelidificution in a humid
Jtuua,ue a far 24 rqara, an aggrOprinte gaggle b sin

Jttern wgs ,JL~“e' in fine gal. J drtffiel tcnylmte was

“.5

“q .7“ ‘ ' 'E , “.1 ‘ ..I' l~"|"‘4.‘n. ".4r'- :o...»"n"‘ ... h ...‘b \‘ .’ l‘t - a
1.2L 0C .1 Lido;- ‘afr 10-633 dud-c..- at} i-=‘II-.~‘--\ «tutu- V v‘d‘o { S 5‘ CB‘Lht-‘irfiiy [Jain-if G.

u ‘1‘ ". . - ~'--.‘~5 - ' ~‘ .. I. 3‘“ n 3" . -. ‘ I 1. !‘~ O ‘ o I.- .- 5‘. .g .l ’ -,- i . \ '
TAB 3.7-. fun, Us :11}; uxfi .1133; 03.3. (“Liv Uzinte :3‘.¢3~":6£1 as} 3 “CG. ‘. u UJ'.‘

L‘ h. u f: _ t ’4 a. .71 . A ‘ . “ _'_J , , ‘ . J-. - I: - , --- 9 fl
blah LJu JLJJJJJ JeJauJuta 30 133 3r “Ann 5 393:3 “‘tcr
_ . . - ., _s _ J.. ." .
:itstfia were Jave1¢333 f3?
., 4., *‘1 . . , ‘4,» ' """ '
4~..‘ 5‘3 79,-“ b‘ 31.3 a! 148 6 Limb-’5. ad" ;J.Q{u{?r at. feifl r1 éién. I‘ve-J 1311816}:

up

warm harixifiaa by Wutai LV-J.- tiaa frum aciat filter

- _g‘. .t ‘I .... v 4 2. WW ,. r ' , i. 3..
1.4 Si 6%; 4.1.; “‘9‘. :3 {3.3- k’wz t‘x‘j '1‘!‘ Sxkuca 5‘31"& ‘1-‘7.---~.-1~g‘;3

, in” d.;\ ‘_‘_ p. . o .‘ _ P‘_ ~..-" , .\ (5. ,_, '_F ‘1; ., u-.
111 u‘JIJClt..J 0,-.LJ..JC'a LAG 1.3;..th CQJJutrJ’Zt (31. has fuel me

H

{a

-. s a ~ -~» ‘ . (h _ ," .-.u- .j ". " - l y" 4' 4 -? 2' .5-';¢' 9|
‘IJQC'L. bf 51 .3.. an: «.3.. 'r- (5.1441454, .uJ. £=..il:)ui4.lei'¢ 'rm . LL‘l .

A:

~ . p A‘, , . ‘ .'. ‘ ‘ .’.-..". '.4‘.,~ . \
5.“; 1.1" 3"“.‘3-3 v. :1" fl: ’. I 16:5. 03! (90' 332A! nLAU'A 1.31 3’: :1

hoist filter yapér to A iLln 532 film. Erct.ln WFE

Pfi-
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:35

- ' Ou‘Ji. " '“ ‘5"W40 $ 4 I V \ ‘ F .'I‘, 1
{-0.775 1' AA. A 1 ...; 1' A p.44. 3.?» -3 {3.3.7251 in 1.. 53.4.»? J.
m 0 \ 1" ".~ 0 Q ‘ .' '
a . 3 uu._fiiktlfln of uroer a

IA:r adicnt 'Aount

 

_A — w ... «m
p“ _v .. , .- .3. 7', d «'3 (A ,.,.
Jhl..-I.'-h;*J‘e di-C :« V,.1{_q

.v:‘.C¢‘LI'~ r}, l? B {2.15

I 1, 3 , ~. rut} r8
.3.“! h. Criolalb ...". kl .16
_ . 3 .... .- J l 1 f!

5’

Ac rculie chloriée 0.15

“-1 3.1!: ..>— .- . A .. ..: "3"“
:.'Lfi§bla.lifi-.l $251512: Cob. ".81.. 1.4%.}.le

p~mwp "...-c ~uO-‘nhflUMD-MOW -..-_“’-.. A W_ “ h ———‘

(3.: mile. 13151}. the

‘ ' ' u: -' (.' J‘ - O"; ‘1 7 . \n‘ffi. :3 A“. . “ 1". '
v 0.13 1.13444. 3.0-“ ‘43. 3.1.»: Of in? L-‘uf. Ami"? A; (A. 31 3363(‘4...U.23

.. ‘- . v 3 . s .. ,‘. ',- p v' so. ". :w . o t' . . - x,” ‘\-‘ -‘ I" .x g ‘b- "‘
acre AAQAAA.I; A0 AAAAetA JVUALGJKKCAHitttiod of tuv

. r
1")
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7‘
Ed.
9‘:
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31'
£
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‘3 It
,A
c F

xvi.” Anti,- 73A. A O‘lt
concentrrtLGn c
ceAwrutAcxs V??? ufiAd y“ effit ..2 fnranti“; VF? re-
A“ceA. 23c Adui‘iun of AlgciAe or c . AAA chloride

vAr 1A-Ay i: zflAcncefi Arcciyitvtion rm: w.s not con-

‘y.q".- .‘ v." .- ._P_‘- "r g. ‘5‘ o.-
Lluaxoi neceAA ry. Aonqb.r no. 2 “.5 eAfA ) A? be

. ‘ ‘ ,. “I ,‘ ... 1.. -. + 3 V, ‘A‘ ,’ "'r- ‘57“: -‘ - s.- 'r a _ 1-.» JI- \ ;;I , ,‘ .
of ifia “AA atncAL.A alt ,chAttcd a rLAAcAiCA in AVA

9»;

J
h 'I +' " *3 v V.“ T“ "q- 'l . in v’ c. _~.~ .l 4 ‘0: a. .v-‘ - 3' ~ "~.
31.1 $31.. 9." 3...; :n ‘33 o 3’ J .- o .L....’..3 "'L: -5 :13 LA 01”; 3:. 3 .2. 0g. 5.3-

O
*5

ficiul offact by allowinfi unrestriatni jiffqu.nn

. ‘7 ~1-.‘-. .""““ - , .“1 .~."
AC 3 U} x.2 o.uxu_l, AACA 10 or can-

the

52

avian reactAAts.

93;_311tratian

 

Gel filtrAtion in Sephsdexlo 9-75, GwlOO, 6-20“
bflé in Ionagmr 501 we: garforuci in glass coAumns 62 cm
in height with an inside fiismetar of 2.3 cm. The exit
orifice wan occluded with glass wool and covared with
a 2 cm luyer of 2 mm 31335 beu$s. :Acxing wus ao-
comgliahmd by a grsfiuul addition of the gal to the
ooluan to inaure Aommgeneity. 3

I."

‘ " ' -' '- v. ’\'¢ h?" x. —- * J -
Agar Ael WAG ErUyfler onm an AA soiuLAon of
“\

IonagAr No. A. The eulidifiad agar was hencgwnizad in

a blender and filtered through a 40 mesh scréea before

Two and one half ml samples of whola serum were
upglied to the tap of the column, fractions were eluted
in phosghAte buffered sAliAc at pfl 7.0 to 7.2. A ro~

ru-

tAtiAg drum frA iion collector fipted nit: A ~ ml

voluxetric siphon colletad portiona of eluate as it
Amergcd from the column. The yrotein content cf each
fraction was determine; by the Polin reaction. The

.. N ..‘3,r,‘,‘ , ‘ ,fi"..,
zroce;1“e for thzs QGtQTHLflJthn was as foA-o a:

‘vd

\

0.5 ml of A Efi potAsulum tartrmte sclution was mixed

with 0.5 m1 at lfi Gas 4. This mixture was fiéded to

 

13. FharmacAA Fine ChAAicAls IAC., New York, N.Y.

rs‘V

59 31 0f «3 b35003 in 0.15 E Ofi. fikia combination can-
(.
33133393 rcssumt %. ”a: but 3 L-s Irapgred by the

fia

-, u" ' ‘H_? ‘1. o =' .‘t. I Q 4. ~ a I‘- .' ‘ a! t ‘ .. .;- ‘
1.3.0.1 Ui')},.t (if 9413-1 1. I- 1 5.!“ Of .-2. a. -. 3310C.- ..‘u ' 7'1. 3 C513"? 36'!"-

Q. ‘ x. "V‘ ’4 .‘ I .- ‘
CIL ‘ '1 ‘erd-JL 1“ silt-06‘» 6-“ .rl\t1)4.:4 w “ :I.

“ 1 ‘ ‘ A "‘ ". ' ‘. v ..2‘.“ ‘ 4‘ ‘C ‘L .1“ " H H. a w?“ '3 v 11 .‘5 er 1-_ a 4 ‘ #‘ ‘ "
ta .6 $31 5.} t ‘ )Ju‘l“. E A’ 5.3. \1 LL '3 ' Efih 113% -'-' a. =‘ ...o‘ u- ‘9‘! o‘er “ l" ,4. b i? 2“ .0 . 31’";
A' 1 "l‘ 3 'I‘ - in ,. *4. ‘ '- q-«u‘ ~ .r' 5" J '3‘. _ u. . e-o -M >10 "5 ...- - v~«_ - ’4»: -
£9! 15’ 3...). ~02. b rUC40 L.;J¢‘.1“(:I -..‘t'v..r“5 . -Vbték 4.! Lo‘nvfiz c'sk-fi-Lm ’5‘ 9“} “$.8l5

:1

lo

.0.) *1 |Q ,- 3,. 3‘ A ‘,~.-4 *‘fi‘ 'D.‘-'E:-'. '0. 1 » ‘ A y A w a. ’ .o, a: 0-1
.1.1 s Lil's”. mail/1 -..:.L‘¢.; ism-LtjiHL-‘T; rig}.&..a«“; &-.'..1.‘.. 1.3 -

O

A . . '0‘ . . ~ A ‘ -‘. A. t" r ...
lCavfi ta bt.L1 .t raga thDergv“8 far 33 min. I»

and was half ml cf (g) cervci as 3 31 5k :33 03313.1

+0 0 p I ‘ r‘{-' ... . ‘ I. J ‘ V “ ‘..I.-
‘Jé .11 ' ’1‘: 53 In J ...” ,J'J ...,Lfi n U 3 :~.C~~ 5-: ‘40., h L a-
Irwin 1371’.) -::;-z':c* . " "of I"

L‘ 2 Q' "_"‘ g‘ ’0 . o A
I: ‘v ... 30.1.3; “Lu.“ If 3. +5n :4) b" I n-n‘ an AA 1‘37““411‘3‘33
W “~“1-

 

‘I‘!¢ f

.p . _ . ... . -. . , M3" .- 1.. .4 . “...-J- °
$13433 resyonae unti-33: cuAuhéfl 'u»1

+

' v~ - -..“ ~‘ ..._ . .1 1 -.«- - - --. - -
chll* ruLeveu erm yhoLa an Lzu-3n by Ln autiae“
I\ h, ‘ 1 ‘ _. .,\ 2 r o- I. ,. w, .' ' \,:, I: r 1‘... 4 at, ., .
0011-162 L; 1.3.1.1 :3 LL 6340* 333...}. c 0 3'.“) “L $3343 CONE. 51:4 It“. 53 .1.” SJ- Bit 1:19
v .x. . . - . . .‘.'~,---~‘ -~.. .. -'
COW” £27.; 1330 b&;$:lh ta.) 2‘}; V ‘Jl 'uOle "i Enllj '.-.-.:10-w\,ly‘

styreaa fails 13a the pr cegure of I;gi, 33.1. (1960).

a

A fluwchcut fer the 3‘932rntion of polystyrene-:ntigan.
ccaju" tL3 is fiinfl in 33Llr¢ l.

.ntiLQLy w 3 I”'U"” frfim 9 ml of uncle :nti-
serum 37 n» Lauitian of l.) 3 of the palys tyrene-
e.=::tir¢3r-. 04:27.35 w 5-133. - .31.“: 33.2.1; for i; ;.:..;;1;:;':- e "I 0.
”5:43 33113111273 2.1.45 twist; sedimented .nd the: {Q‘Llrf'z‘nmfitfiilt

fluid was s 33 for “331313. 330 rcs;n was washed

%
n
I
A
3:
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’41

F.
R“" CF'CHEC‘R fumnfii R

(:3 HNOaj-

 

 

 

 

 

 

‘" " X "’ N01 ‘JX
HQOH
V
r _
R CH-CH Q’ R+CH<H—+- Q’
E MONO; /
HCL \‘1
Cr *- NHI J x

 

 

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R——»C{4-CHA~$2‘
/
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FIGURE 1. Flowaheet for the preparation of poly-
styrene-antigen conjugates (Sahon, 1959).

. H " : . .‘ ‘ . - .5 '= ‘i ' J 1%
. .43 ,1, ”..2 L . 9" .U W a 5: :~_ _‘ o. .1 '9‘, ‘1... u¢q1 . n _. , A 9.. l v- ," P w§y‘
"t “'0. 'n! as). .uJQ‘Lfi» ". 'hrltoéml'v' 3.. '-'~b~’ miJuQC n.~ £358.35 .La'u Uiéz

file 0339191 by flu? eating the 333331 aeéimafit in 5 ml

:1

, z a” 1 - Vr‘ ‘? . . ‘ - > ‘ -. ' ‘ .— ,‘ 0" Wm ‘r, .. .- . «
Of '“ "“ 5 F‘ 05 3 “3E"w’ “‘"Bflm L? D”

*flfiv 1.1515511.

;

2i 3 l. The an 31 9% Cf $33 mixture 333 3.313ted to
500 $0 4000 3 afier fit

.. . 1". .1 - -‘ \« IN I '1'. 3 '7‘ ’V n J
Ii :11; r3031 3.35218 1.. .2122, 1:313 313256.: 1.3:: 1.1.5.3 (3:331:23...-

:3?
T1
(3
5.»
p

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‘33 the Eu: cxnrt333 fltil 3:3 afjusted to pH 7.5
I,unCE. €36 salutimn was C“UP£1t

in $100111,

’1
12:"?
if.)

...*!' ' _hl' f '1

.3.. I ., I y .. K '. 't I. '1 . =- J- . - ‘u-A-_ ‘ .1 ’ a ‘0. I" “s; . -,- ‘3. 3r ,0 u-t -. 1
.zs:.£.5.~1f.'32'j' Lie-24V} 5-}? 15:23 Lti uu.?.1...1.h'3d by Eng-€54.31 '11? 138:3...Q1-‘éld"
_, \.-' a- “1‘ J I: s. 7. ‘th‘ . ' .. .

tinhtlun. 333 d - wool ”t3 3 ti3e, an 333 as3ree of

..-4 4m 1 , n -u.-n'.~ ...:3 a
3353 33.3 £321? 539 v3r3333 parxflgd 3333333

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b D ‘I ‘ J 1.} "~
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11-1=1-~=:~ a .n :3. —. ~ - 1‘. 3' . ....“
.1. u .L.... w . MG3MEJ— 1... 5.7.! fdr T‘.I.t.a~L-Q 1:} (.1 {1.333 L 3 524011
h,~. 1-4. ".' 1 '- n v ‘h-L up - -=" . ~0..- ‘ I K q 4
£333 tLC 333335rrene-3333533 c3333; L63.
Q OP
_: —..- a-m ..... w _ .... v --—
11 ' O a! fi _' '0 a h
. 330c1.5113 c'nuzt3ana
..2: 1. r a. 991.23.. . .‘. ‘ 9
rev: 1" 53:2; 3333:: 332.123 (.5133) p I:

 

 

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‘1 - u. “ '1': : “ I' r' ’ u“-‘- ‘ 1 ' ' ."‘u ‘. 7,.

f; .w 11204.3: Cam, 5,1, 31‘“: "1.: T: CC:.-.V&'.‘.t30£1.;.l 0.5318213.
f 3 . . 1.. ‘ “, _
dg‘a3ntiflcr3m frqm "~r33“*e cachfio

 

r“

13» 332*. .4~ , ”4 .* Y” V H v,ux .
11. 23.13:"...32013 «sf-9 .;%?'s.-A.Q.-.J_3 «Lu-Co, Maw 1.3373" poi.

i p ‘ ‘ ts _ L L
Cflléfllugg_?"9fi*ffi ang LILLtngerLLiL

 

Zena electroyhoresls in celluloL. :cetate ma:-
brer Les 1% a perfLrLed in a L..LLon mi:rLtLon chLmer
using L V0}Lm coratvnt voltht, / conLtLLL current power

1; 12 I 21/2 cm LLIIH‘OLL LOLLLLL fiém-

eugyly. Oxoid
LrLLL strigs were LLLlLyed LB t3 3 LuLLLr*inL Leaium.
A 0L.LA:1”‘" b rbitoue hiffer Lt pH 8.5, a 0.07 wvs
uLLL Ls Leaerlbad by Gwen (1336). The composition of
thiL bafrer in given in Table 5. A buffer system,
fiiflcfifitinucufi with r6" r to its pfi,'u Ln; ionic com~
punition LLB £180 afiglayai (flalfiberg, 1359).

Five miLrLIitLr L LplLL were dinLLLLd 3:83 a

, ...." .3... ,n » L - : an, fi“ 9; - \.- "a .
l Lhfld Lipet-L onta L bufiL: ingrLgLLtud cLLlulOLe

If).

r,
‘4

LCQtuta Ltriy. S Lyle LleIL-LLLL Lax f cilitthd

{J

9 being L rn.1Lr LcrLLL tLL OJtLQLG fo fLr rLLLrVLir
rer_~*‘1fi1 2r to tLL Liruction 0f LLLility Lnfi LirLctly
Ler the CLLLLLL. iLe 6:;Lrle LLL Lyyliei LL Ln evan
liLL uging the ruler Lfiga as L Luide. 2 CLnLtuLt
Larsen 0f 1 mL/Ltrip LLL Ljpliafl far 2L0 min at 4 C.
Erom r=Lnul LlLL WrL in1ti= llyi erparLtni into L
exm'zylra t0 indicate. fix} EX‘L-‘Bl‘at Of album-Lin migl‘iitifin.

Following elQC*“0“‘OfeSL the protein LLL fixed

 

120 ’31): VLIEC'llCL L73. 1a 139" tCriBG LLCa’ C5113 313.230 1:34.. ”1133. :11.

stained with either 0.9% T033333 3, 3.331% 31330333.
.22 Light Gre n L? or 00333513 Blue 3-253. Serum
yrotein dietr i‘a 3tion 3:3 me eured byv Jaye a double

beam rnnorfiing 333 intah+2 in; r3£13ct3nce 3333130-

TZ;LE 5. Comyeaition cf 03 1'3 b.? rbit m3 hm far-

 

'- ‘- r- ¢~ ‘ A _‘ “.‘
if:;_;I'C3dJ.3I-S ,‘ 1:07.31 ,zt
" 1’ — 43 a‘ 1-1...- 4‘

D- J- A \9 3:8 1 ‘8) 7"“: :rtall 5.1%,}:

1
8413133 list-t: 0.333g
fliS‘SillCd $1;th q‘ .3 a 31'? q 1900.331

W ‘ w— w - -~ M 9-0%“ '- “man-”~mh—chodovufl“

1;“: “who ‘1yrfizitaa‘3 q

I
t" ‘V‘ "
a7 L - ... 5
*“ m- -”9”----~~—r '.W‘uv

74X: 531 3333:13333r3313 wra rerformed in 3

AB

Q3 3333 3i3r3tian 33 zzbwr 33135 3 V0333 cons tutat 01:
$333 33333 33;;1r. 155$ 003331 3 x l in. 03,33 micro»
33039 3 1333 nmrvcfi 33 3E3 333; ortinr 333-33. 233
bvrzcrs 3:3 gal were the 5333 33 th 33 de3cribe£ tar
13333331303r.530r3313. $30 3-3r333.‘r 833 133 acre
$13333 ifito 3 cantr+lly 130 .aced 533313 bt3in 331 331b~

4

.25 In: J 31139 far 1 :0

in!

30333 to 3 censtant e"*3*3t of
313 3t 4 C. 3333 333301 bLAe 3u3 13333333.;233 i3t3 one

A ‘9... .. ‘0 41‘ u. w ' . - ‘ ‘ 4' -’ In. , ‘_ ‘ -! ‘-.
3-ui3e 30 3333‘:;33 tufi extant 31 3133313 33333»;on.

.«- n

...-1 w ,2. ....3 3 ‘ ‘ h .. m .. .. ’ "
P311035n3~513+3332355313. $33 313333 3:33 imp
. . i 1* ...q . J. I. r' 5.. *. 4 ‘m a Q o c 1
Eerueu in 63 51:01: 1 333.53 3353 35 {5: tan prafl3-5.
‘4‘ fl ‘ -- - hy- C.‘ q: -.' .... O ..-. nu '- .- u‘ . - v" I ° . ”l. 1\ ",.r\ -_1-
T.’ 1: €1.11; #33 $133!} 01'“; 5'1 by? CV3.-. '3 J7 £51113. 1;} L”! (H. 2,3,5. 1%. .131:

v ‘ 1. 5—5 ‘ ;J ‘1‘.“ - 42'5'1" 1‘1“ . ‘2 '.1‘
.5 "the 1‘3? 1" 5°C}? ”4'1“ rat: ibaeat “Ah—L A. L"l&( ,: I!» .5};pr bsbf'l‘ Q

 

ImmanuelectrfighfirPSiB was e3rfo :33 folloM135

at ~ 1

EiraohleLu mcuifi03tioa (Eiracmzf 311, 13 E0} of tha

st
5
m

k“ ‘-

ficheifiegger 3roce are (fiche 133 n“3r, 1"; :5? ). 3313 313 med

5 x 1 in. 31333 microsoOpa 331333 were layerefi with

2 ml of melted. bufferea flgvr at pH 8.6. 5 Fh3530n

igrqticn chmmber 3:3 mflagtefi for 31139 3003? P333103
by 1333rt135 3 6 . 51/: L1. ylexi51533 c5sette 3cr033
the bridge. T30 r033 cf faur 311363 each caulfi be run
b‘gx‘u; "“1 1., cafin3¢finn W33 3333 t? t”? buffer

r33 3rvo 3 v13 filt3r puper wicka. i V3533 power saurcc
3;:31131 3 0023 n't current of 1.25 mn/ 31133 Car 103
minutnt-C.

' . r .3--.» .. ..."? .. x.- ,- .
3531:13A2xtxn5353 v3r053; bufaar a 3333 3:? 33¢

. .. ,9. 2 my . "tr ‘ ~
313553 fur e5ect3op5eretzc 33

'(‘J

:r53i33. The c33303itisn
of the tro buffers, 10.. electr3de vessel and ag r, is
giV3n in 93318 6. Herthiolato (1310,939) wag flfidfid
t3 £53 buffers to rev rd tne croyvfih of b ci3r15.
A.p3rif13& 333133 333 prenvfifir fr3:n Eifco B;cto
3553 3y 331i31£3335 33 3313333 25 asluti3n in 3 thin

... -‘ .... ..f .‘..&u‘ ,' |Q J- r. ...v‘ .5. ‘1. ‘ w \ T." ;-n- - 1‘. 1
t3.5 553 0593155 gt 55tc 53 .55; c¢u33. 33336 05338 3ere

rinaaa extenaively in diat Alca water to rerove neluble

anstit1231a ufld were "weaé with bszer 33 the muggnrt-

120““tifluo s verfinal
1cfroghnrtsia.

Constituenta iggr Electrode

1
I

31- J”l b:_r3 iturio acid 13.33 13.53

:09 111113.. 3131131311 b.:.-r‘.;ii1.u‘i¢ {£3.1C1{§?.{303 11:53.13

- . — , . . “L f) ., -. Q 5‘" ' P
0113113 luct313 5.343 12.jag
315111183 water 3.3. 3d. 133 13L

%* v ‘— — o-u-Inn-m—Cuooi-w-«o ”cwnwoom-fl-flvb‘ W .1 _ v _— “ .— W

 

 

: 1Jr {M i {01111.3 333333 33 a $3163 fur catting
tne . r0 11353 b13133 gnu L1unrh fer a 3313 and anti»
3crum s .310311 T3 2 33 dimfieter 333313 b33153
were 3031319333 4; mm from £33 anodic and of the 31 fie
anfi equifiiat nt frcm 33‘h 31333 of a cunt» lly loauted
2 mm 313 e .“tiucrum tr01us. 230 microliter 3333158
were Md 2d 10 9 03 b sin and QJDagctad to 3133 t10 3<1hore-
31:3. T123 1;! 311‘ in 1.113 5;:‘3‘331211131 trough 1.:11‘: than rm 1'36}

3y aucmo1 and the r3331v133 1.11‘3113 w a 33131. 311363
rare incub2tad at 21 C far 43 to 73 303:3 in 6 in. fiiaw

meter Eetri fiizhes. Irra Wei 13).» n 1.3 fiifitxi from

4,1 --- ' --. -. - 1 -.,,. A t . ;‘ QC. ' I
t3m 01 by r33edtel 3 2:33 in idouufimte thiarmfi a“ 1:3.

6

'\~‘.’

”t -.’n‘ 'x" ‘-

‘l
I
In t.

.fl. 1‘ t
N. o

'.I' .1
J (b

k

‘

V‘ ‘_"!
I Of

J di‘ri

Q

r “g

1

't’ 1
'1

L1

3 3 ‘5‘

_-
4.,. ...
.'- #‘ov

f:

’3‘??? I”?

"1|, I71? 3'.-1
341.111? JIJ L \L

Mo 0 Tlnc+’,.uc;psir

Zone electruphor sis in cellulose ace ace mem~
333303 333 eLILVIMpu uixh 301 '1311 serum from gefLWf'fie
and convauti 0311 0310331“ of different 3533. Represenw
tativc usasltometrogrums from these analyses are shown
in 3Q are 2. The serum protein distribution was similar

9

from thnse groups 393 -pite the quani itatw M13 63098

33103 exieved among i1.civic wzl fractions. Four principle

electrc;horetic Camponcnts 33 a separatofl in fihe con~

H

tjnu‘ IS burbiton e buffer system: albumin; alpha, beta
3-1 3“"'=—"70r3'7u. ivnntitutivsly, the most yrominent
coxjcnunt wag vlbumin which, because of its 1153 net
char 9, 3:: tbs foremost anodio oomycnent of whole 59 w
um. "he 31; rd bet mu ficculins yore not aprreciably
yfl:3?;i~d 333 313 not ciffer m rkcély in csncantratian.
5361333 527:7:‘133—51133.11.11in 1-133 hi 51313? V’s-111313.18 in conccn~
tration 31035 hickens of the different 5: 193. Its
Kubilif? 3:3 ”rzigrin tcly an 316 in t} a cor tinaous
haffor. Prob bly due to ita higher concen«
tration, a greflter yercantsga of 53333-513b311n in
serum frcm conventional and older 501133 had 11 03 ~

s A 0 ‘ 4 ' I . . Q- I .
thOCZC mobility. An.3ndfit3331nvfl amosnt of protfiln

racord3d in the ban”n—r10t1111 r15ion 333 £36 to the

...8

Ch

62

.nnouoano mmuuaumm uaonhocnoo noun ma n unnouoano

Hwnoapnobnoo caolhauuom scum ad 0 unuuuoano mouuaucm uaouhdc

30¢ Bonn ma m nunmxoano Hmdoauumbdoo uaonhdunoa noun ma 4 .mmma
anououuac no udoxoano quoapdo>Qoo can monuahow Bonn auhmm Hwauou
no namohonaoupomam mdou Ho mwdacuoomu oauaoaopamnun .N ampuHu

 

 

 

 

 

 

 

 

 

‘

pres ence of nan~g3nm.9 ~glcozlin protein from th :2nple
23m Jlicu+1on point.

When 3 d1: oo-t¢n.o3s b 3 130.3 buffer system was
employed,a pre~albumin fro ction was caper ;ted in adfl1~
tion.to the 4 fra :OtiOLS alrea4y no oted. Th e nrmerical
designation given unfier each fraction in Figure 2 de~
notes its ap3roximate yer cent composition in whole
serum. The per cent composition of electrOphoretic

fr3 ctiana of normal serum from different groups is given

 

in Table 7.
T‘BLE 7.h 7330 cent cfi““0~4t'on of tb a major
electro Hr ti c erc.3033 uf n33331 chicken
881114.30
Q
Chickens A 33 b g

0-. mmw~w wa- - ‘ w _ w-

4D-d3y-old canventiongl 49 12 13 23
4O-d3y-old garnfree 53 1? 14 23
90—d3y-old conventional 41 l“ 10 37
90-dmy-old £6? izfree 4% 10 12 27

v- v—- —v ww~v v- w— _v

eerun .nlbumin

*Symbolaa A a
a a a.lphu-globulin.
b a beta-globulin
g a gummfi-globulin

These resulta indicafie that albumin and gamma-
globulin were 333nt3t3tiley the most variable electro~

ishoretio components in.normal scrum. Gamma-globulin

64

leVels were fiturox_mLL ly e1ual in the so rum f; on three

990919, 1.6., 40—6uy-01d :errfree azli conventional and
90—day-old germfrcc chicks. A coyre390nding similar-

ity was fit en in the albumin levels f om these grou.9sa.
In cunt‘aat, the gamma glcuulin level in the serum
from 9.-day-ul& con entional biris was 899:39 cifiuly

'D

, - +-' ‘.~A‘ I'v n" , _. _'v 1 .9 ‘3..." . ‘ -\ . "- ‘, '- :‘ ~~
9, .2“ “-911 3.9L)“. c..:.-..:z~ :93? .99.:3. ho sxtgzlfiicmzc (33.. -

in.
\
’1

ii

f.n

-9 . .9 ‘ .... . -, + ... , "-“"a". '- ', . ... ' _.L 9 . '

afit'EACCJ «U18 13.0 a; u b... in «.911 TC;-.’.TH.V=:J (3031C 9:1? vrutufn‘m Of
A U . 1 ; - " . - ' ‘ 1 t 'o . . ‘ ' - .-._ . wt 4‘ .a y 1 ,-

a:.L LiaaQ :: 1“; z) x)”: r --._:'oo-Q :r ...L log-1.5.: . Jun a3 '43"... if) ' L’ ‘Vv. I3 “0" .C.(‘ a to

.._ . . . - n 4— .
-‘ ". «3 'V‘. ”x 4-4‘ ; «g- ~ 4 399-. 0 a * filo, 1‘45." '\ ‘.‘4- 11+ 4 a 1-: f A“; «.15
'l “‘4" u!» 0‘. ."--.“\3_ 9" “t: i ‘. ‘O‘r'v (“J-x ‘ c|mi.-"V Cw L ‘VI‘5-. “*U‘LS G IflA“, Cal" ’
' «L

.- ‘ g -. Q q Q . .
' 1" .‘\ I‘ ‘ifl s n b‘ .3 . '

v 0
I _ ‘_ ‘ o, (\- ~ 9. ‘\ 0 ..o‘r-L :‘5‘ '3 ‘." a“ 4-W‘
(. $.3'1-1u-mrf ’ Vb 3".- U’ ‘Ijuv‘n-‘n‘? '1 «41.4.38 ”\f-ci "eggs .04. tut? ‘.r .W.’ 'ir U-t-Ln
(.
\. ‘ -' a. \I-- . - -" a'yvzz’ ‘3‘ -: .‘ A. ". u. u 1. .7. I: 1“."
\..LL¢\, vi ‘JLAJsxhia vac ‘A'..~...J...xé.b.,.~\.li£.
"‘ ' .-' {'7' ”I’d .2 wag u. - n “Aw, ._ .‘ .v“.- ~.-~~~ 1-" ‘3 ~1 ,, M -.'n -/ l
I)VA‘—\’ ... ‘v dc ‘J d;I—J ‘1‘- ;a ”-1.0 v. \_‘.'3 1s:- 4 .L. -4 . v \r‘ 1“. *6 ‘o-;) . I-‘t ’34.."-

.'- . ...-‘_”':- — Jv- . uq-- n ' P. v v‘. ‘u -. 3 .-‘§‘g ' J. t . A A, ...,g, 1 1,. -
41L fiu.uatI‘ 90 9430?].{16 c.3309. bfif Qm$¢n1 «.101; G... ROI-..-...-.a. S..:I"'L m

" , ‘5 J . , “'2 . .9 _ .. 9‘ . . - ._...", H . ,. p,. ,I ‘ ’.. ,p - ’ _ ... —L A‘ A v. . ‘ I:- f a :1] ' I... 7 .“
$3.1. UtJ-L..L; infifilk (’11-: ;a'.- 2. SC 5;..gu. CL’:LV€.‘.;‘..&.L}LL: .1. Ct‘fihCELQQ o-1.3u;.
" " .' . ' , J: ~ - 4 . . ..- .;..- ..e .-.- a1..._:. .. 4:!

a .- 1 , ‘ . -‘v I . L. f'. 1 .
(wiuua. ) ., 2..! J Lu 5.! E’Lthl’; UghuA'LbLC mi“. kLJ..r.I.-*.L-i\.)a1 OJ.

_L .9 'i '_ 4 . . fl“ _, , .' . 7 ... _, 1 - _.. n .43,“ ., . \. ' my. ,, (‘1 .. .7
‘z'.’ -‘.- it 5.. * L2; :J l. -. in .L .1.'_;'.','..1 ‘~. U” ‘1 fhfld“ .- 0; u Lav: o~ 1a> A. let-'1‘: city—«l \J 5a. 2...! . ...} (I VE§.~1

-’ n ' \‘ “W" (\V '~ "I: a t I". ~ 11".!- ""VW ‘ 71..
\LJ-d'oluslbb Cy‘ 'J' 'JJlLllib .1...~ v "3!. 1. . t H U. L93 J-‘J‘lal ,. «L ‘4.

.. .-s . “‘1 M. m +
531;... Lyn L-‘g‘uJLgA 3...“; 'v‘: ”1.5 ink; LA)... L- 3.3M.) ...LL} 3.13 1.53.9.1. 118 ;.-"".B .9403 14
~ \ rs

V , .0 , .- -.2 .7 .' , ’. ,,.. 9 1.. . ...... _.-..- S .
9999;“L4L ;:wc.;0u obgcyveu. éureé com9c9:n : were

present in 990 ux9n mx-5loaulin r;3‘on, “Li G 9hich

. .. ... .’ ‘ . '.:.‘. ,. ' , , 9., . 1 ,.‘ - $1....
Viilid bb-.~1IL1.?‘.1 '01.}. ~53 c. CKI‘UOJ ‘7 -...LL;.t‘3"bz (JO-$14.0 {15 la. 140

._ ' . . _L .9 K. 3 ' .1... ,,!._. ,‘ '.. “ 1.,“ .. :
(3\I$.L_’_‘\II‘.L..-. LC: 'L’Lil J grega‘ . v 1.1.1 nut? b3 {sci-“g. if) "Lilia; 1313;340:310

_ '. 4‘. '_”’ fi _ f .t... n “ "t .‘ 3, , +.«‘__‘ _..,j“ .. ~. .'
Tue 06 in}. #5:“; 5.913;.» L4. ...; » 0&4. Gib-C32; 90 u: u £39....“ p... J 91:1 $3
5.4!;

~ ‘- ' ... . ‘1 -‘ g c .-\ . l '00 0‘ r« t ‘ I W. .‘ C
suown my lmmunoc ctha.uq:uuza (Jigumc «.1. la cuntras

65

Plate A.

 

Plate Bo

 

919993 3, Lgdr gal eiectrouhornsis of nor
4o-duy—Old gurmfree and convenfiional ohicxens. Plate i
is next: £13m 599 free 091033.

1 afie B is a rum.from
oonvuntional eulogs.

mal serum from

m
{A

t it 595510 a"bh ‘ 123 in cullu;oa a ucetaiu, serum
9:952151355115 mad 9 oathofiio mahilitg in u5u‘ 551.

2.11 bill}: 53.» \.A.%vu.)”hu.. 5.): L23 03.. 1191122213211 25¢:er

fr .Jngs" 5: can‘ugtianzl cLicks is ahagn in

Pl3Hre 3 (915 B). Era tic: finiti an in thia figure

'.a ‘n-‘0 . "- l. ' +’._ 'IA ‘, 1|" . nu-a ‘ o \
5-3.; I’Zu. 9.2? ‘3;ch {113 .111 0.1.5.3 zal‘xcah‘fig. EfflzffiAh.‘ 9" bi”; 11159

, ." ., - 2'- ‘ - .-. .- a L" . - O *.
‘9. ' --- . .. v . '.r . 4:”‘5 . r . %
"'-‘ \ ...; r.‘ ‘.X- “’32:? -. “-..... C".- no' “m CL ‘L’JL‘a ‘ ‘u {1+‘\J“. Have-1.1 afiec “‘ 0-
“5.2.7! ? . ":2 3.". -.< -. nwc‘ v ”91'1"! “PW-‘10 “17W :O-i‘no 3' 7 ’ 1 '1
' -----~ A‘ thi -‘~ 0- 1‘) ,'..'oL-.~’;l.‘d '31"an U 34cc! .a—E .. d‘u .. O .1... H-uol-Iubb, ) M24 lat-1'..-

Q \ 'w
' l

p! a,“ . ...~ “I‘ u '.- -"4-' " . \ '
;_ 4.:1 3.4.4.... ...}; it. .3”) tong" 1001.15.11.13 :2, L; :1 £3" {kill-bigo;bil3.

 

‘

xv. I! ‘I . 0‘” . ' § _g c Q
59:99.92. 32.27391 -3311 fifiwfifi y-cld {Stiwflfrefi and con-

fl

‘Vemtiun L cgicha 9;; 95393333 '9 i; u £9193 ;.9u02;uis.
255:2 anu,r“1~ 9L13i¢1 acnp‘riw :r t: En 9.59 v”th

‘ W’TC‘. ‘ 29111:? 1;“ :;‘C‘~ '1'; s :a ”31., E 5 9:29:12 of 1?”: 1,-
:: e: :2 3 ‘7‘.“1‘3 L"? i“"*‘3. .. c ““‘u, “i L“ 1; ;*' 21993.9(; .-*3;E~:v35;:mn

Mm . , 1 ..u, u... 4, '~ .- ‘ !l_ “I ‘ '. 4' o»- 4' i‘ ‘I d "i 'Q -
‘f 2:20... I: ~ ‘ -‘;- 2";qu ..QJ“ ni— 1"!!31‘» '1' ~- -C~¢:I_f -Cn-k‘ 0L33’épa: h “.1 {0‘19}; ‘3' c~z:~ 61:3 18

fihfiwn in ffigwre 4. i 2925:? of 21 529 9 .t5 frtci~i€in

area 99*9 vnr2nhlv 9r935nt, net all of which,vure

fauna in.evary sarvn *fi"lf 55. This £15535 is 5n at-

('9
”:1
~. 3
61-
(+-
0
H

egr-a 99 tm liley rtiigsmin 053908 tion

01’ swarm from this group was revefilmi by 1:90 yrocmlcrea
ea¢lm M2. Only 099 of t.9 are cfivyuner to v;;c 5901?..05,
bct3?; (line 14), the ot ;“er? ware tbserv d Lnfc1 t.h

canfiiaicna of 9'11 ac'Qm 5:513:19. TIG ceyiharticn

41.4.

t”!
O

’2...)
Q

*4
3'.“:

7“.

#9;

I. ..gysra 4 Fey.

"
L! (“2922 an“ the“ -t
Jy‘l.'.:"'..“ 4.

albumii

—
HQ. .Qt- . u‘ I .3 ‘
(I pal-3.2; tx- .o‘ H .-' z.“

"

_ .- ‘aa-u u. Q-QM- ‘

'1
r. V:
a.) arm-....

'1 3 1:1

*J2.

.naouoano Unofipnmbuoo gonna—T3 noun Igo- ...dlug
Ho Idumouonnoupooamonug 333300 4 3 ac:

O

 

 

 

 

 

 

 

 

¥

is t t = 23332? 3 3 a n L _.. ¢ 3:“

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

inCn to each precipitin are 15 not for the purpoee or
final 0.119131 icrxticn but hiker is intandéd as am aid
to 831 111 oriCEtaticn.

“111:” 211011., ... 1(11116 1) xiv-:3 the most; 1:011. 21321

I I: 4 .Ir'. ' “-" ‘fl 3' ‘5 4’ “' l - ‘1 "- '1 Fa ' '
c-1-t-n 1rc e1ucxdei 1rum 1 11311111“ 110110 1 1b

.-' . , '1 I. r .v ‘ J- r I" t f 3“ .3.. .. .
turmlakpl <10 ’33.. 5.3L ” c .. 1.1-1.3113 11.133 and “L 139 b:‘ min.

F" " ‘U\""$ "I." "i '=-.“¥3.‘- my' ~, W .1‘.‘ --.t""vr'.l ... 1“]? 6‘ 1-" "a. 3 " 0‘? "‘111 "\1“‘ -
11.6153 $.11. 11hr; Lr x.- be Irv-£3 “A {dip-’5 if) LIA-3 Jr do '-' ‘1 «3.0.24. '44.? "3.1-3.1- C q LA“;

f).

E’

;+

l.

i ‘
.

d”

All of th yhtl-globulias were E
11 1% in 1111, unler V a cr:5% or ageinfiing 1111 of

RI

, .-. Q’-. l. can '1. «N. 4~ I '
the all?! 353...?) g'TUCifit 162 Q Ju'.

25
f3
’5‘
H; 4
O
...:
n
c
\4‘3
7"
E-
5.
Q:
.5

. . a : a” -9 ~ .. -: ‘~. 1w!» .11»-

Law" A» *1; 03-57 Vlaib¢3 lurid ? :1“: I «L ,‘-:J- r, l- .59": a»): U «23.3115

~1- . .0 ' 1_ + av‘ . ‘ 0‘ . | 4-3 3! o. # ‘ ‘--.‘ + J I. ' - -‘ -- Hal-A ,
fixf1ereau 211111. “11111tuu1vcly, thfise c1: c1t3
~" -—~ ' \ .' ’ I" 1’. '4“ 1' n.’ ‘ f _ " - . '. - -'~ “ ‘o ‘ -
aype1rux .0 1% 1110: c112 1+.31.: c1 t113 rcgign.

{Elmir dare-’0 11:11 area {were also £12311 1:131:21? the (1:1-
oer-.1111; (:13... 1.1:: 113:?) cfa. :1. 1311111.. 53:11 7111 1-. €311

J

v-§ 91“" s . ‘ v fi -' '.- “‘ . ‘. ‘1- i ’r __1 '_ T :‘Q‘ 1 - ‘ll . &' ‘.

{3’. 1 1131- C531; Inna: :3 (111163 :3 . 11*“). 9) £1.00 1143.11 f.1b'3‘9'3 c.3-
o .--_ a l. . - -‘ a. ' :2. «a; n7. ' '-,w.~ ‘ ‘.‘v -. ”3 ‘ .r. ‘ - 1

Air}??? ‘3“; :.~: b¢*I“J 01. 154.6 {-13.511141 ru¢3zi.bn 111d 6:1 98! 1:183 film'-

w. ‘ y“ ‘ ‘ tn \- ‘1
'i‘w" ("V i g "t O t: 12?, 5d». 731.151,) 1': 0156 O
..

1t 1111t 4 iiffflrext greciyitin 111:.f3 rare

_ - .3 -‘: ufi. .*~ 1"»3 ,-. q 1 ‘1 . ‘ . t ' «1 ft.” n1. P. I a ‘ M. - y" ‘."- 'hl “.. __
Ox :1 {2‘ PJ‘}; {‘1 i443 V ' “ a ;'-'L ;;A*'- f} 2:, 7'“ ‘_" \“ "“13", ..v' ‘V G 2.! II‘ '.V’! ‘ :qu‘IJ-ln U com.
‘A
.1 . ‘3 1-...3 13.11,. wt: 1.2,, f *1) 2,
1" VAL“ «nut :13 ' u..; Alma-12-)“ :‘J‘m‘t ...J'h {'J.;.‘Al . ._r —JZ. ($4.30 3 l) 5.1111 in.» “-51.8

4‘»

3333333 in each 33333 333333. Zing 3333 precipitLtad
in 3y33tricnl, ruaghly 13331131 3333 which 0303333 333
fleccxfiing 1133 a: 33b.33 .333 33. «,gnd e3 Lsiicgllyo
Lina tr 0333 in :h3 3333 sf 313332-3 anfl 4 (11333
12 and 13) 33v 3339L103t3£ by manageeifiu pretaxn
pracigit3t~3n 3393 the 333133333.trc "fh. Kevarhhelaaa,
thrsa 3033593233 33?: rugulsrly obaervcd in 3L3 majarity
cf 3*~‘33.

03 LL: 6 3:03 333033333333 in the bcfigl regzon,
5 were cozsiL r33 basal 00“"033333 333 1 was a betag.

‘.
11333 13, 16 333 17 iaantify 333  bet31-l, 2 3:33 5 com.

.. «than ua.‘ J. 2 O I 4 6 g ‘. 3
3333333 333.3033333y. 'heaa com M3333 nts 3333 V3333

U
3.3
‘¢

yrsacnt in a3ffe we 3 803332, 311 mi t3c; were not

‘ n,‘ c‘ .¢ :5 w 3' - " ‘ ‘ '-c "' . 9r ,3 '*-- 31 ‘5‘?" 'n

flea-":33: 3. i3. ‘ 3 03.111 1?} 61.1611 §J=§3‘1.hal $333313". ,3 . 33373.3 3311233 3132.8
4" ..‘fi' - .33 “'9 "

d 333331t o LnsM :3 bncuane 03 333 class 3‘ro3i mi t3.

3330333 "raoi'3333 lines.

'\

annsto3ca13, find crowfiing 0! a
$hfi b0t31~4 had a aflxxfln Junctinn with the betaE-B and
bfital-B comgomazta. Th3 mast 3‘013nent 3 re in 333
betfilr racism :2: 3 £33 ed by precifiitation 03 :33 be "tn 1-5
com;o&efit, (lire 1%). This component 393 9393933 in
all sarux: rd 1.3 preci;.;‘t .te 1d3ntified this raglan
fer 5333331 srien 3*1on.

TLQ W “2 333133 333 0303:: 133 by 3 313373 pra-

p§.t' +3 3:130: £03333 from tLe betz3-2 oompm mint (line

20). T315 line was usually f3inu but 333 prusant in

4". y.) 13" ’31‘1 ~ I ~.1. w- Bi
Quaint n-4-5 - J 3 $. .

_. _. n‘ -.o, .7 L .1. g» *»
gym-(31,4. ow LC“! 1. MAE}

...!

s '- 'rr‘ '3' .~- N
CC.2“D 'H ‘15-. lat...“ {Li-(L b ‘C ’ LT
‘ . A C " N ' .u ""“" Q l: ' 7
E: f; ...‘zarh'! (l;ff'1‘).i in‘l'm-H.

.. .M 49‘}-
, 3;..rsqrcnt 3' bee mam

I f 1 G o
' (It Y \ V 7'- ---\‘° "* Q“ g b'}1+ - " '1 1' 1' ;."€1,"~1‘:1 t
0f 1 to.) a. £ _. ‘1 infill A4 ml M. .1;th ,au v ' ._ .' J. C -h ‘15:]. tu. ‘19
.-.« 7 ‘ -... n 3"» .~~. r" w- w A ~.-. -: .~.~ .~~,- 'H .3.—«1.-
..a- .- i x 4-0 0 u .t O .3 I 0'.) Liv, tf'.» ‘.h (a J» a. z. r‘. ad. ~31 wanna 0 .. 4111' Hoe-
.. -0 ‘ 4- c- Q I .1 a." J‘fld-v , - - c. A ‘4 :- u o d * u' ti *
“*‘ ‘. N V A» ‘ A u .1. :".. 9- ,‘. - ~ "’ , P' *‘ V1
4-4:?- ‘t at“. l in ' :0 1“ ~Hu v J» a 0'” #23 ‘.J‘EJJ ' L&.o uh Ard- c w-OAU 1 “‘ \s-Jv .5 ... bio eaé H
V w i .- IL. wv # I I ‘ ‘- (5.! C ~~ ,1 C‘. v 1 1 I r— \fiv
n w. .v I. l“ A V A: r.- - 'V- . '“ ~ t‘ . 9
£.:!Ci him! .3 C I H L “-13 3.: .‘ '9 I" ::_i ”3 Jan ‘54 J. M’JL la“J~&nL-bu’> - ndy). Hall“,
I p'I‘ ‘ ‘ ‘ —- k : .- ...; . . - 0‘ . . an
A 1 i I '1 ~ 1 r . - A“; ‘ ... r) ~ » :X , . -‘. ' . I
val... ..~..~ ' erJ'U-J. ab .3 {AA uLtZ'i .x... 9184.). giantjiu‘J... .Lv ‘C’k-th
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SULMIJ ARY

Zone electroghoresis in cellulose acetate and in
agar gel reve2led the qu1litative and eusntititive
similLtrities of ncrr2 l ser m proteLn patterns from
40-dcy-old gernfrce and convent 1onal chickens. In con—

if Mre1ces were ob= erved in a1011.in

C1

trhst, quantitative
end gamma—globulin fractions of serum from 90-day-old
geimfree and conventional ch ckens. Gamma-globulin
levels in n rmnl serum from 40-d: y-old germfree and
conver tionz; l and from 90-day-old gernfrec chicken were
similar. This fraction was considerably higher in

norms scrim frcn 90-dey-old conventional animals.
Immunoelectrophoresis confirmed these findings but re-
vealed some apparent differences in the antigenic com-
ponents of normal serum from.4O-d2y-old gernfree and
conventiongl chicks. Because of their vsri able pres ence,
however, the 4 alphal components which were found in

the serum from conventional but not germfree chicks

are not considered to be sufficiently reliable to serve
as a means of differentiating seruns from these two
groups. Gel filtretion of normal serum from 40-day-old
conventional chicks yielded 2 protein containing peeks
in contrast to 3 obtr lined by Bards s (19 63) from serum

of much older chickens. It appear's, therefore, that

118

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Ibram5ff, P., and H.R. Tolfe. 1955. Precigitin fro-
duction in Chickens. XIII. A Quantitative
stafig of t2e “ffeot of ‘izu‘t\n5or5 InjiW ion
of Two 3;: Luna J. Inmrmml. 11394-1131.

iitken, D., an ”. Eulligan. 1962. fuflfititative Fre-
01,1315 Stuujes on Foxl "5&13335 to Bovine
55253 $155515 ::;nd Bovine Gaza dGlobuli.. Im—
“255'0'r g3 29§~ 505.

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and Ex erIm5nt2l “5213913 of J OLble Dfi.ffusion
F5531 itfin P510 niana in G513 5 r1 its $351105tion
to Ch:m ctarluflt‘an of “n+1322 Imuunologyizz
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flucti 1022 in 05105555. ”I". T}3 0““5 5nt5 of
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B5neaiot, . C. Laraar, 5:1 E. Hik-thh. 193 3.

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m“”forul UJnthficis uni $535 LHI;51503r55hic and
Titracxlt IJ,H1 Char ovariatioa of Chicken anti-
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Brandt, L. ., R. Clea» and ”.C. [5135 5. IS 61. The
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Scrum Proteins of the Chici:5n. J. :.01. Chem.
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