AN ELECTROPHORETIC AND CHEMICAL FRACTIONATION
STUDY OF SERA FROM RATS IMMUNIZED AGAINST
THE NEMATODE, NIPPOSTRONGYLUS MUR IS

By
Stanley Edward L eland, Jr*

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Bacteriology and Public Health
1953

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DEDICATION
The author wishes to dedicate this thesis to his parents, Mr*
Stanley Edward Leland, Sr., and Mrs. Kathryn Miklas Leland*

358105

ACKNOWLEDGMENTS
The author wishes to express his sincere appreciation to Dr.
William D. Lindquist who, after the untimely death of Dr. Phillip A*
Hawkins, has acted in a supervisory capacity during the remaining course
of this study.

The author is indebted to Dr. Hans A. Lillevik for offer­

ing his knowledge of the electrophoretic techniques and certain chemical
aspects of the problem.

To Mrs. Stanley E. Leland, Jr. the author is

indebted for her untiring aid in preparing the manuscript.

Acknowledg­

ment is also due to Michigan State College for providing the author
with the opportunity to make this study.

VITA
Stanley Edward Leland, Jr.
candidate for the degree of
Doctor of Philosophy

Dissertation:

An Electrophoretic and Chemical Fractionation Study of
Sera from Hats Immunized Against the Nematode,
Nippo strongylus muris.

Outline of Studies:
Major subject:
Minor subjects:

Parasitology
Biochemistry, Animal Pathology

Biographical Items:
Born, August 1, 1926, Chicago, Illinois
Undergraduate Studies, Thornton Junior College 19^3-^5
University of Illinois, 19*+7-^9
Graduate Studies, University of Illinois, 19^9-50, Michigan
State College, 1950-53
Experience:

Graduate Teaching Assistant, Michigan State
College, 1950-53* Member United States Navy, 19*+5-^6

Member of The Society of the Sigma Xi, The American Society of
Parasitologists, The Midwestern Conference of Parasitologists.

AN ELECTROPHORETIC AND CHEMICAL FRACTIONATION
STUDY OF SERA FROM RATS IMMUNIZED AGAINST
THE NEMATODE, NIPPOSTRONGYLUS MURIS

By
Stanley Edward Leland, Jri

AN ABSTRACT
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Bacteriology and Public Health
Year
Approved by

1953

Stanley Edward Leland, Jr.

1

The purpose of this study was:

(l) to investigate the changes in

the host serum protein components which occur as a result of infection
with Hippo strongylus muris (2) to study these changes progressively
through the hyperininrunization period and (3) to determine the conditions
that cause .t h e s e serum protein changes*
Hyperimmunization was attained by subjecting the rats to increasing
numbers of larvae at two week intervals.

The actual sequence of injec­

tions was 1,000; 2,000; 5*000; 10,000; 20,000; 50*000 larvae.

Serum

analysis were made on various days between the injections by means of
the Perkin-Elmer Tiselius electrophoresis apparatus and fractionation
with sodium sulfite.

The following serum changes were noted in the

infected animals when compared with uninfected litter mates*
(a)

The total protein increased as the number of larvae in­
jected was increased*

(b)

The beta and total globulin increased as the number of
larvae injected increased, and diminished when the num­
ber of larvae injected was reduced, or when the larval
injections were stopped.

(c)

In five out of six two week intervals between injections
the beta globulin of the infected animals was slightly
higher after two weeks than after on9 week following
injection.

The beta globulin increase was electrophor-

etically determined in veronal buffer at pH S.6 and
phosphate buffer pH

This minimized any anomalous

effects that might occur at a particular pH or with a
particular buffer*

Stanley Ed.nra.rd Leland, Jr.
(d)

2

The albumin/globulin ratio decreased as the number of
larvae injected was increased and continued to increase
when the larval injections were halted*

(e)

The most striking serum protein changes occurred around
seven days after the 50»000 larval injection*

(f)

The gamma globulin content showed no significant increase
or decrease*

(g)

The mobilities of the protein components showed a de­
crease in the majority of infected serum samples,

(h)

Serum from rats, which had received injections of the
supernatant from the larval suspension, was essentially
normal.

This indicated the material in the inoculum

other than the larvae did not cause the observed serum
protein changes*
These serum changes are interpreted as responses of the host to
injury or trauma by migrating larvae, although, the possible existence
of anantibody can not be ruled out.

Since rats were bled only once

duringthe course of the experiments, serum depletion changes were
eliminated*
Living larvae, saline extracts of living larvae, and ground larvae,
were used as antigens and exposed to immune sera,

Antigen-antibody

combination, as indicated by the precipitation of a serum fraction, was
not observed.
Utilizing bovine albumin as a standard, a modification for deter­
mining total protein of rat sera by the biuret reaction was established,
A modification for fractionating rat sera into albumin and globulin,
utilizing a 2k percent sodium sulfite solution, was devised.

TABLE OF CONTENTS
CHAPTER
X.
II*

PAGE
Introduction and Historical Review......... . . . . .

Materials and Methods
A.

III.

. . .........................

Experimental Procedure

........

. . . . . . .

1
8
8

B. Infection Technique.........................

11

C. Chemical Fractionation and Analysis..........

12

ID* Electrophoresis . . . .......................

16

R e s u l t s .................................. ........

22

A. Preliminary Experiments * ....................

22

The ability of an immune host to reduce
the parasites• egg-production...........

22

The ability of immune serum to produce
precipitate.........

23

The ability of the immune host to resist
reinfection ................... . . . . .

23

B* Preliminary Electrophoretic Analysis. . . . .

23

C. The Analysis of Sera Two Weeks After
Each I n f e c t i o n ............................

24

Experiment I ............................

24

Experiment l a ..........................

31

D. The Analysis of Sera From Older Rats One
Week After Each Infection...................
Experiment II
E*

* ...............

44
44

The Analysis of Sera From Young Rats One
Week After Each Infection...................

55

Experiment I I I ...........................

55

CHAPTER

PAGE
P.

CompositeResultsof Serum Protein Changes

Gr* AbsorptionStudies
17*
V*
VI.

....................

Discussion......................
Summary

. .

. .

• * * ...............................

Bibliography.......................................

69
7^

~[G
84
87

INTRODUCTION AND HISTORICAL REVIEW
The nematode, Nippostrongylus muris. was first described by
Yokogawa in 1920 as Heligmo somum muris.

The adult worm (3-4 mm in

length and 0,085-0.1 mm thick) is a natural parasite in the small in­
testine of the wild rat*
In 1922 Yokogawa worked out the life cycle in detail, but he over­
looked one of the larval molts which was later observed by Lucker (1936),
Thus, N. muris undergoes four complete molts and does not differ in any
essential respect from the development reported for other skin-pene­
trating nematodes*
The third stage filariform larvae actively penetrate the skin of
the rat*

They are then carried by the blood stream to the lungs and

here, break through the alveolar epithelium.

After a period of develop­

ment in the lungs the larvae migrate up the trachea, down the esophagus
and through the stomach to the small intestine where they develop to
maturity*

The females begin to lay eggs 5 to 6 days after infection.

The eggs require a 4 to 5 day developmental period outside the host
to reach the infective third stage*
Africa (1931) and Schwartz, ALicata, and Lucker (1931) first
reported that rats, after recovery from an initial infection, are releutively resistant to subsequent infections.

Furthermore, Schwartz et al.

noted that many of the larvae from the second infection were retained
in the lungs of the rats.
Chandler (1932) concluded from his investigations that the resist­
ance acquired as the result of:

(l) previous infections, (2) inoculation

2
with the living larvae of a related worm (Longistriata) and (3) inocurlation with killed larvae, is similar in nature in each case.

He

states, 11It appears not to he due to the development of a local lethal
effect on the worms in the intestine, hut to a more general effect,
acting through the blood, which tends (1) to interfere with reproduction,
(2) to cause a stunting in growth, (3) to inhibit development to the
adult stage, (4) to prevent successful establishment in the intestine,
and (5) to bring about the elimination of worms already established.1*
Graham (193^) showed that the degree of acquired resistance de­
veloped is associated with the size of the initial infection, i.e., the
heavier the primary worm burden, the greater the resistance developed.
Repeated exposure to increasingly large numbers of larvae at weekly
intervals resulted in marked resistance as judged by egg counts.
Chandler (1935a) *n one ou* of four experiments was able, by the
injection of immune serum, to demonstrate passive immunity as evidenced
by the effect on the prepatent period, number of worms established, egg
output, rate of inhibition of egg production, and rate of loss of worms.
Since he was unable to duplicate the passive transfer in the other three
experiments he attributes the abnormally high egg and worm counts in
the controls of this positive experiment to some unknown factor.

These

abnormally high counts in the controls, when compared with the lower
counts of the rats injected with immune sera, suggested passive immuni­
zation.

In experiments with parabiotic twins in which one of the pairs

was immunized either before or after being joined to its mate, Chandler
(loc. cit.) showed, that although proof of a good intermingling of blood

3
was obtained, no immunity at all was conferred on the previously unin­
fected member of the pair.

Chandler thus states, nThe conclusion is

inevitable, therefore, that the immunity is local, a property of the
intestinal mucosa.11
Chandler (1935*>) showed there was a progressive increase in the
resistance to superinfection as the interval between the two infections
is increased from J to 1^ or 15 days.

After the fifteenth day the re­

sistance developed was gradually lost until by the thirtieth day there
is even less resistance than was manifested after an interval of 7 days.
This resistance was demonstrated by a falling off in numbers of worms
acquired, a gradual decrease in the percentage of worms which succeeded
in passing the final molt, and a gradual decrease in the amount of growth
of the worms.
Chandler (1936) found that worms could resume egg-laying or could
grow and begin egg-laying when transferred from the intestines of immune
rats to those of normal rats.

Chandler feels this supports the hypo­

thesis that the immunity of white rats to H* muris is due to the develop­
ment of anti-enzymes which prevent the worms from digesting and assimilat­
ing the host*s protein.
19hen Spindler (193&) transferred young and adult worms by duodenal
tube to the intestines of normal rats he found that the rats were immune
to a second infection by the usual cutaneous route.

Since only small

numbers of larvae were recovered from the lungs he concluded that lungmigration was necessary for inhibition of development in the lungs, but
that intestinal infection by itself could give a local intestinal immunity*

k
Sarles and Taliaferro (1936) studied the migration and development
of the parasite in normal and hyperimraunized rats at short intervals
during the first week of the infection.

In hyperimmunized rats the above

authors found the worms were retained and killed, to a small extent, in
the skin, and to a larger extent, in the lungs; of those that migrated
to the intestine, many were delayed, and upon their arrival, the major­
ity not only failed to grow or produce eggs hut failed to remain.
By means of intraperitoneal injections of serum from hyperimraunized
rats these authors were able to demonstrate passive immunity as mani­
fested by:

a slower rise in the number of eggs passed, a smaller

maximum egg count, a smaller total number of eggs passed, a smaller
number of worms in the intestine and a slightly smaller size and paler
color of the worms*
Sarles

(193 ^) demonstrated in vitro antibody action as evidenced

by invariable precipitate formation of four types:

(1) cuticular (with

infective larvae and lung-stage only), (2) excretory, (3) oral, and (h)
intestinal, and sometimes the decreased activity (of infective larvae
and lung-stage) and the inhibition of development (of larvae).
the work on Fippostrongylus, BlackLock, Gordon, and Fine

Before

(1 9 3 0 ) showed

that death of the larvae of the myiasis-producing fly, Cordylobia
anthropophaga. in the skin of immune guinea pigs was associated with a
precipitate in the gut and around the larvae and that the precipitate
in turn was due to the formation of precipitins by the immune host to
the hemocoel fluid and excreta of the larvae.

In addition, Fourie

(1936) described hyaline masses closely applied to the surface of

5
Oesophagostonrum columbianum which no doubt was a precipitin reaction.
Similar reactions have since been noted by Otto (19*K)) with Ancylostoma
caninum. Manss (19U0 ) and Oliver-Gonzalez (19*10) with Trichinella spiralis.
Oliver-Gonzitlez (l9*+3) with Ascaris lumbricoides and Hawkins and Cole
(19*+5) with sheep strongyles.
Sarles (1939) found the degree of passive immunity produced varied
with the dosage, interval after infection, and the inherent potency of
the serum*
Taliaferro and Sarles (1939)

& study of the cellular reactions

in the skin, lungs, and intestine of normal and immune rats after infec­
tion with H. muris concluded that immunity against the worm was largely
dependent upon humoral factors with secondary cellular cooperation.
The precipitins and possible other humoral factors produced the follow­
ing effects on the worms

(1) immobilization (2) formation of precipitates

in the alimentary canal and around the body orifices (3) stunting and
(4) the occasional

killing of the worn.

The antibodies also localized

the irritating excretions and secretions of the worm and brought about
more intense inflammatory responses.

The same authors pointed out that

during the intestinal phase of the infection the worms pierced the
epithelium and ingested antibodies (as evidenced by intestinal precipi­
tates) and deposited antigenic secretions in the lamina propria*

Thus

the worm in the intestine had intimate contact with lymphoid tissue and
the blood and lymph.
Thorson (1951) incubated larvae in normal rat serum.

The larvae

were then removed and the normal serum presumably containing their
secretions and excretions was injected intraperitoneally into rats.

The

6

rats were then infected with larvae and after S days the worm burden
was determined*

The injected rats showed a significantly lower worm

burden than did uninjected controls, indicating that protective anti­
bodies were formed against the secretions of the worm*
A few references are available which indicate changes occur in
the serum proteins during parasitic infections*

Ikejianl (1946) found

an increase of globulin and a decrease of albumin in rats infected with
Trypanosoma brucei and Trypanosoma eouiperdum* Lysenko (1951) asso­
ciated a high gamma-globulin with the presence of ablastic activity
and a low gamrna^-globulin with no ablastic activity in rats infected
with Trypanosoma lewisi*
In contrast to the many electrophoretic studies that have been
made in the field of bacteriology, there have been relatively few in
the field of parasitology; and of these studies that have been made,
most seem concerned with protozoan infections such as those of Cooper
(1946) Lysenko (1951) Dole and Emerson (1945)*
In summarizing the review of the literature cited above the fol­
lowing statements can be made concerning the activities of this host—
parasite relationships

(l) after recovery from an initial infection

rats are relatively resistant to subsequent infections (2) acting through
some medium in the blood the host Influences reproduction, growth,
development, maintenance of the worm (3) the degree of acquired resist­
ance of the host is associated with the size of the initial infection
(4) resistance to super infect ion is highest 14 days after a previous
infection (5) worms regain their egg-laying potential when they are
transferred from an immune rat to a normal rat (6) resistance qualities

7
can be passively transferred by the serum of immune rats (7) immune
sera is capable of precipitate formation when exposed to larvae and (8)
the resistance of the host appears to

be in

response tothe excretions

and secretions of the worm*
The purpose of the present
in the serum protein components

study was (l) to
which occur

investigate the changes
as a resultof infection

with H* muris (2) to study these changes progressively through the hyperimmunization period and (3) to determine the conditions that cause these
serum protein changes*

MATERIALS AND METHODS

Experimental Procedure
In the present study certain advantages and disadvantages were
encountered in regard to the host and parasite used.

One disadvantage

that arose in the preliminary investigations was the limited amount of
serum that could be obtained from an eight week old rat*

Even when

rats were bled to death there was insufficient serum available for the
desired determinations and, in addition, the rats were no longer avail­
able for later comparative studies*
encountered, namely:

At this point another problem was

if the rats were partially bled, how much blood

could be removed without hindering the normal physiology of the rat?
A number of workers have shown that protein depletion either by restrict­
ing dietary protein or by plasmapheresis results in an altered electro­
phoresis pattern /TShow et al* (19^5)* Benditt et_ al. (19^9)t Chow, B. E*
(19%), Chow et al* (19^17 ♦
In order to assure a sufficient amount of serum the following pro­
cedure was utilized*

Bats of the same strain, age, and sex were divided

into two groups (control and experimental) by including half of every
litter in each group.

When determinations were to be made three rats

from the experimental pool and the three corresponding litter mates from
the control pool were bled completely and discarded.

In this way suf­

ficient serum was usually available for the various determinations and
the possibility of detrimental effects due to blood depletion was
eliminated*

9
The advantage of working with this host-parasite combination lies
in relatively low cost and maintenance of the host and the ease with
which large numbers of the larvae can be cultivated#
In the present work rats were subjected to increasing numbers of
larvae at two week intervals with the exception of absorption experiment
IV in which case the rats were subjected to larvae at one week intervals.
The actual sequence was 1 ,0 0 0 ; 2 ,0 0 0 ; 5*000* 1 0 ,0 0 0 ; 2 0 ,0 0 0 ; 5 0 ,0 0 0
larvae.

Thus, an eight week old rat inoculated with 1,000 larvae was

eighteen weeks old when 5 0 ,0 0 0 larvae were administered.

This rather

long period of time in the life of the rat meant that in any conclusion
drawn concerning serum protein changes would necessitate a consideration
of normal physiological development during this period.

There is evi­

dence from the data on rats and cats, of which the young were studied,
and from data on developing chick and pig embryos that there are major
changes in the serum patterns with age and development (Moore et_ al.
19^5^)*

Brandt et al. (1951) ia the case of normal chickens was able

to show quantitative changes in some of the serum protein components
as the birds matured.

The uninfected controls in the present work which

were litter mates of the infected group served as a check on this point
since they underwent the normal growth process in the absence of N* muris.
The rats used in the experiments were either the Wistar strain which
were bought as weanlings or the Sprague-Dawley strain which were raised
in this laboratory.

The rats were kept in an isolated room away from

other animals in order to minimize the chances of contaminating infec­
tions.

They were housed individually in most cases and in wire-bottomed

10
cages*

Food and water were available at all times*

Throughout the

experiments the rats were maintained on a pellet ration^ which con­
tained, as recorded by the manufacturer, a guaranteed minimum analysis
of 22,00 percent protein, 3*00 percent fat, *+*00 percent fiber and
45*00 percent nitrogen free extract.
When serum was to be collected, the rats were anesthetized by in2
jecting intraperitoneally 0*05 to 0*10 ml* of Halatal * If anesthesia
was incomplete, ether or chloroform was used in addition to the Halatal*
The rats were immobilized after anesthesia by stretching them back down
on a board using heavy rubber bands looped around the legs and attached
to nails appropriately placed in theboard*

The blood was then drawn

from the heart, placed in sterile plugged test tubes and the tubes slanted
to give an increased surface from which a larger serum harvest was pos­
sible.

The slanted tubes were left at room temperature from 12 to IS

hours.

Under aseptic conditions the serum was then removed with Pasteur

pipettes, placed in centrifuge tubes, and centrifuged at 2,000 R.F.M.
for 30 minutes.
placed in tubes*

The clear serum was removed with Pasteur pipettes and
Determinations were made immediately when possible to

minimize any serum changes due to storage.

If measurements could not

be made immediately the serum was stored at 4°C,

^Miller's Eaties - Manufactured by Battle Creek Dog Food Co,, Battle
Creek, Michigan.
2

Halatal, Jensen-Salsbery Laboratories, Inc*, Kansas City, Missouri*

11
Infection Technique
Prom initially-infected rats 7 to 12 days after infection feces
were collected in dropping pans containing moistened paper toweling to
prevent the feces from drying.

The feces were thoroughly mixed with

water to the consistancy of a paste and an equal volume of charcoal
added and again mixed.

The charcoal-feces mixture was then set out in

petri dishes in the form of a patty which was one-half inch from the
edge of the petri dish and did not tou&h the lid.

The petri dishes were

then stored at room temperature from one to three weeks in suitable
closed containerst which contained moistened paper toweling to prevent
the cultures from drying out.

The filariform larvae were isolated with

a Baerman apparatus and repeatedly washed with sterile distilled water
by centrifugation.

Five hundred to one thousand units of penicillin

and 0.05 &a* of streptomycin were added per cc. of suspension#
The larval concentration was determined by making serial dilutions
of an aliquot of the concentrated suspension until 50 to 200 larvae were
counted in 0.05 cc. of the last serial dilution in a Scott counting
chamber 3 m

In this manner the number of larvae in the original larval

suspension could be calculated.

The suspension was diluted so that the

desired number of larvae for injection were contained in 0.1 to 0.25 cc*
of solution.

After the dilution the larval concentration was checked

by making another count*

^Counting Slide Scott No. 4099-A for hookworm (3x1 l/2 inches) Arthur
H. Thomas Company, Philadelphia, U.S.A*

12
The larval suspension was injected subcutaneously by means of a
tuberculin syringe.

After all larval injections were made the remain­

ing larval suspension was allowed to settle and the supernatant drawn
off.

This supernatant material was examined under the dissecting micro­

scope to insure the absence of larvae.

The larval-free supernatant was

then injected into rats in an amount equal to the volume of the larval
injection.

The injection of this supernatant material provided a con­

trol on the non-larval portion of the inoculum and showed its influence
on the serum proteins.
Chemical Fractionation and Analysis
Total protein determination.

The total serum protein was deter­

mined with the aid of Weichselbaum's biuret reagent prepared according
to Wolf son et al. (19^8).

This method necessitates a rather involved

standardization but allows determinations to be made on small amounts
of serum and the time of analysis is much shorter than the Kjeldahl
method.

The standardization was carried out in the following manner.

Serum from 10 to 15 rats was collected (as described above), pooled and
thoroughly mixed.

From this pooled sample, standards were prepared by

using normal saline as a diluent.

Thus four or five standards ranging

from undiluted (about 6.00 grams per 100 cc. serum) to zero protein con­
centration were prepared.

The protein concentration of the pooled

sample was determined as follows:

The total nitrogen was ascertained

by the macro-kj el dahl method according to Cradwohl (19^3)*

Non-protein

nitrogen was determined by the method of Folin and Wu (1919) and was

13
subtracted from the total nitrogen*

The difference represents protein

nitrogen which was multiplied by the factor^- 6.25 to obtain total pro­
tein.

Therefore, knowing the total protein concentration of the pooled

sample, the protein concentrations of standards could be calculated
and plotted.

Each of the standards (of known protein concentration)

was analyzed for total protein by the following biuret method:
1. Into a 10 ml* graduated mixing cylinder, 0.4 ml. of serum (or
standard) was pipetted by means of a Kahn pipette. From a burette
7*6 ml. of distilled water was added. The solution was mixed well
by inversion taking care not to cause foaming*
2* To another graduated mixing chamber 4.0 ml. of the diluted
sample and 4.0 ml. of biuret reagent were added and mixed well*
3* A blank was prepared with 4.0 ml. of distilled water and 4.0
ml. of biuret reagent*
4. The solutions were allowed to stand 30 minutes for complete
color development and then read on the photelometer^ at a wave
length of 525 millimicrons (green filter). From the data obtained
a standard curve was constructed on semi-log graph paper*
In addition to the pooled rat sera, a series of pure bovine albumin^ solutions diluted to known protein concentrations were analyzed by
the above method.

It was found that the standard curve obtained from

bovine albumin although not superimposible was very close to that of
the pooled rat sera as is shown in Figure 1*

^The conventional procedure of converting nitrogen determination to
values for whole protein by multiplying by 6.25 is quite arbitrary
(Jager at al. 1950)* The experimental conversion factors determined
for various protein fractions of human serum have been found to vary
from 6.10-3.40* (Armstrong et. al. 1947) •
5Cenco-Sheard-Sanford Photelometer, Cat. No. 41000, Central Scientific
Co., Chicago*
c
The author wishes to express his thanks to Dr. M. C. Ziporyn of the
Armour Laboratories, Chicago for supplying the Bovine Albumin Solution.

14
Since the two materials reacted so similarly to the biuret reagent
and since the bovine albumin is more readily available it was used as
a primary standard throughout the course of the experiments, The author
felt the use of the bovine albumin curve was both justified and desirable#
Salt fractionation.

In selecting a method for chemically fraction­

ating serum the author was faced with a number of complicating factors#
A few will be mentioned in order to justify the final decision#

Cohn

and Wolf son (19^7) and Petermann et. al* (1947) showed that "serum album­
in" as determined by the classical Howe (1921) sodium sulfate method,
actually included both albumin and alpha globulin.
are encountered in some of the other methods#

Certain disadvantages

Globulin precipitation

by methanol as devised by Pillemer and Hutchinson (1945) involved work­
ing between 0° and 1° C. with occasional erratic results.

Popjak and

McCarthy (1946) used magnesium sulfate to separate albumin and globulin,
but this method involved a delay of twelve hours before filtration.
The method of Milne (1947) required an overnight delay for precipitation
of globulin#

The quantitative immunochemical reaction of Chow (1947)

employed biologic material of unstable titer and uncertain composition.
A method suggested by Wolfson et. al. (1946) appeared to overcome
these difficulties*

These authors utilized sodium sulfite at a concen­

tration of 26.33 which precipitates globulin from serum*

Campbell and

Hanna (1937) ^sed sodium sulfite but their method was adjusted to give
results approximating Howe*s fractions*
In preliminary determinations for the present study an attempt was
made to utilize the method as described by Wolfson et al. (l94g).

it

15
was soon evident that although this method was satisfactory for human
serum it gave poor results in the case of rat serum when checked by
electrophoretic analysis#

Hence, it was found necessary to modify

this method for application to rat serum analysis#
This adjustment was carried out in the following manner:

a

pooled sample was analyzed by the Tiselius electrophoretic method and
the total protein determined by the method described previously.

Us­

ing solutions of sodium sulfite ranging in concentration from 20 to
28 percent (W/V), the amount of albumin fraction was determined upon
aliquots of the pooled sample according to the following procedure:
1# Into a 10 ml. graduated mixing cylinder or tube at room temp­
erature, 0.4 ml. of pooled serum was pipetted by means of a Kahn
pipette.
2# To the serum
ml. of sodium sulfite solution of appropriate
concentration was added and the two components mixed by inversion#
3* After the development of maximum precipitate (about ten minutes)
two ml. of Span7-ether reagent^ was added and the mixture gently
shaken. This was followed by centrifugation for 5-10 minutes at
2,000 R.P.M.
4. After centrifugation, a pipette was carefully inserted through
the Span-ether layer and beneath the packed globulins and 4*0 ml#
of clear centrifugate transferred to another cylinder. This was
found to be best accomplished by slanting the tube to separate the
precipitate from the wall of the cylinder#
5# To the clear centrifugate 4.0 ml. of biuret reagent was added
and the components mixed well by inversion.
b. A blank was prepared with4.0 ml. of sodium sulfite and 4.0 ml#
of biuret reagent#
7# After a period of 3^ minutes the protein content was determined
by reading the color development on the photelometer and consulting
the standard curve described above#
Since the absolute values of the pooled sample were known from
the electrophoretic and total protein determinations, the proper sodium

7 Span 20 (Sorbitan monolaurate) Batch 438-c, Atlas Powser Co., Wilming­
ton, Delaware.
SWolfson et al. (1948).

16
sulfite concentration for use in rat sera was that salt concentration
which yielded results in agreement with the electrophoretic analysis.
It can he seen from Figure 2 that 24 percent sodium sulfite (under
these conditions) appears to he the critical concentration wherein the
glohulins are separated from the albumin in pooled rat sera.
The total serum globulin was obtained by subtracting the quantity
of serum albumin found from the total amount of serum protein determined.
The A/Or ratio was obtained by dividing the serum albumin value by
the total serum globulin value.
Electrophoresis
All electrophoretic analyses were carried out with a Perkin-Elmer
Model 3S Tiselius Electrophoresis Apparatus.

Unless otherwise indicated

the buffer used in the present study was a barbiturate (veronal and
sodium veronal) solution of pH S.b, 0.1 ionic strength and made up at
25°C.

This buffer gave good resolution of the alpha globulin and reveal­

ed somewhat more total globulin than phosphate buffer.

There was also

better separation of the delta and epsilon boundaries from the gama
globulin peak (Longsworth 1^42).
From preliminary studies it appeared that the best resolution of
rat serum proteins was obtained by operating at 7*5 milliamperes for
7.200 seconds (veronal buffer).

Therefore, these conditions were chosen

for standard procedure to be used for the duration of the experiments.
However, since the experiments were carried on over an extended period
of the ratfs life in which the serum components changed with growth of

17
the rat, it was soon evident that these conditions would require some
alteration*

These alterations in the standard conditions are, therefore,

indicated in the ’’Results” section and unless so indicated the above
standard conditions were employed.

Ullhere alteration in the standard

procedure was necessary, the corresponding control was altered indentically.
The Perkin-Elmer manual for the model 3^ Tiselius apparatus gives
the technical procedure for analysis.

In the present work this was

followed with the following modifications!
Dialysis. Two ml. of serum was

diluted with four ml. of buffer

and placed into a dialyzing membrane of seamless regenerated viscose
process cellulose^.

The sack was suspended from a clamp on a ring stand

and then lowered into a cylinder of buffer revolving on a turn table.
Care was taken to lower the sack into the buffer to the depth where
there was just enough buoyance to insure constant mixing of the con­
tents within the sack*

In this manner the solutions inside the outside

of the sack were in continual motion.
a cold room at a temperature of 4°C.

The dialysis was carried out in
The protein solutions were dia-

lyzed for two to four hours against 100 mis. of buffer, followed by
four to six hours against 100 ml. of fresh buffer.

The protein solution

was then equilibrated overnight (sixteen hours) against 300 ml. of
fresh buffer.

The equilibrated serum was clarified by centrifuging 15

.,0' *
minutes at 2,000 R.P.M. in the cold room (4 C.).

9visking Corp., Chicago, Illinois

18
Photographic Film*

The film^ was developed 4 minutes in D19*^

developer (total darkness) followed by 10-15 minutes in hypo and 15-20
minutes wash in running water*
Analysis of Electrophoretic Patterns*

Two-fold enlargements of

the descending patterns on negative film were projected from an enlarger
upon plain paper and traced (longsworth and Maclnnes 19^0)•

The base­

line was drawn in such a manner as to connect the minimum values of
concentration gradient.

In assigning areas to the various components

of the serum, the method of Tiselius and Kabat (1939) w&s followed by
drawing an ordinate from the lowest point between adjacent maxima to
the base line.
TP
The areas were measured with a planimeter • Moore et al* (19^9)
had discussed the importance of obtaining accuracy and precision when
using the planimeter for determining areas of serum protein.

Thus,

each component area from a pattern was traced three times such that
three readings did not differ more than five planimeter area units.
The average of the three readings was used for calculation.
By relating the total area of the pattern to the total analyzed
protein concentration by the biuret method, the protein concentration
of each component was determined from the size of its area in terms of
the fractional part of the total area.

■^Kodak Contrast process ortho 3 1/4 x 4 l/4 inches.
Kodak Company.
No. 4236 Compensating polar planimeter, Keiffel and Esser Co.,
New York*

19
The serum protein component mobilities were calculated as recommend­
ed by Longsworth and Maclnnes (1939) from the measured distance in
centimeters between the initial boundary and the ordinate dividing the
area of the component in half.

This migration distance (d) was sub­

stituted into the following formula of Longsworth (19*40) along with
other pertinent data for the determination of mobility*
u =

d q K sp
i t m

The potential gradients were evaluated by supplying data in
following formula:
Potential gradient = -- ^--*1 sp
Where:

d
q
t
i
m
K sp

=
=
=
=
=
=

distance migrated in
cross-sectional area
time of migration in
current in amperes
enlargement factor
specific conductance

cm*
of the cell
seconds
of protein

— conductivity cell constant
resistance of protein in ohms

the

20

A

COM PARISON

PROTEIN
THE

TOTAL

MEASUREMENTS

BIURET R E A C T IO N

BOVINE
7.0

OF

ALBUMIN
RAT

BY

OF

AN D P O O L E D

SERA

CONCENTRATION

(GM

6.0

3.0

2.0
<g) BOVINE ALBUMIN
(g) POOLED RAT SE RA
80
60
90
70
50
PH O T E L O M E TE R
READING

FIGURE I

100

21

SELECTION

OF

SODI UM

SUL F I T E

CONCENTRATION

5jO

450

GMS. ALBUMIN

4.00

ELECTROPHORES I S

3.50

ALBUMIN

3.00

2.00

21

23

25

PER C E N T

SODI UM

FIGURE 2

27

SUL FI TE

29

RESULTS
Preliminary Experiments
The purpose of the preliminary experiments was to establish a
direct connection between host response seen by earlier investigators
and the present study.

Results similar to early investigations, when

obtained under the conditions of the present work, render possible a
more valid correlation between serum protein changes and the various
manifestations of immunity observed previously-

Thus, the following

experiments were carried out to insure that the proposed larval sequence
would duplicate the results of earlier works
(1)
production.

T*16 ability of an immune host to reduce the parasites1 eggFour rats were subjected to increasing numbers of larval

injections at two week intervals until they were able to tolerate
50,000 larvae*

During immunization (60 days) the egg production curve

was essentially the same as recorded by other investigators ^Graham
(193^) » Chandler (1936)7•

The

production reached a peak five to

eight days after the initial infection and then decreased to less than
200 eggs per gram of feces after 16 to IS days-

Despite the repeated

infections of increasing numbers of larvae, the eggs never reappeared
in amounts equal to the initial response but finally decreased until
quantitative measurement was no longer possible.

Thus, it was assured

that the sequence of larvae administered produced a resistance in the
host as manifested by the reduction of eggs.

23
(2) The ability of immune serum to produce precipitate.

Larvae

were incubated in immune serum utilizing the method of Sarles (1938).
In place of the 0.1 percent mercuric chloride solution employed by
Sarles, 500 units of penicillin and 0.05 S®* of streptomycin per cc.
of serum were used to control bacterial growth.

Oral, excretory, and

intestinal precipitates were observed in agreement with Sarles (193^)*
Figure 2a shows a filariform larva incubated in the presence of immune
serum.

The precipitate is visible in the excretory pore and in the

vincinity of the pore.

The immune serum used for incubation was ob­

tained from a rat which had received the proposed sequence of larval
injections.

This assured that the sequence was adequate for a produc­

tion of the antibody responsible for the precipitate.

Larvae incubated

in normal serum produced no precipitates.
(3) The ability of the immune host to resist reinfection.

To

test protection produced by the larval sequence against reinfection,
two mature litter mates were injected with 50»000 larvae.

One had

previously received the larval sequence, while the other had never
been infected and served as a control.
third day following the injection.
death as verminous pneumonia.

The latter animal died the

Necropsy established the cause of

The other rat which had received the

larval sequence showed no visible adverse symptoms.
Preliminary Electrophoretic Analysis
To ascertain the optimum conditions of electrophoretic analysis
the following determinations were made:

2k
An electrophoretic analysis was made on normal and immune serum
in phosphate buffer (the immune serum was produced with the proposed
larval sequence).
in Figure 3

The patterns from these preliminary runs are found

a comparison of the control with the infected samples

shows an extrao rdinary increase in the globulin component for the
latter.
Several electrophoretic nans were made on normal rat serum in
veronal buffer.

Somewhat better resolution of serum proteins was

obtained in this buffer.

Figure k shows two sets of patterns from

normal rats in this buffer.
From this preliminary work it was established that;

(l) at the

selected infection sequence the egg production of the worm was decreased
below quantitative measurement; (2) the immune serum from the infection
sequence was able to produce the precipitates previously described by
Sarles (loc. cit.); (3) rats which had received the sequence were pro­
tected against lethal effects of 50,000 larvae, whereas, controls died;
and (4) the electrophoretic patterns of the immune rats showed striking
increases in serum globulin production.

Figure 2a
Filariform larva incubated in immune serum
which shows excretory precipitate

26

ELECTROPHORETIC PATTERNS OP RAT SERA SEVEN DAYS AFTER THE
FIRST 50,000 LARVAL INJECTION COMPARED WITH CONTROL

Descending

Ascending

Infected
Rats
(Pooled)
pH 7.4; Phosphate buffer (sodium phosphate 0.02M and sodium chloride
0.15M); 10,800 sec.; Pot. Grad. 5*2 volts/cm.; 2.25$ protein.

Control
Rats
(Pooled)

pH 7»*+5 Phosphate buffer (sodium phosphate 0.02M and sodium chloride
0.15M); 10,800 sec.; Pot. Grad. 4.9 volts/cm.; 1.7®& protein.
FIGURE 3

27
ELECTROPHORETIC PATTERNS OP NORMAL RAT SERA

Ascending

Descending

(Pooled)
Veronal "buffer at pH 8.6, ionic strength 0.1, for 7»200 sec.; Pot.
Orad. 7*3 volts/cm.

"sr.1
(Pooled)
Veronal buffer at pH 8.6, ionic strength 0.1, for 7,200 sec.; Pot.
Orad. 7.8 volts/cm.
FIGURE k

28
The Analysis of Sera Two Weeks After Each Infection
Experiment I.

From the work of Chandler (1935^) it was demonstrated

that resistance to superinfection increased as the interval "between in­
fections was increased during 7 to. 1^- days*
resistance gradually decreased*

From the fifteenth day on,

Thus, the fourteenth day after the last

infection represented the time of maximum resistance*

This was mani­

fested "by a falling off in numbers of worms acquired, a gradual decrease
in the percentage of worms which succeeded in passing the final molt,
and a decrease in worm growth.

Since the immunity was strongest lH days

after infection, it seemed logical to examine the serum at this time*
Consequently the various determinations in Experiment I were made, in
most cases, 1*4- days after the last larval injection*
Tables I and II are composites of the data obtained from electro­
phoretic and chemical fractionation analyses of sera in Experiment I.
Figures 5* 6* and 7 show graphically the pertinent data of Table I*
Examination of Figure 5 indicates, with the exception of the deter­
minations following the infection of 1,000 larvae, the total protein
was consistently higher than, the corresponding control*

Tiselius and

Kabat (1939) also observed that on immunization an increase generally
occurs in total serum protein as well as total globulin and antibody
fractions*

An interesting significant fact was that the highest total

protein concentration occurred seven days after the last
injection*

larval

It suggested that although the host*s resistance was high­

est lU days after infection, the greatest serum protein alterations
were occurring earlier.
From Figure 5 ft ^ y

This observation led to Experiments II and III*
noted that rats injected with the super­

natant material from the larval suspension fell well within the normal
range*

This indicated the material in the inoculum other than the

larvae did not cause the increased total protein.

29

Figure 6 shows a marked increase in the total globulin as the
number of larvae injected was increased; and a decrease of total
globulin following a decrease in the number of larvae injected*

The

highest total serum globulin value was noted, again, seven days follow­
ing the last administration of 50*000 larvae.

However, the total serum

globulin of rats injected with the supernatant of the larval suspension
was within the normal range*
Figure 7 shows the beta globulin fraction (electrophoretically
determined) was higher than the corresponding control in every case*
The gamma globulin component showed no significant increase (Table X)*
Figures 8, 9* 10, and 11 represent electrophoretic patterns from
which the corresponding calculations of Table I were made*

They show

the variation in serum protein components with the number of larvae in­
jected*

The patterns of Figure 8 indicate there is little difference

between the infected animals after administration of 1,000 larvae and
the control animals*

(This was also found to be the case in Experiment

la which followed).
In Figure 9 ^ e difference between the infected and control animals
thirteen days after the 20,000 larval infection is quite marked*

It

may also be noted that in the infected animals the mobility of the pro­
tein components was distinctly slower than that of the controls. Table II
contains the calculated mobility values from Experiment I*

The most

striking difference between the control and infected animals was the
increase in the component area which corresponds to the beta globulin.
With the increase of the beta globulin, the gamma globulin tended to
become less resolvable from the former at the standard time of 7*200
seconds.

This plus the decreased mobilities in the infected animals

30
tended to produce poor resolution of the globulins.

To improve resolu-

tion photographs were also taken at 10,000 seconds.
Figure 10 shows the patterns as they appeared four days after the
second injection of 50,000 larvae taken at both 7*200 and 10,000 seconds
(see Table I for sequence of injections).

At 10,000 seconds the gamma

globulin separated better from the beta globulin and was distinguishable
as the slowest migrating component.
globulin (Moore 19^5)*

Hat serum contains little gamma

In veronal buffer at pH 8.6 the gamma globulin

electrophoretically separates from the salt boundary so that it is pos­
sible to more accurately determine total globulin.

But in this buffer

the beta and gamma globulins do not separate as sharply and are some­
times not as well defined as in phosphate buffer at 7-^ (Moore 19^5)•
Thus, in veronal buffer the gamma globulin appeared as a slight shoulder
on the beta globulin peak, which is in agreement with the patterns of
Moore et al. (19^).

According to this description of the gamma globu­

lin, the globulin increase may be attributed to the beta component.
Later in Experiment III, serum electrophoretically analysed in both
veronal and phosphate buffers corroborated this statement (Figures 19
and 20)•
The pattern in Figure 11 from pooled sera of rats number 25a and
27a shows the changes that occurred seven days after the last injection.
(See Table I for injection sequence).

In these rats the total protein,

total globulin and specifically the beta globulin reached the highest
level of all measurements taken in Experiment I.
The rats which received injections of supernatant from the larval
suspensions showed normal patterns.

An example of such may be seen in

31
the pattern from rat fy-5 in Figure 11*

Inspection of Table I also shows

that the various serum components were normal in these rats*
Body weights were measured throughout Experiment I and Table III
shows the hyperimmunization process produced a decrease in body weight
when compared with the uninfected controls#
Summarizing the results of Experiment I it may be noted that
measurements, taken 1*+ days after the last injection, showed a steady
increase in the total protein and total globulin as the number of lar­
vae injected was increased*

More specifically the beta globulin was

consistently higher in the infected animals*

Of the determinations made

at four, seven,and fourteen days after the last infection of 50,000
larvae, the greatest changes were noted seven days after infection.

As

mentioned, Experiments II and III were the result of this finding.
Experiment la. From the results of Experiment I it was found that
after the 1,000 and 2,000 larval injection, there was little or no
change in the serum components when compared with the controls.

Since

after the administration of 1,000 larvae the host exhibits a rather
strong resistance to reinfection as manifested by reduced egg produc­
tion, it was decided to repeat the serum analysis l4 days after the 1,000
and 2,000 larval injections#

The results are reported in Table IV.

The

findings substantiate the results of Experiment I In that there was no
significant difference in the serum protein components at this level of
infection between control and infected animals.

TABLE I.
Rat No.

Age (Days)
Last
Bled
Infected

No. of
Larva©

ELECTROPHORETIC AND SALT

By Salt Fractionation (Gtau jo)
Total
AIL.
Glob.
Jkj£
Protein

-

58
58

1,000
Control

5.10
5.52

2.97
3*51

2.13
2.01

1.39
1-75

7,8.9
10,11,12

59
—

72
72

2,000
Control

6.47
6.03

3-73
3.90

2.74
2.13

1.36
1.83

37.38,39
40,41,42

73
—

86
86

5,000
Gontrol

7.67
7.03

4.31
4.90

3.3b
2.13

1.28
2.30

25,49,27
28,29,30

g7
—

100
100

10,000
Control

7.70
6.75

3.67
4.19

4.03
2.5b

.91
1.64

13,14,15
16,17,18

101

n4
114

20,000
Control

7*96
6.47

3-50
3.61

4.46
2.8b

.78
1.26

19,20,21
22,24,47

118

128
128

50,000
Control

7-70
7.22

3.30
4.03

4.4o
3-19

•75
1.26

31,32,33
34,35,36

132
—

142
142

20,000
Control

7.57
b.75

3.92
4.57

3.65
2.18

1.07
2.09

5a, 51

146

150

50,000
Control

7.54

—

25a,27a
43,44

165
—

172
172

50,000
S

8.21
6. b5

3.30
3.85

_ _

181
181

s

6.20
b.82

1.2,3
“1.5,6

46
45

45
—

—

—

S = Supernatant

—

4.91
2.80

—
.67
1 .3s

.^

4.10

2.72

1*51

33

FRACTIONATION ANALYSIS OF EXPERIMENT I

Alb.

Total
Glob.

By Electrophoresis (Gm.
Globulins
Alphap
Beta
Alpha2

Gamma

a /g

2.97
3*58

2.09
1.98

.44
.65

.40
*39

.96
*73

*32
.26

1.42
1.81

3-57
3*52

2.91
2.52

*71
•91

.83
.48

1.17
.69

.20
*52

1.25
1.40

4.31
4 .9s

3*39
2.07

.80
*79

.80
.42

1*57
•72

.18
*15

I.27
2.40

4.06
4.^3

3*64
2.52

.64
*54

.86
.58

1.62
1.05

•53
•31

1.11
1.68

4.4o
3*53

3*65
2.90

*57
*73

*57
.62

2.02
1.13

.48
.40

1.25
1.22

3*59
4.07

4.14
3*1S

•7*
•63

.70
*67

2.23
1.4i

•ft
.44

.87
1.28

3.7S
4.08

3.7S
2.67

1.14
•76

•77
*53

1.62
.84

.40
•50

1.00
1.53

3*25

4.24

1.06

.98

1*57

.71

•76

3*51
3*97

4.70
2.68

*92
•74

.80
*37

2.79
1.25

•19
*33

*75
1.48

3.84
3.41

2.36
3*37

*71
1.28

•31
.60

1.09
*96

.25
.64

1*59
1.01

3*+

TABLE II.

Rat No.

MOBILITY CALCULATIONS OF EXPERIMENT I

Alb.
Alpha^

1.2,3,
^,5,6

MOBILITIES*
Globulins
Beta
Alphag

Gamma

6.10

5.51

4.83

3.31

2.63

7,3,9
10,11,12

5-37
6.22

4.71
5-71*

3-95
4.88

3.04
3.54

2.38
2.96

37.3s *39
4o ,4i ,42

5.04
5.10

4.35
4.16

3.62
3.59

2.57
2.S3

1.74
2.08

25,49,27
22,29,30

6.03
4.36

5.24
3.99

4.55
3.3^

3.48
2.51

2.47
1.81

12.14.15
16.17.15

4.21
4.85

3.73
4.35

2.97
3*73

1*93
2.94

1.32
1.91

19,20,21
22,24,47

3-56
4.64

3.02
4.06

2.53
3-56

1 .7s
2.86

1.12
2.24

31.32.33
34,35.36

3.59
5.17

3.1S
4.60

2.82
4.06

2.34
3.31

2.08
2.76

5a, 51

3*26

2.79

2.18

1.54

.90

25a, 27a
*13,1+4

4.17
3.73

3.6S
3.02

3.06
2.56

2.23
2.05

1.49
1.47

*+6
*+5
♦Table figure X 10~5 cn£ volt-1 sec-1

35

THE

RELAT I ON

INFECTED

OF

TOTAL

PROTEIN

TO

INFECTION

Q

UNINFECTED

Q

SUPERNATANT

(

llJ

m

lAl
o
8j00

-i

o

o

oc
CM

PROTEIN

(GM.%)

04

UJ

7100 -

_j

TOTAL

*>

a:

5.00
40

60

80

100

RAT

140

120

AGE

FIGURE

( DAY S)

5

160

180

36
th e

5.00 -

relation

of

total

I
I
ELECTROPHORESIS—
SALT FRACTIONATION
INFECTED
A

globulin

to

I
—

UNINFECTED
SUPERNATANT

(GM.%)

4.50 *

?

TOTAL

GLOBULIN

4.00 -

3.50 -

300 ~
4

2.50 -

R

2.00 100

RAT

120

AGE

FIGURE

140

( DAYS)

6

NFECTION

37

THE

RELATION
•

OF

i

IN F E C T E D

^

U N IN F E C T E D

Q

SUPERNATANT

0

BETA

GLOBULIN

i

I

TO INFECTION

I

i

3.00

o
o
o

<
>
a;
<

#-

cm

IO

a*

9

/

s
UJ

'-2J50I.

o ><
o ac
om <

O <
o>

o4 0C
o
IO

*

ZD

m

o

UJ

o?po|-

O <
o >tr
o <
#

o
o <>
o# ac
o <

/

O

GJ

/
o <UJ
>
o a:
o* <
o

ca

o

<
HUl

o
o
o

CD

/

<
>
ac

m

UJ

o n5
r
o<

o -I
1.00^'

ml

UJ
<
o >
o ac
<
CM
i

< *

J

)

A

<£
o

p -

✓

9

o-o—<3
■JL«

40

60

X
80

100

RAT

AGE

120

140

(DAYS)

FIGURE 7

160

-l_l
180

ELECTROPHORETIC PATTERNS OP EAT SERA lk M T S AFTER THE
1,000 LARVAL INJECTION COMPARED WITH CONTROL
Ascending

Descending

pH S.6; 0.1M Veronal "buffer; Ionic strength 0.1; 7*200 sec.; Pot.
Grad, not available; 1.70$ protein*

pH S.6; 0.1M Veronal buffer; Ionic strength 0.1; 7,000 sec.; Pot*
Grad. 8*4 volts/cm.; 1.8*$ protein.
PIGURE 8

ELECTROPHORETIC PATTERNS OF RAT SERA 13 M T S AFTER THE FIRST
20,000 LARVAL INJECTION COMPARED WITH CONTROL
Ascending

Descending

Infected
Rats No*
13,14, 15.
pH S. 6; 0*1M Veronal buffer; Ionic strength 0*1; 7*200 sec.; Pot
Grad. 7*4 volts/cm.; 2.98$ protein*

Control
Rats No*
16,17,18

pH 8*6; 0.1M Veronal buffer; Ionic strength 0.1; 7,200 sec.; Pot*
Grad. 8.4 volts/cm.; 2.1S$ protein.
FIGURE 9

ELECTROPHORETIC PATTERNS OF RAT SERA FOUR DAYS AFTER THE
SECOND 50,000 LARVAL INJECTION COMPARED AT
7,200 AND 10,000 SEC.
Ascending

Descending

pH 8.6; 0.1M Veronal buffer; Ionic strength 0.1; Pot. Grad. 8.6
volts/cm.; 2.77$ protein; rats No. 5a and 51.
FIGURE 10

41

ELECTROPHORETIC PATTERNS OF RAT SERA SEVEN DAYS AFTER THE
LAST 50,000 LARVAL INJECTION COMPARED WITH SERA FROM
A RAT WHICH RECEIVED INJECTION OF SUPERNATANT

Ascending

FROM THE LARVAL SUSPENSIONS
Descending

pH 8.6; 0.1M Veronal buffer; Ionic strength 0.1; 7214 sec.; Pot
Grad. 8.4 volts/cm.; 2*74$ protein

J w La
W ▼Wl

Supernatant
he

^ i/ 1
|

m

pH 8.6; 0.1M Veronal buffer; Ionic strength 0.1; 7 *200 sec.; Pot.
Grad, not available; 2.22$ protein.
FIGURE 11

42

TABLE III.

TEE RELATION OF BODY WEI ©IT TO INFECTION
(EXPERIMENT I)

Level of
Infection

Age (Days)
When
Weighed

Uninfected
Control

44
44

21
21

87-9
89.4

1,000
Control

58
58

21
21

129.1
137.9

2,000
Control

72
72

17
18

152.8
177.3

5,000
Control

86
86

l4
15

20b. 9
214.9

10,000
Control

100
100

11
12

225.8
239.1

20,000
Control

114
114

9
9

248.2
261.2

50,000
Control

128
128

6
5

252.?
270.0

20,000
Control

142
142

3
3

238.7
269.0

Number
of Rats
Weighed

Average
Body
Weight

*3

TABLE IV,

Rat
No*

ELECTROPHORETIC AND SALT FRACTIONATION
ANALYSIS OF EXPERIMENT la

Age (Days)
Last
Bled
Infected

Level
of
Infection

By Salt Fractionation (Gm. %>)
Alt.
Total
Glob.
A/G
Protein

55,56
59, bO

88
—

103
103

1,000
Control

5.53
6.37

3.4o
3.45

2.13
2.92

1.60
1.18

53,54
57,58

104

117
117

2,000
Control

b.28
7*04

3.39
4.31

2.89
2.73

1.17
1.58

By Electrophoresis (Gra. %)
Globulin
Alpha2
Alphai
Beta

Gamma

Hat
Ho.

Alb.

55,56
59,60

3-36
3.30

2.17
3.07

.62
1.36

•59
•50

•73
•79

.1^
.46

1.55
1.07

53,5^
57,58

3*56
3-61

2.73
3. **3

.83
.90

.*3
.64

•92
1.46

.49
.34

1.03
1.65

Total
Glob.

A/G

MOBILITIES*
Globulins
Alpha-p
Beta

Rat
No.

Alb.

55,56
59,60

5-25
5.27

4.61
4.56

4.06
3.9*

3-27
2.83

2.67
2.17

53.5*+
57,58

3.62
4.82

2,97
4.11

2.55
3.49

1.95
2.64

1.25
1.89

Alphai

♦Table figure x 10~5 cm2 volt*"^ sec”-*-

Qa-mma

44
The Analysis of Sera From Older Rats One Week After Infection
Experiment II. Results of Experiment I suggested that the serum
changes might be more pronounced seven days after injection although
resistance was highest l4 days following injection.

Therefore, the pur­

pose of this experiment was to study the serum protein changes seven
days after each larval injection.
At the time this experiment was set up, the only rats readily
available were those considerably older than used in Experiment I.

The

rats in Experiment I were initially infected with 1,000 larvae at 45
days of age while those of Experiment II received the same initial irw
fection at 120 days of age.

As a result of this wide age difference

it is difficult to make direct comparisons between the two experiments.
Therefore, the results of Experiment II may be considered as an isolated
investigation and an amalgamation with the other experiments will be
included in the nDiscussion” portion of this thesis*
Tables V and VI contain a composite of the electrophoretic and
chemical fractionation analysis for Experiment II.

Figures 12 and 13

illustrate graphically portions of Table V*
Table V demonstrates that although the differences were not large,
the total protein was higher in the infected animal than in the cor­
responding control in every instance.
Figure 12 graphically shows that the total globulin increased
markedly as the number of larvae

is increased.

In Figure 13 the beta globulin concentrations resulting from larval
injections are plotted.

Only points which were obtained from the analysis of

the best resolved patterns are included in the graph.

The beta globulin

concentration was increased in the infected animal in every case with
the highest point following the 50,000 larval injection*
Figures l4 and 15 contain electrophoretic patterns which compare
the early part of the hyperiromunization process with the latter part.
The pattern from the 2,000 larval injection showed little difference in
the concentrations of the various components when compared with the
corresponding control (Figure l4).

Again it was noted that the compo-

n e n t mobilities of the infected animals
compared with the control.

decreased markedly when

Examination of the patterns in Figure 15

shows the infected animals (bled seven days following the only 50,000
larvae) exhibited a marked increase in the beta globulin peak and a
decrease in mobility.
It has been shown (Luetscher 19^7) that certain pathological changes
affect electrophoretic patterns. In the necropsy examinations of Experi­
ment I it was noted that the lungs of the infected animals were greatly
enlarged.

Therefore, in Experiment II it was decided to take weight

measurements of the lungs throughout the course of hyperimmunization.
The results of these measurements are recorded in Table VTI.

When the

lung weight is converted to per cent of the total body weight^ the in­
crease in the infected animals was noted in some cases to be four-fold
(Rats 68 and 78, Table VII).
Grossly the lungs of the infected animals were enlarged, highly
infiltrated with scar tissue and contained a considerable number of
petechial hemorrhages.

weight
body weight

3lung

_

*

cent of body weight attributable to the lungs,

46
On microscopical examination the air sacs were found generally to
contain an exudate composed of (in order of their prevalence) erythocytes, polymorphonuclear leucocytes, lymphocytes, and monocytes.

This

pathology cannot be totally disregarded in evaluation of electrophoretic
patterns.
Summarizing the results of Experiment II it was noted that in the
infected animals the total protein, total globulin, and specifically
the beta globulin were increased when compared with the control.
ing hyper immunization the lungs increased as much as four-fold by
weight (percent of total body weight).

Dur­

TABLE V.
Rat
No.

Age (Days)
Last
Bled
Infected

61,62
71,76

133

63,b*+
72.77

l4s

65,6b
73,74

161

67,68
78,79

176

69,70
75

190

—

—

—

—

144
144
155
155

No. of
Larvae

2,000
Control

5,000
Control

169
169

10,000

183
183

20,000

197
197

50,000

Control

Control

Control

ELECTROPHORETIC AND SALT

By Salt Fractionation (Gin* %)
Total
AIL*
Glob.
A/G
Protein

b.65
6.38

4.10
4.18

2.55
2.20

1.61
1.90

6.55
5-7^

3.7^
3.58

2.81
2.16

1*33
1.66

6.85
6.39

3.78
4.12

3.07
2.27

1.22
I.83

6.55
6.47

3.10
4.10

3*45
2.37

.90
1*73

6*74
6.22

2.73
3*56

4.01
2.66

.68
1.34

FRACTIONATION ANALYSIS OF EXPERIMENT II

AIL*

Total
Glob.

By Electrophoresis
Globulins
Alpha!
Beta
Alpha^

Gamma

a /g

3.82
3*50

3*13
2.8g

*91
1.28

•^9
*50

1*39
.90

*35
*27

1.12
1.21

3*76
3*19

2.79
2.55

l.l4

.40

•86

.21

1.3^
1.25

3*36
3*73

3*^9
2.66

•9*+
1*13

.62
•31

1.49
1.10

•35
*19

*96
1.40

3*1^
3.82

3-4l
2.64

-

-

--

2.90
3.4o

3.84
2.82

1.03
1*33

.42
.42

2.06
.80

mmmm

—

*39
*3S

.92
1.45
*76
1.20

1*9

TABLE VI.

MOBILITY CALCULATIONS OF EXPERIMENT II
MOBILITIES*
Globulins
Beta
Alpha^

Rat
No.

A16.

61,62
71.76

3.64
5-92

3.06
5.17

2.65
4.43

1.99
3-3!

1.37
2.65

63,64
72,77

3.01
5.90

5.00

4.10

3.05

2.15

65,66
73,74

3.84
3.69

3.27
3.95

2.61
2.42

1.88
1.64

1.23
.94

67,68
73,79

4.72
3.72

4.27
3*20

__

2.89

2.46

69,70
75

3.48
4.33

3.81
3.69

2.31
3.1S

1.74 ■
2.59

Alpha^

* Table figure x 10"-* cm2 volt"*3, sec”1

Gamma

—
1.09
1.99

50

THE

R E L A T I O N OF T O T A L GLOBULI N TO I N F E C T I O N
INFECTED
U N IN F E C T E D
SUPERNATANT
ELECTROPHORESIS
SALT F R A C T I O N A T I O N

laj

tu

04

O
iaj

oc
3
cc
to

h-

LftJ
oc

300 o

120

csl

140

1
160

RAT

1
200

1
180

AGE

FIGURE

(DAYS)

12

220

51

T HE

RELATION

T

OF

BETA

GLOBULI N TO I NFECTI ON

r

INFE CTE D
UNINFECTED

3.00-

3

O

o t>r
o<
o
r>

CD

O

•

o

2.00H

Ui
CD

UI
o <>
o
o o<c
o

UJ

<

O >
o 0C
o <

<
O >
o
o <tr.
lO

<
O >
O 0£
o<

o<
o>
o4 QC
o<

0

0

CM

1.00

o

-

c

/
'•o

120

140

X
180

160

RAT

AGE

FIGURE

(DAYS)

13

200

220

52

ELECTROPHORETIC PATTERNS OF RAT SERA 11 DAYS AFTER THE
2,000 LARVAL INJECTION COMPARED WITH CONTROL
Ascending

Descending

Infected
Rats No*
61,62.
pH S.6; O.IM Veronal buffer; Ionic strength O.l; 7*200 sec.; Pot*
Grad. 8.4 volts/cm.; 2.22$ protein.

Control
Rats No.
71,76
pH 8.6; O.IM Veronal buffer; Ionic strength 0.1; 7,200 sec.; Pot.
Grad. 8.4 volts/cm.; 2.13$ protein.
FIGURE l4

53
ELECTRO FHORETIC PATTERNS OP RAT SERA SEVEN DATS AFTER THE
ONLY 50,000 LARVAL INJECTION COMPARED WITH CONTROL
Ascending

Descending

pH 8*6; O.IM Veronal "buffer; Ionic strength 0.1; 10,000 sec.; Pot*
Grad. 8.6 volts/cm.; 2.27$ protein.

Rat No.

^B

pH 8.6; O.IM Veronal "buffer; Ionic strength 0.1; 10,000 sec.; Pot.
Grad. 8.2 volts/cm.; 2.07$ protein.
FIGURE 15

54

TABLE VII.

Rat
No.

THE RELATION OP LUNG- WEIGHT TO INFECTION
(EXPERIMENT II)

Level of
Infection

Body
Weight

Lung
Weight

Luns wgt.
__
Body wgt. x 100

259
246
271
278

3.05
2.09
1*71
1.34

1.18
.85
.63
.48

237
2*18
208
256

2.60
3.10
1.28
1.59

1.10
I.25
.61
.62

266
264
272
287

4.10
4.80
1.80
1.50

1.54
1.82
•66
.52

292
293
295
317

4.15
5.36
1-17
1.3s

1.42
1.83
.40
.44

215
277
267

*
*
*

Infected
Infected
Control
Control

6l
62
71
76

2,000
2,000

Infected
Infected
Control
Control

63
64
72
77

5,000
5,000

Infected
Infected
Control
Control

65
66
73
74

10,000
10,000

Infected
Infected
Control
Control

67
68
78
79

20,000
20,000

Infected
Infected
Control

70
69
75

50,000
50,000

—
—

—

...
—

■—

—

* Weights not available.

55
The Analysis of Sera Prom Young Rats One Week After Each Infection
Experiment III.

Results from Experiment I showed the greatest

serum protein changes to occur seven days after the last injection of
50.000 larvae (see Table I for injection sequence).

The purpose of

Experiment III was to determine the serum protein changes that occurred
seven days after the last injection in order to compare these changes
with those observed l4 days after infection.

When Experiment III was

initiated, the rats were approximately the same age as those used in
Experiment I.

Therefore, a better comparison between the two experi­

ments was possible.
Tables VIII and IX are composites of the results obtained by the
electrophoretic and chemical fractionation analysis of Experiment III.
Pertinent data of Table VIII are presented graphically in Figures 16,
17, and 18.
Consistent with the findings of the previous experiments, the
total serum globulin showed a progressive increase as the number of
larvae was increased (Figure 16).

The most significant increase in

total globulin was again noted seven days after the only injection of
50.000 larvae.

Figure 16 shows that from a determination made 21 days

after this 50,000 larval injection, the total globulin had dropped
considerably when compared with the determination made seven days after
the injection of the same number of larvae.
The beta globulin was higher in the infected animal than in the
corresponding control in every case (Figure 17) ♦ The most outstanding
increase occurred seven days after the 50,000 larval injection.

56
Twenty—one days after this injection of 50,000 larvae the beta globulin
had decreased considerably from the concentration it had reached seven
days after injection.
In Figure 18 the albumin to globulin (A/G) ratios for Experiment
III are plotted.

The A/G ratio gradually decreased as the number of

larvae increased.

This was not surprising since the globulins were

already shown to increase in the infected animal.

In addition, in

almost every pathological state there is a relative or absolute decrease
in serum albumin (Luetscher 1947) • Examination of Table VIII substantiates this observation.

The influence of these two developments would

result in an A/G ratio decrease.
Figures 19 and 20 contain the electrophoretic patterns from in­
fected rats l40, l4l, 142 and controls 143, l44, 145.

Analysis of

identical samples was made in both veronal and phosphate buffers.

As

previously mentioned the veronal buffer separates the gamma globulin
from the salt boundary, whereas the phosphate buffer shows better
resolution between the beta and gamma globulins.

Since the objective

was to confirm the finding that the beta globulin component was in­
creased by the infection, the use of both buffers presented the logical
approach to analysis.

Figure 20 shows that in phosphate buffer the beta

and gamma globulins were resolved somewhat better than in veronal buf­
fer (Figure 19).

Electrophoretic data and calculations for the serum

runs in both buffers are recorded in Table X.

It was found that in

both buffers the beta globulin was approximately doubled in the infected
animal after seven days when compared with the uninfected control, where­
as the gamma globulin was not significantly increased.

57
TABLE X.

Rats

Total
Protein

IMMUNE SERTJM ANALYZED ELECTROFHOEETICALLY IN VERONAL
AND PHOSPHATE BUFFERS (Gm. f)
Alb.

Total
Glob.

Alpha^

Globulins
Alphag
Beta

A/G
Gamma

Veronal Buffer
I
C

I
c

7 .2 1
6.90

7*21
6*90

2 .4 7
3.71

4 .74
3.19

.59
.6 4

2.1 0
1 .0 2

.63
•53

• 52
1 .16

3.32
3 .74

Phosphate Buffer
3.89
.94
.47
3.16
1.1 3

1-53
.8 2

•97
.7 2

.85
1.1 8

1 . 1+3
1 .0 2

1= Infected
C — Control
Figure 21 shows the serum pattern changes 21 days after the only
50.000 larval injection*

It was noted that, although the beta globulin

was still somewhat increased, the over-all pattern of the infected an­
imals was approaching normalcy*
Table XI indicates that idien the lung weight was converted to per­
cent of the total body weight, the increase following the injection of
the 50,000 larvae was noted to be as high as a six fold increase over
the controls (Rats 1^4 and 289, Table XI).

Twenty-one days after the

50.000 larval injection, these weights had dropped indicating the maxi­
mum change had been reached and the tendency was now toward normalcy.
Thus, in Experiment III it was ascertained that:

(1) the total

globulin increased as the number of larvae was increased; (2) the beta
globulin specifically was increased when analysed in two different buf­
fers; (3) changes in serum proteins were most outstanding following the
injection of the 50,000 larvae; (4) the lung weight (converted to percent

of the total body weight) was increased as the number of larvae was
increased and (5) after injections ceased this lung weight receded*

TABLE 711X. ELECTROPHORETIC AND SALT
Rat No*

Age (Lays)
Last
Bled
Infected

116-17-18
119-20-21

No* of
Larvae

By Salt Fractionation (Gm. $)
Alb.
G-ldb.
a /g
Total
Protein

5S
—

65
65

1,000
Control

5-98
5.32

73

80

—

—

—

—

—

—

80

2,000
Control

6.32
6.05

3*22
3*83

3*10
2.22

1.04
1.72

152-53-54
155-56-57

87
—

3k
3k

5,000
Control

6.20
6.03

3.16
3*47

3.01+
2.56

i.o4
1.36

128-29-30
131-32-33

101
—

108
108

10,000
Control

6*77
7*42*

3*16
3*85

3*61
3*57

.88
1.08

ll46-47-*J6
1^9-50-51

115
--

122
122

20,000
Control

5*97
6.o4

2.65
3.42

3*32
2,62

.80
1.30

l40-4l-42
ll+3_41ul+5

129
—

136
136

50,000
Control

7*21
6.90

2.8k
3 *88

4.37
3-02

•65
1.28

158-59-60
161-63

129
—

150
150

50,000
Control

7*25
6.60

3*38
3*85

3*87
2.75

.87
1.40

13^35-36
137-38-39

* Hemolysis.

6o

FRACTIONATION CALCULATIONS OF EXPERIMENT III

Alb.

Total
Glob.

Alphap

3*55
2.85

2.43
2.47

—

3*30
3*57

3.02
2.48

2.30
2.92

By Electrophoresis (Gm. * v ....
Globulins
Alphap
Beta
Gamma

A/G

*39
—

.86
—*

.36
—

1.1+6
1.15

•56
•66

.bl+
.51

1.27
.80

.60
•55

1.09
1.1*4

3. to
3.01

1.02
.7*

.1*5
.1*6

1.1*9
1.20

.1*5
.71*

.82
•97

3*36
3.9s

3.1*1
3.1*1+

1.16
--

*39
—

1.1*7

2.75
3*25

3*22
2.79

•52
—

.90
—

1.50
—

—

2.47
3*71

4.7H
3*19

1 .1*3
1.02

•59
•6**

2.10
1.02

.63
•53

•52
1.16

3.62
3.63

3.63
2-97

1.10
.93

•71
.36

I.5I*
•9S

•31
•29

1.00
1.22

•79

•35 “
—

•30

•99
1.16
.85
1.17

61

TABLE IX.

MOBILITY CALCULATIONS OF EXPERIMENT III
MOBILITIES*

Rat
No.

AIL.
Alpha

Globulins
Beta
Alphag

Gamma

1.93
—

l.*41

3.52
3.69

2.63
2.96

1.9*4
2.*42

2.70
3.75

2.03
3.2*4

1.19
2*53

*36
1.7S

3-56
—

2.96
—

2.56
—

1.96
—

l.*48
—

1U6-U7-US
1*49-50-51

u .05
—

3.60
—

3-23
—

2.72

2. *42

l*40-*41-*42
1H3-U4-U5

3 .SB
5-3“+

3.35
4.77

2.9*+
*4.01

2.21
3.03

1.86
2.31

158-59-60
161-63

U.72
4.72

4.06
4.00

3-17
3.32

2.07
2.20

1.26
1.52

116-17-18
119-20-21

3.70
3.*46

3.09

13*4-35-36
137-38-39

*4.61
*4.76

*4.0*4
*4.17

152-53-5*4
155-56-57

3-5*+
*4.36

12S-29-30
131-32-33

—

♦Tahle figure x 10”5 cm^ volt“^ sec~l

2.62
—

—

—

—

62

THE

R E L A T I O N OF T O T A L G L O B U L I N TO I NF E CT I ON

r
&oo -

i

r

INFECTED

^

UNINFECTED

Q

ELECTROPHORESIS — —
SALT

9

FRACTIONATION

4.50

*9
2

4.00

Z>
CD

O
e>
3.50
r -

<
ho
\—
300

x

5

Z5 0

2.00
X

40

60

80

RAT

X

X

X

100

120

AGE

FIGURE

140

(DAYS)

16

160

180

63

THE

R E L A T I O N OF B E T A GL OBUL I N

INFE CTE D

Q

UNINFECTED

Q

TO I N F E C T I O N

2.50

O

2.00
Z
Ui

=>
CM

-1
Ui

e>
1.50

ID

CM

UJ
1.00

.50

40

60

80

100
120
M
RAT
AGE
(DAYS

FIGURE

17

160

180

6U

T H E R E L A T I O N OF T H E

i

2.00

i

A/G

i

RATIO

i

TO I NFECTI ON

i

i

r

««

**

I.SO a

-

<

cr

9

1.00

»««
<
o >
o
o 5

CD

o UI
o
o <
>
DC
<
~ -1

-1

<

.50
INFECTED
UNINFECTED

^

o

e l e c t r o p h o r e s is —

SALT

4*

*

——

FRACTIONATION —

l0 RAT ,l°ASE 'fgAYS)

FIGURE

18

65

ELECTROPHORETIC PATTERNS OE RAT SERA SEVEN DAYS AFTER THE
ONLY 50,000 LARVAL INJECTION COMPARED WITH CONTROL
Ascending

Descending

pH S.6; O.IM Veronal buffer; Ionic strength 0.1; 7*200 sec.; Pot.
Grad. S.5 volts/cm.; 2.40$ protein.

pH S.6; O.IM Veronal buffer; Ionic strength 0.1; 7*200 sec.; Pot.
Grad. S.6 volts/cm.; 2.30$ protein.
FIGURE 19

66

ELECTROPHORETIC PATTERNS OP RAT SERA SEVEN DAYS AFTER THE
ONLY 50,000 LARVAL INJECTION COMPARED WITH CONTROL
Ascending

Descending

Rats No*
l4o,l4l,
142.
pH 7.4; Phosphate buffer (sodium phosphate 0.02M and sodium chloride
0.15M); 12,000 sec.; Pot. S-rad. 4.9 volts/cm.; 2.40$ protein

pH 7.4; Phosphate buffer (sodium phosphate 0.02M and sodium chloride
0.15M); 14,^100 sec.; Pot. Grad. 4.9 volts/cm.; 2.3<$ protein.
FIGURE 20

ELECTROPHORETIC PATTERNS OF RAT SERA 21 DAYS AFTER THE
ONLY 50,000 LARVAL INJECTION COMPARED WITH CONTROL
Ascending

Descending

pH 8*6; 0.1M Veronal buffer; Ionic strength 0*1; 7*200 sec.; Pot.
Grad. S.5 volts/cm.; 2.42$ protein.

J

U

J

.

pH g.6; 0.1M Veronal buffer; Ionic strength 0.1; 7.200 sec.; Pot.
Grad* S.7 volts/cm.; 2.20$ protein.
FIGURE 21

68
TABLE XI,

THE RELATION OF LONG- WEIGHT TO INFECTION
(EXPERIMENT III)

Rat
No.

Level of
Infection

Control
Control
Control
Infected
Infected
Infected

119
120
121
116
117
118

_ ml
— __—

Control
Control
Control
Infected
Infected
Infected

137
138
139
134
135
136

Control
Control
Control
Infected
Infected
Infected

155
156
157
152
153
15k

Control
Control
Control
Infected
Infected
Infected

131
132
133
128
129
130

Control
Control
Control
Infected
Infected
Infected

1U9
150
151
146
1*7
148

Control
Control
Control
Infected
Infected
Infected

143
l44
145
l4o
l4i
142

Control
Control
Infected
Infected
Infected

161
163
158
159
160

1,000
1,000
1,000
,-,w
——

2,000
2,000
2,000

...

5,000
5,000
5,000
...
...
...

10,000 .
10,000
10,000
...
—

20,000
20,000
20,000
...
— -

50,000
50,000
50,000
...
...

50,000
50,000
50,000

♦Lung showed congestion.

Body
Weight

Lung
Weight

Luns
I 100
Body wgt.

134
Ijk
143
135
150
137

•75
•95
1.38*
1.05
1.00
1.07

0.56
0.55
0.97
0.78
0.67
0.78

150
155
172
l44
166
181

1.00
1.01
.91
1.31
1.40
1.42

0.67
0.65
0.53
0.91
0.84
O.78

186
187
237
169
223
228

1.06
1.34*
1.17
1-35
1.73
2.80

0.57
O.72
0.49
0.80
O.78
1-23

250
248
242
250
235
200

1.22
1.23
1.20
1.67
2.38
3.53

0.49
0.50
.50
0.67
1.01
1*77

269
274
293
246
234
266

1.30
1.10
1.50
5.00
2.10
8.62

0.48
0.4o
0.51
2.03
.90
3.24

326
265
230
300
275
256

1.26
1.20
1.10
3-75
7.05
7.40

0.39
0.45
0.48
1*25
2.56
2.89

255
216
264
283
284

1.00
1.30
2.73
3.06
3.40

0.39
0.61
1.03
1.08
1.20

69
Composite Results of Serum Protein Changes
In Experiments I and III
Since the determinations in Experiment I were made two weeks after
each injection and those of Experiment III were made one week after
each injection, the serum protein changes were more completely demo ro­
strated when the results of the two experiments were plotted on the
same graph.

In Figures 22 and 23 the total globulin and the beta

globulin, respectively, are plotted against infection from both experi­
ments*
The total globulin (Figure 22) of the infected animal showed a
gradual increase as hyper immunization proceeded.

In the early stages

no consistent pattern of changes occurred, i.e. measurements at seven
days after the last injection were not consistently higher than those
taken two weeks after the last injection or vice versa.

However, when

the four electrophoretic determinations were made 4, 7, 13, a^d 21
da^s after the 50,000 larval injection, the highest total serum globu­
lin was found to be on the seventh day.
The composite graph of the beta globulin determinations in Figure
23 shows there was a higher concentration of this component in the in­
fected animal throughout hyperimmunization.

Of particular interest is

the fact that in five out of six intervals between injections the beta
globulin of infected animals was slightly higher after two weeks than
one week after each infection*
Thus the composite of the two experiments, tends to substantiate
the fact, that a gradual increase in total globulin and specifically,
the beta component, occurs as the hyperimmunization proceeds.

70

THE

R E L A T IO N

OF

TOTAL

G LO B U LIN

TO

IN FE C TIO N

( C O M P O S IT E )

^

UNINFECTED

Q

ELECTROPHORESIS — —
SALT FRACTIONATION

—

INJECTIONS 14 DAYS APART

TOTAL

wGLOBULIN

*

IGM.%)

4.50 -

INFECTED

d
0

♦ 2 1 D A Y S -H

p-

ipoo

zpoo

spoo

10,000

20p00

LARVAE

FIGURE 22

SOyOOO

71

THE

R E LA T IO N

OF BETA

GLOBULIN

TO INFECTION

(C O M P O S IT E )

I

T

I

INFECTED

©
UNINFECTED
O
NJECTIONS 14 DAYS APART

b
•21 DAYS— H

IPOO

2,0 0 0

5P00

10,000 eopoo sopoo
LARVAE

FIGURE

23

72
Absorption Studies
The purpose of the absorption studies was an attempt to identify
the globulin increase as antibody formation.

The principle of Tiselius

and Kabat (1939) ws-s applied to the present work#

The procedure con­

sisted of comparing electrophoretic patterns of immune serum before
and after exposure to prepared antigens.

If the specific antibody

were present in the serum and combination with the antigen occurred,
the antigen-antibody complex might be removed as a precipitate. . The
electrophoretic pattern after exposure would reveal the absence of all
or a part of a component.

A component removed in this manner was con­

sidered to be antibody by these authors#

Experiments of this type are

important in establishing the relationship of antibody to serum proteins.
In the present studies larvae and various larval preparations were
used as the antigen and were exposed to normal and immune sera.

Larvae

to be used as antigen were processed in the following manner:

(l) the

larvae were washed five times by centrifugation in tap water,

(2) this

was followed by two washes in distilled water (3) the larvae were then
placed in a 0.5 percent mercuric chloride solution for one-half hour
(4) five washings with sterile saline followed (5) five hundred units
of penicillin and 0.5 gms. of streptomycin were added per cc. of larval
suspension (6) this was then either ground with a tissue grinder, or
incubated in the immune serum or saline.
Taliaferro and Sarles (1939) believed the secretions and excretions
of the worm and mechanical damage were the effective inflammatory stim­
uli rather than the worm itself#

These authors believed the precipitins

73
producing the precipitate of Sarles (1938) were in response to these
secretions and excretions.

The work of Thorson (1951) also seems to

indicate antibodies are formed in response to the excretions and secre­
tions of the worm.

As a result of this previous work, active filariform

larvae were incubated at room temperature in immune serum for six days
(Absorption Ho. I).

Ina s much as samples of the same serum were used

for analysis both prior and following absorption, removal of any com­
ponent would be reflected as a change in the albumin/globulin ratio*
Thus, an increase in the A/G ratio following exposure of antigen to
serum would indicate absorption of globulin.
tion experiments are found in Table XII.

The results of the absorp­

Ho significant change in the

A/G ratio was observed as a result of absorption*
Absorption experiment II was a duplication and expansion of ab­
sorption I.

Immune serum was analysed electrophoretically at the time

of bleeding, and after exposure to living larvae for three days.

By

comparing the A/G ratios (Table XII) of immune serum at the time of
bleeding and after six days incubation at room temperature without
larvae, it was evident little alteration occurred in relation to this
time interval.

A comparison of the A/G ratios of three day old immune

serum and immune serum in which larvae were incubated for three days,
showed no significant change*
Absorption experiment III was a duplication of absorption I with
certain additions namely;

normal serum was analysed before and after

exposure to living larvae; and immune serum was analysed before and
after exposure to saline in which larvae had been incubated one week.

7^
This absorption experiment netted the following results!

(1) no change

in the A/G ratio was observed when normal serum was exposed to living
larvae for six days (2) exposure to living larvae failed to absorb any
protein component from immune serum in a six day incubation period*
(3) a larvae free saline extract, in which larvae had previously been
incubated for one week, failed to absorb any protein component from
immune serum*
The results of Absorption IV are not included, because addition
of veronal buffer to the serum after exposure to the antigen resulted
in precipitation of much of the albumin (observed electrophoretically).
In Absorption V immune serum was exposed to ground larvae and
analysis was in veronal buffer pH 8*6 and phosphate buffer
The addition of the phosphate buffer of pH 7»^

pH 7*^*

the serum after ex­

posure to ground larvae resulted in precipitation of protein while on
this occasion no precipitation occurred on addition of veronal buffer
of pH S. 6. Only the results of serum analysed in veronal buffer are
included in Table XII.
Summarizing the results of the absorption experiments (Table XII),
no significant change in the A/G ratio was observed following exposure
of immune and normal sera to the various larval preparations*
for the defined conditions no absorption of antibody occurred*

Thus,

75

TABLE XII.
Ah sorp­
tion
Exp. No#

RESULTS OF THE ABSORPTION EXPERIMENTS

♦Serum Age (Days)
Before
After
Absorp­
Absorp­
tion
tion

Antigen Used

A/G Ratio
Before
After
Absorp­
Absorp­
tion
tion

I-Immune

1

7

living larvae

0 .5 8

0 .6 6

1 1 - Immune

1
6

mm

Immune

none
living larva©

0 .7 7
0 .7 0

O .5 6

IIl-Jfcrmal
Immune
Immune

1
2
2

10
10
2

living larvae
living larvae
saline extract

1 .5 s
0 .6 1
0 .6 1

0 .6 9

V- Immune

3

2

ground larvae

0.72

0 .6 5

6

1 .6 2

o*6h

♦These figures represent the age of the serum when determinations
were made*

DISCUSSION

Qualitative or quantitative alterations of the serum components
are often encountered in diseased animals.

Qualitative changes are

manifested in electrophoretic patterns as new or altered peaks which
have specific mobilities.

Seibert and Nelson (1942) reported that

the first serum protein changes in tuberculous rabbits were a rise in
the alpha globulin fraction and the appearance of an unknown component
designated as the ’’X” component.
Kabat (1939)

The ”TH component of Tiselius and

Van der Scheer (1941) is an example of a new peak ap­

pearing in the electrophoresis patterns as immunization increases.

On

the other hand, numerous workers J^edwich and Record (1940)» Bjornboe
(194*0 , G-jessing and Chanutin (1947&)7 have shown quantitative increases
in various serum components associated with the diseased condition*
In the present investigation no new serum components were de­
tected in the infected animals.

However, significant increases in the

globulin fraction were consistently observed in the latter stages of
hyperimmunization (after the 5,000 larvae injection).

Increase in

globulin with a decrease in albumin appears to be the characteristic
serum alteration in many diseases.
by:

The rise in globulin may be caused

(1) the possible formation of antibodies (2) alteration of the

relative production and utilization of albumin and globulin, or (3) a
compensatory rise in an attempt to maintain osmotic pressure (Martin
1946).

77
Some of the more pertinent references which support the above men­
tioned causes of globulin increase will be discussed*
Tiselius and Kabat (1939) showed conclusively that the gamma
globulin increase was due to antibody formation.

These authors removed

the increased gamma component of immune serum by exposure of the serum
to the specific antigen.

Other workers have established relationships

of antibody to plasma proteins*

Wyckoff and Hhian (1945) Seibert and

Nelson (1943), Bj^frnboe (1944), Boyd and Bernard (1937)*> Heidelberger
(1939) proposed that antibodies are serum globulins modified in response
to the presence of an antigen*
Chow et al. (1948) found in dogs depleted of protein by a proteinfree diet and by plasmapheresis, that albumin and gamma globulin were
decreased, whereas, alpha and "other globulins" remained unchanged.
Zeldis at al* (1945) showed, following plasmapheresis, that both
alpha and beta globulins continued relatively elevated during the early
portion of the recovery period.

They suggested a rapid formation of

these components from tissue reserves as well as a rapid synthesis
from dietary protein*
A compensatory mechanism may be in operation which results in the
maintenance of constant serum osmotic pressure.

Cohn (1942) states,

"Although albumin comprises but 60 per cent of the plasma proteins, it
accounts for approximately 80 per cent of the osmotic pressure."

Since

osmotic pressure depends upon the number of particles and not upon their
weight,

it has been proposed that the globulin concentration compensates

for any decrease in albumin.

Bj^rnboe (1944) suggested there was a

7S
relation between the albumin and globulin concentrations which kept
the colloid osmotic pressure constant or varying only within certain
limits*
For the conditions as defined in this investigation, no absorption
of antibody was detected when immune serum was exposed to the various
larval preparations*

The absorption studies presented here have not

necessarily exhausted all the possible conditions for maximum antigenantibody combination*

If the increased beta peak of the immune serum

could be reduced by exposure of the serum to some larval preparation,
it would be highly indicative of an antigen-antibody combination and
would tend to establish the beta globulin increase as due to antibody
formation.

This was not established*

An increase in beta globulin

in the diseased animal has been noted by other workers ^Seibert and
Nelson (1942), Bj^rnboe (1944), Stern and Reiner (1946), and Martin
(1946)7*

Fell et al* (1940), Enders (1944) and Kekwick and Record

(1940), have demonstrated that some antibody is formed in globulins
other than the gamma fraction*

In fact, the latter authors found that

immunization with diphtheria toxoid leads to the formation of two anti­
toxins which are associated with the beta and gamma globulin fractions
respectively.

From the above references, it seems plausible that anti­

body can also be associated with the beta globulin fraction.

Before

one excludes the increase as due to antibody production, a thorough
study of the relative amounts of immune serum and antigen necessary for
a antigen-antibody reaction to occur would be necessary.

This would

tend to eliminate possible negative results due to zone effects.

79
Furthermore, the electrophoretic method used was not capable of resolving
protein concentrations much “below 0.002 percent in the presence of and
relative to another protein in a concentration of approximately two
percent.

Therefore, absorption of antibody at a concentration below

this limit would not be detected.

Thus, the antibody absorption phase

of the problem awaits further study and perhaps refinement of technical
methods.
It does not seem likely that the rise in beta globulin noted could
be attributed to an alteration of the relative production and utiliza­
tion of albumin and globulin.

Zeldis et al. (1945) found, following

plasmapheresis, all plasma proteins entered the blood stream even when
food was not given during the first 24 hours after depletion.

In addi­

tion these authors noted that both alpha and beta globulins continued
relatively elevated during the early portion of the recovery period.
This suggested a rapid formation of these two globulins from tissue
reserves as well as a rapid synthesis from dietary protein.

To achieve

these results removal of from 50 to 190 percent of the blood volume and
its continuous replacement with a non-protein physiological solution
and the washed cells was necessary.

It would appear that any condition

which resulted in an extensive and prolonged loss of plasma protein
might reflect itself as a beta globulin increase.

During the migration

of the larvae through the lungs, there is capillary damage and hemor­
rhage.

It seems doubtful, however, that the amount of serum lost, as

a result of larval damage, reaches the proportions of plasmapheresis.
In addition, four to seven days after the last larval injection when

so
the larvae are most active in the lung and when serum protein loss
would he the greatest, the total protein was not decreased, but showed
a significant increase*
was minimal.

This would seem to indicate that plasma loss

The histological studies of Taliaferro and Sarles (1939)

would lead one to believe that blood loss due to larval damage is not of
the proportions encountered in plasmapheresis*

The acceptance of these

facts tends to rule out the observed serum changes as due to an altera­
tion of the relative production and utilization of the serum proteins*
Since the animals in the present work were subjected to only one
bleeding for analysis, changes in serum patterns due to blood depletion
can be eliminated.

Dimopoullos (1952) showed in chickens, which were

bled weekly and from which one third of the total blood volume was re­
moved, that the albumin/globulin ratio decreased steadily.
Thus, from the information available at the present time, one must
conclude that the increase of globulin in the present work was due to
injury or shock by the larvae to the host.

The injury to the host was

considerable when one considers the large numbers of larvae that had
the opportunity of migration through the tissues.

The data concernix^

lung enlargement reported in the "Results11 section indicates that con­
siderable pathology existed.

It is entirely possible that the observed

globulin increase was from a reaction by the host to the disturbance in
the lungs produced by the larvae and that this increase for the most
part was not due to antibody production.
Further evidence in favor of the globulin increase being due to
trauma from the larvae and not antibody production, lies in the fact

SI
that early in the hyperimmunization process little increase in globulin
was noted.

Yet these animals at the same time exhibited strong immun­

ity as demonstrated by the decrease of the parasites* egg production.

References that follow substantiate the fact that serum components
are altered due to shock or injury.
Moore and Fox (1950) demonstrated that mice traumatized by tourn­
iquet for two hours produced a large quantity of a serum protein com­
ponent having a mobility similar to gamma globulin.
Ferlmann et^ al. (1943) compared electrophoretic patterns of normal
serum from calves with those obtained after the animals were subjected
to burns.

The serum of the burned animals showed a slight decrease in

the albumin/globulin ratio with an increase in the alpha globulin
fraction*
Grjessing and Chanutin (1947a) dipped one-third of the clipped body
of rats into hot water at 75°C for *10 seconds.

The serum changes were

an increase in the alpha and beta globulins and decreases of albumin
and gamma globulin.
Grjessing et al. (1947b), using animals injured with sulfur mustard,
turpentine, and heat, found an increase in the percentage distribution
of the alpha globulins and a decrease in the albumin.

The lipid combined

with alpha and beta globulins also increased in injured animals.

How­

ever, Grjessing et, al. (194S) reported severe injury in goats produced
relatively small changes in the distribution of protein components of
whole serum or its fractions.
Leutscher (1947) states that electrophoretic patterns have proved
to be characteristic not of the specific disease but of the host*s

82
reaction to infection or injury*

The various changes are frequently

proportional to the severity of the physiological disturbance and may
vary with the duration or stage of the disease, with nutritional factors,
with loss of plasma proteins, and with involvement of certain organs
such as the liver.

An examination of the results obtained in the pres­

ent work reveals that serum and lung changes are, indeed, proportional
to the number of larvae injected which in turn is probably dependent
on the severity of the physiological disturbance*

That a specific

organ was involved in the disturbance.was demonstrated by the increased
lung/body weight ratio and microscopical changes in the lung*
Before one attributes the larval activity in the lung as solely
producing the traumatic response, one must consider the lung as a pos­
sible site of antibody formation.
phovitellin containing

Banks e£ al* (19^8 ) noted when phos-

^as injected into rabbits, that it was rapidly

bound by the liver and the lung.

Haurowitz (1950) believes this find­

ing supports the view that these organs are fundamentally involved in
the process of antibody formation.

The fact that large numbers of

lymphocytes are found in lungs of the infected animals can not be com­
pletely ignored.

Antibodies have been extracted from lymphatic cells

and it has been proposed that a portion of the antibodies arise in these
cells /White and Dougherty (19^6), Ehrich and Harris ( 1 9 ^ 2 Talia­
ferro and Sarles (1939) point out that one of the differences between
the immune rat and the initially infected rat is that in the former
many worms do not reach the intestine but are retained in the sxin and
lungs.

This would seem to indicate that these stations (skin and lungs)

S3
are actively involved in the resistance displayed by the immune rat*
Prom the foregoing information one might conclude that if antibody is
involved, the lungs may be the site of antibody production or be one
of the locations wherein the antigen-antibody combination occurs.

It

would, in view of the great response found, seem unlikely that the
beta globulin increase was due to antibody formation by the lungs*

A

more tenable explanation would be to attribute the globulin increase
to trauma due to the massive larval doses whereby the lung is the focus
of maximum shock.

If the trauma explanation is conceded, the existence

of an antibody still cannot be eliminated.

It is conceivable that the

antibody is present in the serum in minute quantities which cannot be
detected by the limited rathods used herein, or on the other hand the
antibody may be masked or incorporated in the greatly increased beta
globulin peak.

The answer to these questions await further study*

One might profitably utilize the electrophoresis-convection appara­
tus in establishing the presence of an antibody.

Cann at al. (l9*+9)

separated bovine gamma globulin into eight fractions using this appar­
atus.

It might be possible with the aid of this apparatus to obtain a

beta globulin subfraction which might be predominantly antibody.
sorption studies would thereby be rendered less complicated.

Ab­

SUMMARY

1*

Electrophoretic and salt fractionation determinations on nomal

and nematode immune rat sera were performed*
2*

Utilizing bovine albumin as a standard, a new modification for

determining total serum protein of rat sera by the biuret reaction was
developed* ,
3*

A new modification for fractionating rat sera into albumin and

globulin, utilizing a 24 percent sodium sulfite solution, was devised.
4.

The hyperimmunization process produced a slight decrease in

body weight when compared with the uninfected controls*
5*

The lung weight when converted to percent of total body

weight was found to increase as much as six fold in the infected ani­
mal when compared with the control.

This lung involvement is consider­

ed as the traumatic center which produces, at least for the most part,
the observed serum changes*
b.

Living larvae, saline extracts of living larvae, and ground

larvae, were used as antigens and exposed to immune sera.

Antigen-

antibody combination, as indicated by the removal of a serum fraction,
was not observed.
7.

Rats were injected with increasing numbers of N. muris larvae

at two week intervals.
between the injections.

Serum determinations were made on various days
The following serum changes were noted in the

infected animals when compared with .uninfected litter mates:
(a)

The total protein was increased as the number of larvae
\

injected was increased.

The beta and total globulin increased as the number of
larvae injected increased, and diminished when the num­
ber of larvae injected was reduced, or when the larval
injections were stopped.
In five out of six intervals between injections the bets
globulin of the infected animals was slightly higher in
two weeks than one week after each injection.

The beta

globulin increase was electrophoretically analysed in
veronal buffer at pH 3.6 and phosphate buffer pH 7*^*
This minimized any anomalous effects that might occur
at a particular pH or with a particular buffer.
The albumin/globulin ratio decreased as the number of
larvae injected was increased and also increased when
the larval injections were halted.
When rats were injected with 1,000; 2,000; 5*000; 10,000;

20,000; and 50,000 larvae at two week intervals, the most
striking changes occurred seven days after the 50,000
larval level.
The gamma globulin showed no significant increase or
decrease.
The mobilities of the protein components were decreased
in the majority of infected serum samples*
Serum from rats, which had received injections of the
supernatant of the larval suspension, was essentially
normal.

This indicated the material in the inoculum

86
other than the larvae did not cause the observed serum
protein changes*
These serum, changes are interpreted as responses of the host
injury or trauma by migrating larvae, although, the possible existence
of an antibody can not be ruled out.

Since rats were bled only once

during the course of the experiments, serug depletion changes were
eliminated*

BIBLIOGRAPHY

Africa, C

1931

M.

- Studies on the host relations of Hippostrongylus muris. with
special reference to age resistance and acquired immunity.
J. Parasitol. 18;1-13*

Armstrong

19^7

-

S. H., Jr., Budka, M. J. E . , and Morrison, K. C.
Preparation and properties of serum and plasma proteins; XI.
Quantitative interpretation of electrophoretic schlieren
diagrams of normal human plasma proteins.
J. Am. Chem. Soc., 69:^16-*4-29*

Banks, T. , Boursnell, J. C., Dewey, H. M . , Francis, G-. E . , Tupper, R*,
Wormall
19US - The use of radioactive isotopes in immunological investigations.
Bio chem. J. *4-3;518-523 .
Benditt, !3. P., Wessler, R. W., Woolridge, R. L*, Rowley, D. A.,
Steffee, C. H.
Loss
of body protein and antibody production by rats on low
l9*+9 protein diets*
Proc. Soc. Exper. Biol, and Med. 70J24O-21+3*
Boyd, W.

1937

)•, and Bernard, H.
- Quantitative changes in antibodies and globulin fractions in
sera of rabbits injected with several antigens*
J. Immunol. 33:111-122.

Brandt, L , W., Clegg, R* E., Andrews, A. C*
1951 - The effect of age and degree of maturity on the serum proteins
of the chicken*
J. Biol. Chem. 191:105-111.
Bj^rnboe, M.
Serum proteins during immunization*
Acta Pathol. Microbiol* Scand. 20;221-239*
Ibid.

19^6

-

Studies on the serum proteins in Hepatitis.
I* The relation between serum albumin and serum globulin.
Acta Med. Scand. 123:393-^1*

Campbell, W. R . , Hanna, M. I.
1937 - The albumin, globulins, and fibrinogen of serum and plasma.
J. Biol. Chem. 119;15-33.

S7
Cann, J, H . , Brown, R. A., Kirkwood, J. .G-.

1949

_

Application of electrophoresis convection to the fractionation
of bovine gamma^-globulin.
J. Biol. Chem. 181:161-170.

Chandler, A. C.
1932 - Experiments on resistance of rats to superinfection with the
nematode, Hippostrongylus muris.
Am. J. Hyg. 16(3):750-782.
Ibid.

1935a

Studies on the nature of immunity of intestinal helminths.
I. The local nature of the immunity of white rats to Hippostrongylus infection.
Am. J. Hyg. 22(1) :157-168.

Ibid.

1935b

Studies on the nature of immunity to intestinal infections.
II. A study of the correlations between degree of resistance
of white rats to Hippostrongylus and interval between infec­
tions.
Am. J. Hyg. 22(1) :243-256.

IMd.

1936

- Studies on the nature of immunity to intestinal helminths.
III. Renewal of growth and egg production in Hippo st rongylus
after transfer from immune to non-immune rats.
Am. J. Hyg. 23(l):46-54*

Chow, B. J.
The determination of plasma or serum albumin by means of
19^7
precipitin reaction.
J. Biol. Chem. 167:757-763*
Chow, B. J., Seeley, R. D . , Allison, J. B., Cole, 17. H.
> The effect of repletion on the plasma proteins in the dog
1948
measured by electrophoretic analysis.
Arch. Biochem. 16:69-7^*
Chow, B. P.
■ The electrophoretic studies on the effect of protein deple­
1946
tion on plasma proteins and the regeneration of plasma proteins
after oral administration of hydrolysates prepared from casein
and lactoalbumin.
Ann. H. Y. Acad. Sci. 47:297-316.
Chow, B. P., Allison, J. B . , Cole, 17.
■ Effect of protein depletion
19%
measured by electrophoretic
Proc. Soc. Exper. Biol, and

H., Seeley, R. D.
on plasma proteins in the dog
analysis.
Med. 60:14-17.

88
Cohen, P. P., Thompson, F. L*
1948 - A comparative study of micro- and macro electrophoretic anal­
ysis of human and rat serum*
J. Lab* and Clin* Med. 335 75-80.
Cohn, E. J.
1942 - The plasma proteins: their properties and functions.
Trans. & Studies Coll. Physicians, Philadelphia 10:1^9.
Coh$, C., Wolf son,
19^7 - Studies
albumin
J. Lab.

W. Q.
in serum proteins. I. The chemical estimation of
and of the globulin fractions in serum.
and Clin. Med. 32:120,3-1207*

Cooper, 0. H., Rein, C. R., Beard, J. W.
19^6 - Electrophoretic analysis of Kala-azar human serum.
Hypergamma-globulinemia associated with seronegative reactions
for syphilis.
Proc. Soc. Exper. Biol, and Med. 6l:179-183•
Dimopoullos, G. T.
1952 - Electrophoretic and serum neutralization studies of sera from
chickens exposed to infectious bronchitis virus.
Unpublished Ph.D. Thesis. Michigan State College, 93 numb,
leaves, 35 figures.
Dole, Y. P., Emerson, K. E.
1945 - Electrophoretic changes in the plasma protein patterns of
patients with relapsing malaria.
J. Clin. Invest. 24:6^i4-b^7*
Ehrich, W. E., Harris, T. R.
1942 - The formation of antibodies in the popliteal lymph node in
rabbits.
J. Exper. Med. 76 5335-3^*
Enders, J. F.
1^44 _ Chemical, clinical, and immunological studies on the products
of human plasma fractionation. X. The concentration of cer­
tain antibodies in globulin fractions derived from human
blood plasma*
J. Clin. Invest. 23:510-530*
Fell, R., Stern, K. G., Goghill, R. D.
1940 - A physical-chemical study of normal and immune horse serum.
J. Immunol. 395 223-2*4-6.
Folin, 0., Wu, H.
1919 - A system of blood analysis.
J. Biol. Chem. 38:81-110.

89
Gradwohl, R. B. H.
19^3 - Clinical Laboratory Methods and Diagnosis.
Third Ed., C. V. Mosby Co., St. Louis
Vol. X. 215-217.
Graham, G. L.
193^ - Resistance Studies with the nematode, Hippostrongylus muris.
in laboratory rats*
Am. J . Hyg. 20(2):352-372.
Guessing, E. C., Chanutin, A.
19*+7a - An electrophoretic study of plasma and plasma fractions of
normal and injured rats*
J. Biol. Chem. 169:657-665.
Gjessing, E. C., Ludewig, S., Chanutin, A.
19^7^ - Fractionation, electrophoresis, and chemical studies of pro­
teins in sera of control and injured dogs.
J. Biol. Chem. 170:551-569*
Ibid.
1948

- Fractionation, electrophoresis, and chemical studies of pro­
teins in sera of control and injured goats.
J. Biol. Chem. 174:683-696

Hardt, C. R.
1943 - Operation and Application of Tiselius Electrophoresis appara­
tus.
Unpublished Ph.D. thesis. Michigan State College. 129 numh
leaves, 101 figures.
Haurowitz, F.
1950 — Chemistry and Biology of Proteins. First Ed.
Academic Press, Inc., Hew York, pp. 287.
Heidelberger, M.
1939 - Chemical aspects of the precipitin and agglutination reactions.
Chem. Rev. 24:323-3^3*
Howe, P. E.
1921 - The use of sodium sulfate as the globulin precipitant in the
determination of proteins in blood.
J. Biol. Chem. 49:93-108.
Ikejiani, 0*
1946 - Studies in Trypanosomiasis. I. The plasma proteins and
sedimentation rates of erythrocytes of rats infected with
pathogenic trypanosomes.
J. Parisitol. 32:369-373*

90

Jager, B* V*, Schwartz, T. B*, Smith, E. L., Nickerson, M., Brown, D. M.
1950 - Comparative electrophoretic and chemical estimations of human
serum albumin; an evaluation of six methods.
J. Lab. and Clin. Med. 35*7€>—S •
Kekwick, R. A., Record, B. R.
19^0 - Some physical properties of diphtheria antitoxic horse sera*
Brit. J. Exper. Path. 22j29-44.
Longsworth, L. G>
1942 - Recent advances in the study of proteins by electrophoresis.
Chem. Rev. 30:323-339*
Longsworth, L. G., Mac Innes, D. A.
19^0 - The interpretation of simple electrophoretic patterns.
J. Amer. Chem. Soc. 62:705-711*
Longsworth, L. G., Shedlovsky, L.t Mac Innes, D. A*
1939 - Electrophoretic patterns of normal and pathological human
blood serum and plasma*
J. Exper. Med. 70:399-413*
Luetscher, J. A., Jr.
1947
Biological and medical applications of electrophores
Physiol. Rev. 27:621-642.
Lysenko, M. Gr*
1951 — Concerning salicylate inhibition of ablastic activity in
Trypanosoma lewis! infection*
J. Parasitol. 371535-5^*
Martin, N. H.
ig46 - The components of the serum proteins in infective hepatites
and in homologous serum jaundice. An electrophoretic study*
Brit. J. Exper. Path. 27:363-368*
Milne, J.
1947 - Serum protein fractionation: A comparison of sodium sulfate
precipitation and electrophoresis.
J. Biol. Chem. 169:595-599*
McMaster, P. D.» Hudack, S. S.
1935 - The formation of agglutinins within lymph nodes.
J. Exper. Med. 61:7^3-^05*
Moore, D. H.
1945 - Species differences in serum patterns.
J. Biol. Chem. l6l:21-32*

91
Moore, D. H., Fox, C. L.
1950 — Correlation of electrophoretic studies and other factors in
the syndrome of secondary shock*
Nature lb5:872-876.
Moore,
1944

D. H., Levin, L. and Leathern, J. H.
— The
alpha globulin fraction of the serumofnormaland
hypophysectomized rats*
J. Biol. Chem. 153:349-353.

Moore, D. H. , Levin, L* and Smelser, G-. K.
19^5& - Electrophoretic and salt fractionation of the serum proteins
of normal and hypothyroid rats*
J. Biol. Chem. 157:723-730.
Moore*
19U9

D. H.,Roberts, J. B., Salonberger, L. W.
- Factors influencing the electrophoreticanalysis of human
serum.
J. Biol. Chem. 180:1147-1158.

Moore, D. H., Shen, S. C., Alexander, C. S.
1945b - The plasma of developing chick and pigembryos.
Proc. Soc. Exper. Biol, and Med. 58i307-310Perlmann, G-. E., Glenn, W. W. L., Kaufman, B.
1943 - Changes in the Electrophoretic pattern in lymph and serum in
experimental burns.
J* Clin. Invest. 22;627-633*
Petermann, M. L., Young, N. F., Eoagness, K. R.,Rhoads, C. P.
1947 - The determination of plasma albumin by chemicaland electro­
phoretic methods.
Proc. Amer. Federation Clin. Research 3:75*
Pillemer, L., Hutchinson, M. C.
1945 - The determination of the albumin and globulin contents of
human serum by methanol precipitation.
J. Biol. Chem. 15S;299-301.
Popjak, G., McCarthy, E. F.
1946 — Osmotic pressures of experimental and human lipaemic sera.
Evaluation of albumin-globulin ratios with the aid of electro­
phoresis.
Biochem. J. 40;789-803*
Sarles, Merritt P., Taliaferro, W. H*
1936 - The local points of defense and the passive transfer of ac­
quired immunity to Hippostrongylus muris in rats.
J. Infect. Bis. 59:207-220.

92
Sarles, M. P.
1935 - The iii vitro action of immune rat serum on the nematodey
Hippostrongylus muris.
J. Infect. Dis. 62; 337- 3118.
Ibid.,
1939

- Protective and curative action of immune serum against
Hippostrongylus muris in the rat.
J. Infect. Dis. 65:183-195.

Schwartz, B . , Alicata, J. E . , Lucker, J. T.
1931 - Resistance of rats to superinfection with a nematode,
Hippostrongylus muris, and an apparently similar resistance
of horses to superinfection with nematodes.
J. Wash. Acad. Sc. 21(12) :259-261.
Seibert, F. B., Helson, J. W.
19^2 - Electrophoretic study of hlood protein response in tuberculosis.
J. Biol. Chem. 143:29-38.
Ibid.,

1943

- Proteins of tuberculin.
J. Amer. Chem. Soc. 65:272-278.

Spindler, L. A.
1936 - Resistance of rats to infection with Hippostrongylus muris
following administration of the worms by duodenal tube.
Am. J. Hyg. 23(2):237-242.
Stern, K. G-., Reiner, M.
1946 - Electrophoresis in medicine.
Yale J. Biol. Med. 19:67-99*
Taliaferro, W. H., Sarles, M. P.
1939 - The cellular reactions in the skin, lungs and intestine of
normal and immune rats after infection with Hippostrongylus
muris.
J. Infect. Dis. 64:157-192.
Thorson, R. E.
1951 - The relation of the secretions and excretions of the larvae
of Hippostrongylus muris to the production of protective
antibodies.
J. Parasitol. Suppl. Vol. 37* No. 5* Sec. 2, pp. 18.
Tiselius, A., Kabat, E. A.
1939 - An electrophoretic study of immune sera and purified antibody
preparations.
J. Exper. Med. 69:119-131*

93
van der Scheer, J. Wyckoff, W. G., Clarke, J. H.

194-0 -

Ihid.
19Ul -

The electrophoretic analysis of several hyperimmune horse
sera.
J. Immunol. 39*65-71* *
The electrophoretic analysis of tetanal antitoxic horse sera.
J. Immunol. 40:173-177*

White, A . , Dougherty, T. F.
1946 - The role of lymphocytes in normal and immune globulin produc­
tion, and the mode of release of globulin from lymphocyles*
Ann. N. Y. Acad. Sci. 46:859-882.
Wolfson, W. Q . , Cohn, C., Calvary, E . , Ichiba, F*
194s - Studies in serum proteins. V. A rapid procedure for the estima­

tion of total protein, true albumin, total globulin, alpha
globulin, beta globulin and gamma globulin in 1.0 ml. of serum.
A. J. Clin. Pathol. 18:723-730*
Wyckoff, W. G., Rhian, M.

19^5 -

An. electrophoretic study of an anti-influenzal horse serum*
J. Immunol. 515359-363*

Yokogawa, S*
1922 -

The development of Heligmosomum muris Yokogawa, a nematode
from the intestine of the wild rat.

Parasitology l4(2):127-l66.
Ibid.

1920 -

A new nematode from the rat*
J. Parasitol* 7*29-33*

Zeldis, L. J., Ailing, E. L., McCoord, A. B., Kulka, J. P.
I5J+5 - Plasma protein metabolism-Electrophoretic studies-Restoration
of circulating proteins following acute depletion by plasma­
pheresis.
J. Exper. Med. 81:515-537*