University of Southern California Dissertations and Theses

Chemical and immunological studies on a heterophile antigen extracted from bovine erythrocyte stroma

(USC Thesis Other)

Chemical and immunological studies on a heterophile antigen extracted from bovine erythrocyte stroma

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content CHEMICAL AHD IMMUNOLOGICAL STUDIES ON A HETEROPHIEE
ANTIGEN EXTRACTED PROM BOVINE ERYTHROCYTE STROMA
) A Dissertation
i Presented to
I
j the Faculty of the Graduate School
I University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
by
Abraham Markowitz
July 1952
This dissertation, w ritten by
....... Abra^Wm Markowitz...........
under the guidance of h%P....Facuity Committee
on Studies, and approved by a ll its members, has
been presented to and accepted by the C ouncil
on Graduate Study and Research, in p a rtia l f u l
fillm e n t of requirements fo r the degree of
D O C T O R O F P H I L O S O P H Y
D e an
C om m ittee on Studies
- f /C h a irm a n
..
ACKNOWrEDaEMBNTS
Grateful appreciation due to all the many friends
the author has amongst the graduate students of the Depart
ment of Biochemistry for their kind consideration in use
of equipment and materials is acknowledged.
Special thanks are acknowledged to Mr, Joseph
Michelson for advice and equipment in many phases of the
problem; to Mr, Alfred Williams, for technical advice and
assistance in the phosphorus determinations and Mr, Burt
Kallman for technical advice and. assistance in the electro
phoretic determinations.
To Mr, Joe A, Lazaroni Jr.» Department of Medical
Microbiology for his innumerable expressions of interest
in the problem, both actively and implied, special acknow
ledgement is accorded.
To the management of Gudahy Packing Company, Los
Angeles, California,thanks, is acknowledged for facilita
ting an ever ready supply of bovine and sheep erythrocytes.
Lastly, to Dr, Wayne L, Simmonds, Department of
Medical Microbiology, sincere appreciation is acknowledged
for thoughtful, considerate and constructive advice from
the inception to the completion of the problem.
TABIE OP GOIsTTENTS
CHAPTER PAGE
I. THE PROBLEM AOT DEFINITIONS OF TERMS USED . . . 1
The problem.......... 1
Statement of the problem ................. 2
Importance of the study . . . . . . . 2
Definitions of terms used .... ..... 3
Heterophile . . . ............... ..... 3
Forssman antigen . ................... 4
Mononucleosis antigen or M-antigen . • . . • 4
Serum sickness antigen or SS-antigen .... 4
Antigen .................................... 4
Haptene.......... «... 5
II. REVIEW OF THE LITERATURE ............ 6
III. PREPARATION AND TESTING. OF M-ANTIGEN. . . .... 10
Preparation of stroma ................. 10
Extraction of M-antigen from stroma ..... 13
Determination of extract concentration . . . . 15
Determination of extract activity ...... 15
Results . . . . . . . . . . . . . . . . 18
IV. CHEMICAL ANALYSIS OF M-ANTIGEN . ........ 22
Materials and methods . . . . . . . . 22
I. Nitrogen .. ......... . . . . . . . 22
II. Phosphorus................ 22
CHAPTER page ;
III. Iron................................ 22|
IV è Sodium and potassium «••••••• 22
V, Carbon and hydrogen.............. 25;
VI* Total carbohydrate . . . . . . . . 25
VII. Lipid determinations............... 24;
VIII# Enzyme digestions ........ 24
IX# Filter paper electrophoresis . . . . 26
X. Filter paper chromatography of acid
hydrolysates . . . . . . . . . 27
Results . . . . . . . . . . . . . . . . . 29
V. IMMUNOLOGY AND SEROLOGY..................... 58
Adsorption experiments . . . . . . . . . . . 58'
A. Dowex-2 colloidal particles . . . . • 59,
B. Dowex-1 colloidal particles ..... 4o;
C.
Bentonite .. .
4o|
D. Kaolin ..........
4l'
1
E.
Alumina adsorption (Fisher)......... 41
F. Norite and carbon black ....... 41
G. Ferric hydroxide , 42
Results 42
I
Absorption experiments with M-antigen and sera
containing heterophile antibodies .... 45
Methods and materials . . . . . . . . . . . 45
Results . . . . . . . . . . . . . . . . 44
I Vi
CHAPTER page|
VI. ..................................... 52
VII. SDMHART ................ 59;
(
61
LIST OF TABLES
TABLE PAGE
I. Dry Weight of M-antigen per Milliliter of
Alcohol ............ 17
II. Protocol for the Assay of M-antigen by use of
the Hemagglutination Inhibition Reaction . . 19
III. potency of M-antigen Extracts as Determined by
Hemagglutination Inhibition Reaction .... 21
IV. Element and Component Analysis of M-antigen on
Undialysed, Lipid Containing Samples .... 30
V. Percentage composition of M-antigen as Calcu
lated on the Basis of Ash Free Material . . . 31
VI. Effect of Enzymatic Hydrolysis of M-antigen as
Determined by the Hemagglutination Inhibition
Reaction . . . . . . . . . . . . . . 55
VII. Filter Paper Chromatography of M-antigen Hydro-
lysates with Regard to Reference Amino Acids. 35
VIII. Protocol for Standard Sheep Cell Agglutination
Reaction . . . . . . . . . . . . . . . . 45
IX . Hemagglut ina t ion T iter s of Inf ec tious Mononuc le -
osis Sera Before and After Absorption with
M-antigen . . . . . . . . . . . . . . 46
X. Hemagglutination Titers of Sera Showing Hetero
phile Antibodies Before and After Absorption
_________ with-M^antigen__. . . . . . .................. 50
LIST OF FIGURES
FIGURE PAGE
1* Protocol on Preparation of Stroma ••••••• 12
2* Protocol on Extraction of M-antigen ...... 16
3. Two dimensional Chromatograms of M-antigen Acid
Hydrolysates . ........... . .. . . . 56
CHAPTER I
THE PROBIEM AND DEFINITION OP TERMS USED
The hemagglutination of sheep erythrocytes can he
brought about by so-called ''normal serum, " infectious
mononucleosis serum and serum sickness serum. As the
situation exists today an agglutination titer against
sheep erythrocytes is not sufficient to differentiate the
antibodies in the three conditions. In order to bring
about such a differentiation it is necessary to follow the
sheep erythrocyte reaction with an absorption analysis
pattern utilizing guinea pig kidney macerate and boiled
bovine erythrocytes as the absorbing agents. The normal
serum hemagglutinins will be completely absorbed by the
guinea pig kidney macerate but are not affected by the
boiled bovine erythrocytes. The serum sickness hemagglu
tinins will, be completely absorbed by the guinea pig kidney
macerate and partially absorbed by the boiled bovine
erythrocytes. The infectious mononucleosis hemagglutinins
will be only slightly absorbed by the guinea pig kidney
macerate and completely absorbed by the boiled bovine
erythrocytes.
The hemagglutination of both bovine and sheep
erythrocytes by infectious mononucleosis serum leads to
2
the conclusion that a similar or identical antigen is
shared by these erythrocytes. Extraction of this antigen
in an Immunologically pure state would make possible the
utilization of this substance in a specific serologic re
action.
I. THE PROBLEM
Statement of the problem. It was the purpose of
this study to extract the mononucleosis antigen from bovine
erythrocyte stroma; (2) to chemically characterize this
antigen; (3) to adsorb this antigen onto a serologically
inert material for utilization in an agglutination re
action; and (4) to utilize this antigen as. a substitute
for the guinea, pig kidney and boiled bovine erythrocytes
in the differential absorption test for infectious mono
nucleosis.
Importance. of the study. The diagnosis of in
fectious mononucleosis is rarely, made, without a serological
examination of the. heterophile antibodies present in the
patient's serum.
The present confusion as to the.significance of a
sheep erythrocyte hemagglutination, titer is partially
ameliorated by the differential abaorption test outlined
in the introduction. However, the differential test
■ -------------------- 5
supplies, at best, a negative answer. In this study the
problem was approached from the viewpoint of obtaining a
positive answer. It had been shown that the heterophile
response in infectious mononucleosis is unique and that a
serologic reaction which would involve a specific system
would be a definite aid in the diagnosis.
Aside from the practical importance, the problem
j presents the more intriguing académie considerations of
I heterophile antigen clarification. The antigen under
I examination has been called Porssman-like and non-Porssman
I without regard to the significance of such terminology, |
Many workers use the terms Porssman antigen and heterophilei
; I
; antigen synonymously. This practice is to be decried sincej
I !
I the Porssman antigen is just one of the group of hetero- <
I I
phi le antigens. The mononucleosis, antigen Is itself a |
I heterophile antigen, quite distinct by all criteria from j
I the Porssman antigen. Therefore, terminology such as I
I
I Por3sman-like and non-Porssman are entirely without signif-i
I I
I icance In an immunologic sense. j
i
II# DEFINITIONS OP TERMS USED
!
; Heterophile. Immunologic ally related substances I
j found in the tissues of animals, plants and micro- |
I
! organisms which are widely separated in the zoological and j
L_botanical systems.._________ J
4
Forssman antigen. Boyd (1947) has stated that the
Forssman antigen is probably not a definite chemical
entity, but a serological conception, a collective term
covering substances which, when injected into rabbits will
stimulate the production of sheep erythrocyte hemolysins,
porssman antigen in this paper will refer to a definite
serologic and chemical entity which is one of the three
known heterophile antigens.
Mononucleosis antigen or M-antigen. An immuno-
1 ogically and chemically distinct heterophile antigen
present in sheep and beef erythrocytes,, reacting specifi
cally with the hemagglutinin,present in infectious mono
nucleosis serum.
Serum sickness antigen or SSr-antigen. An immuno-
logically distinct heterophile antigen present in guinea
pig kidney, and. beef.and sheep erythrocytes, reacting with
the hemagglutinins present in serum sickness serum.
Antigen. In those instances, where the Porssman,
serum sickness and mononucleosis antigens are referred to
they will be called antigens, (in compliance with common
usage) in spite of the fact that these fractions are pre
sumably not complete. antigens in that they may not induce
antibody formation if injected alone into an experimental
animal.
Haptene. An Immimologically active substance
which by itself will not induce antibody formation if in
jected as such into an experimental animal. Serologically
it will react with antibodies induced by the injection of
such a substance coupled with a protein carrier.
CHAPTER II
REVIEW OP THE LITERATURE
Paul and Bunnel (1952) pointed out that in in
fectious mononucleosis there was an increased titer of
anti-sheep hemolysins and agglutinins over and above any
"natural** occurring heterophile antibodies. These workers
were of the opinion that the antibodies present were
Porssman antibodies, but subsequent workers showed that
these antibodies were not completely absorbed by guinea
pig kidney tissue, the accepted criterion for the presence
of Porssman antibody. Bailey and Raff el (1955) demon
strated that the antibody of infee tious mononucleosis
could be absorbed almost completely from the serum by sheep
and bovine erythrocytes, and not to any extent by guinea
pig kidney. From this,. they concluded that the hetero
phile antibody of infectious mononucleosis was not the
same as the Porssman antibody.
Davidsohn and Walker (1935) indicated the need for
future work by stating, that the sheep erythrocyte contains
at least two antigens; the Forssman antigen and the hetero
phile antigen that reacts with .the antibody found in in
fectious mononucleosis.
In 1936 Stuart, Pulton, Ash and Gregory suggested
7
that the substance which had been known as the heterophile
antigen of the sheep erythrocyte was composed of various
antigenic components. In a later publication, Stuart,
Griffin, Wheeler and Batey indicated that cold alcoholic
extracts of sheep and beef erythrocytes did not absorb the
hemagglutinins of infectious mononucleosis sera. They con
cluded that the part of sheep and beef erythrocytes which
does absorb such antibodies was not alcohol soluble.
Knowing that the guinea .pig kidney did not com
pletely absorb the heterophile antibodies of infectious
mononucleosis whereas the bovine erythrocyte did, David-
sohn (1938) set up his differential diagnostic test,
Tomcsik and Schwarzweiss (1948) were able to ex
tract, in hot alocholic solution, the mononucleosis antigen
from both beef and sheep erythrocyte stroma. The immuno
logic ally active fraction was thermostable, soluble in hot
80 per cent ethyl alcohol and could be diluted to two
million parts and still inhibit hemagglutination of sheep
erythrocytes by the antibody of infectious.mononucleosis.
In a subsequent publication (1949) these authors
presented evidence . that the mononucieosia antigen was of a
polysaccharide nature, related to the same . . group of sub
stances. as the human group .A material and the Porssman
antigen, and that glucose or glucosamine plays an im-
8
portant role In the haptene structure. Controlled diges
tion with trypsin and pepsin engendered no loss of im
munologic activity, and in general, the fraction showed no
evidence of protein nature. They also stated that their
purest preparation of M-antigen was contaminated with
approximately 2-6 per cent of serum sickness antigen, and
that a complete separation could not be made.
The presence of an elevated heterophile antibody
titer has been reported in many disease conditions,
carpenter, Kahler and Reilly (1950) investigated the
heterophile antibodies in acute leukemia, a disease which
at times may be clinically difficult to distinguish from
infectious mononucleosis. They found that although there
wa 8 an increas e in the he ter ©phi le ti ter, the ant ib od le s
were absorbed with guinea pig kidney. This finding, in
e 8 senc e, has been subs tant iat ed by the work of Goldman,
pishkin and Peterson (1950) and Southam,. Goldsmith and
Burchenal (1951),
Viral hepatitis is. another disease which has been
reported as bringing.about the elevation of the hetero
phile titer , The evidence is somewhat contradictory as to
the frequency of its occurrence, but wherever it has been
reported as.being present there is unanimous agreement
that the antibodies formed are.absorbed by guinea pig
kidney* Leibowitz (1951) performed absorption tests on
thirteen patients with hepatitis and elevated titers; only
one patient showed the absorption pattern which is char
acteristic for infectious mononucleosis# This finding has
also been made by Eaton^ Murphy and Hanford (1944)*
Of interest here is a report by Watson, Johnson,
Kahn and Stone (1951) showing a high incidence of hepati
tis in subclinical cases of infectious mononucleosis*
Schultz (1948) studied the heterophile antibody
titer in diseases other than infectious mononucleosis*
She found titers of 1:56 or higher in Hodgkin's disease,
monocytic leukemia, myelogenous leukemia and tuberculosis*
She did not perform any differential absorption tests on
these serums, therefore making the findings of somewhat
limited significance*
CHAPTER III
PREPARATION AND TESTING OP M-ANTIGEN
Preparation of Stroma
Bovine blood was collected in large quantities (as
much as 10-20 gallons per time) using 10 per cent sodium
citrate made up in physiologic saline as the anti
coagulant* The blood was collected in quart jars using
approximately 30 ml* of sodium citrate solution per quart *
The plasma was separated from the erythrocytes by
centrifugation in the basket centrifuge at 1800 RPM for
thirty minutes and then washing the erythrocytes three
time8 with cold physlologic saline *
The washed and packed erythrocytes were lysed with
ten volumes of carbon dioxide saturated water; the pH
adjusted to 5*5 with 10 per cent hydrochloric acid and the
lysate put in the cold at 0-5®C for twenty-four hours*
The supernatant from the stroma which precipitated in the
cold was removed by aspiration, its pH readjusted to 5*5
with 10 per cent hydrochloric, acid and returned to the
cold for another twenty-four hours during which time a
second batch of . stroma settled . out* This was collected as
before, the supernatant being discarded*
The precipitated stroma was concentrated by passing
11
the suspension through a Sharpies supersentrlfuge (24-
25,000 RPM) at an intake flow rate of approximately 500 ml*
per hour. The packed stroma was removed from the barrel of
the centrifuge and stored in the cold.
The effluent from the Sharpies outlet was saved,
its pH readjusted to 5.6 with 10 per cent hydrochloric acid
and put in the cold at 0-5®C for twenty-four hours during
which time an additional batch of stroma precipitated.
The supernatant was removed as before and discarded, the
precipitated stroma combined with the stroma being stored
in the cold.
The collected stroma was then washed with carbon
dioxide saturated water by centrifugation in the basket
centrifuge until the washings were free of coloration by
hemoglobin. It had. been found that carbon dioxide satu
rated water was preferable to saline for washing since the
former promoted packing of stroma as compared to solubiliz
ing tendencies of the saline.
After washing and packing the stroma was lyo-
philized and then stored at 0-5®C until used in the ex
traction procedure. A typical protocol is shown in Figure
1.
The entire process is flexible to some extent.
The described procedure of cold sedimentation of the
PROTOCOL ON PREPARATION OF STROMA
12
packed bovine erythrocytes
(COg saturated water)
Lysate
(pH adjusted to 5.6
and put in cold at
[-5°C )
Supernatant
(pH adjustment
and cold storage)
Precipitated stroma
Supernatant
Discard
Stroma-
Supernatant
(Sharpies super-
centrifuge)
Stroma
(pH ad justment
and cold storage)
Supernatant
Discard
Stroma-
Wash and pack
Lyophilize
FIGURE 1
13
stroma and removal by way of supèr-centrifugatlon is not
rigid but may be varied depending upon time available and
absolute amount of stroma desired* In general, it may be
stated that the more times the supernatant is treated to
pH adjustment and cold precipitation the more stroma may be
realized (within reason) from a particular batch#
Extraction of M-antigen from stroma
Fifty grams of lyophilized stroma.was treated with
2500 ml* of acetone with constant agitation for twenty-
four hours at room temperature* This was accomplished
with a Magnamix apparatus# The acetone was removed by
filtration, discarded, an equal amount of absolute ethyl
alcohol added to the stroma and the stirring process re
peated for twenty-four hours. This step was repeated for
an additional twenty-four hours with an equal amount of
alcohol after removal by aspiration of the previous
alcohol which was discarded.
After twenty-four hours the alcohol was removed by
filtration, discarded, and-two liters of absolute ethyl
alcohol added to the residue and the mixture refluxed for
three hours, In order to obtain efficient extraction of
the stroma the refluxing mixture was constantly agitated
by a mo tor-driven stirring rod inserted through the
14
condenser canal. After cooling and settling of the stroma,
the alcohol was removed by aspiration, discarded and
another two liter portion of absolute ethyl alcohol added
and ref luxing continued as before for three more hours.
This alcohol was removed by the same method as above, dis
carded, and 1500 ml. of 80 per cent (by volume) ethyl
alcohol was added to the residue. Eefluxing was carried
out for exactly 15 minutes, timing starting at the moment
the first drop formed at the condenser tip. The time is
critical inasmuch as Tomscik. and Schwarzweiss (1948) have
determined that maximum activity is obtained at this
period, above and below giving lesser yields.
The 80 per cent ethylalcohol extract was stored
in the cold overnight during which time a fine reddish
brown precipitate appeared which was removed by centri
fugation. The alcohol was then removed by evaporation
under reduced pressure with the temperature controlled so
as not to exceed 60®C. The dark brown residue was taken
down to apparent dryness and then refluxed for one and one
half hours with 200 ml. of absolute ethyl alcohol. This
extract was removed by decanting, discarded, and 200 ml.
of 80 per cent ethyl alcohol was added to the residue
which was refluxed for exactly 15 minutes, timing ac
complished as indicated above. This last 80 per cent
16
ethyl alcohol extract constituted the M-antigen solution.
The process is diagramed in Figure 2.
The entire extraction procedure with minor var
iations of technic was a duplication of the procedure
given by Tomscik and Schwarzweiss (1948).
Determination of Extract Concentration
The determination of weight of M-antigen per
milliliter of alcohol was made by drying aliquots of the
extract in tared porcelain crucible in a dessicator over
anhydrous sodium.silicate in vacuo. These values were
later checked with data obtained by drying over PgOg
in vacuo (Table I).
Determination of Extract Activity
The immunologic potency was determined by the
hemagglutination inhibition reaction using 0.3 ml. of
two.-fold, serially diluted M-antigen, 0.1 ml. of in
fectious mononucleosis serum containing four, hemagglutinin
units, and 0.1 ml. of a 2 per cent suspension of sheep
erythrocytes.
The highest dilution a particular infectious
mononucleosis, serum could be diluted in a total volume of
0.55 ml. and at ill agglutinât e 0.1 ml. of a 2 per c ent
16
PROTOCOL FOR EXTRACTION OF M-ANTIGEN
Lyophilized Stroma
Extract
discard
Extract
discard
(2 1. 100^ ETOH
reflux for 3 hours),
Extract
discard
Residue
(1500 ml. 80^ ETOH
reflux for 15 minutes)
(2 1. acetone at room temperature
with stirring for 24 hours)
Residue
(2 1. lOC^ ETOH at room temper
ature with stirring for 24 hours)
Residue
(2 1. 100^ ETOH at room
temperature with stirring
for 24 hours)
Extract
discard
Residue
Residue
(2 1. 100^ ETOH reflux for
3 hours )
Extract
discard
Extract
(Evaporate ETOH)
Residue
(200 ml. lOqgg ETOH
reflux for 1.5 hours)
Extract
discard
Residue
discard
Distillate
discard
Residue
Residue
discard
(200 ml. 80J5 ETOH reflux
for 15 minutes)
Extract (save as M-
antigen)
FIGURE 2
17
TABEE I
DRY WEIGHT OP M-ANTIGEN PER MILLILITER
OP AIÆOHOL
; s :
Dry Weight in Mg./ml.
of 80^ ETOH
Over Over Anhydrous
Na silicate
iS i iSi
0,580 0.578
0.261 II
0.561 0.560 III
0.540 0.543 IV
0.580 0.580
18
suspension of sheep erythrocytes was considered as one
unit# Pour units was one-fourth of that dilution#
The immune serum was added to the respective dilu
tions of the M-antigen solution and permitted to stand at
room temperature for one hour; after which time the sheep
cell suspension was added# The rapid method of obtaining
the end point was ei^loyed «herein the tubes were centri
fuged for three minutes at 1500 RPM# The reading was made
by gently tapping the bottom of the tubes and observing
for clumps of red cells# The highest dilution of the
M-antigen which inhibited the hemagglutination of the
sheep erythrocytes by the immune serum was taken as the
potency level (Table II)#
In order to determine the heterophile singularity
of the M-antigen extract the identical procedure as above
was employed using four hemagglutinin units of serum sick
ness serum#
Results
The average yield of lyophilized stroma was ap
proximately one gram per one hundred mi 111 lit er s of packed
erythrocytes#
Five M-antigen extracts in all were prepared from
three batches of stroma during the period of the
19
TABEE II
PROTOCOL FOR THE ASSAY OF M-ANTIGEN BY TJSE OF THE
HEMAGGLGTINATION INHIBITION REACTION
Tube M-antigen Saline Serum Sheep cells
in 0#3 ml# ml# ml# ml#
1 undilute - 0#1 0#1
2 undilute 0#3 Q#1 0#1
(1) (2) (3)
3 0.3 ml# from tube 2 0#3 0#1 0#1
4 0.3 ml. from tube 3 0#3 0#1 0,1
*^(4)
n — 0.3 0.1 0.1
(1) Dilutions of M-antigen are made in saline before the
constant amount of serum was added# The serum con
tains four hemagglutinin units#
(2) Antigen dilutions plus serum are permitted to stand
at room temperature for one hour.
(3) After the sheep cells were added tubes centrifuged
for three minutes at 1500 RPM.
(4) The test was usually run for sixteen tubes, the last
tube being a control#
20
experiments* The dry weight of M-antigen per milliliter of
alcohol, with one exception, did not vary more than 40
gamma from preparation to preparation, all averaging
around 0*56 mg. per milliliter (Table I).
The potency of all the fractions, with the same
exception as above, was well over a 1:3,000,000 dilution
with two extracts reaching a potency of over 1:7,000,000
(Table III).
Heterophile singularity of the M-antigen prepara
tions was shown by no inhibition of the ability of serum
sickness serum to hemagglutinate sheep erythrocytes.
21
TABLE III
POTENCY OF M-ANTIGEN EXTRACTS AS DETERMINED BY
HEMAGGLUTINATION INHIBITION REACTIONS
Preparation Number
I
II
III
IV
V
7.000.000
2. 000.000
3.700.000
3.800.000
7,100, 000
Potency is defined as the minimum weight of
M-antigen in mg ,/ml. expressed as a dilution factor and
which completely inhibits hemagglutinin in the test
system.
CHAPTER IV
CHEMICAL ANALYSIS OP M-ANTIGEN /
Materials and Methods
I. Nitrogen
The method employed was the micro-K jeldahl pro
cedure outlined by Kabat and Mayer (1948), The alcohol
was evaporated from aliquots of M-antigen extract prior to
digestion,
II. Phosphorus
The method used was a modification of the Piske-
Subbarow technic, (Werkheiser, 1948), using ascorbic acid
as the reducing agent instead of 1,2,4-aminonapthosul-
fonic acid and a brief heating period of ten minutes for
maximum color development and stabilization.
III. Iron
The method of Kennedy (1927) was used.
IV. Sodium and Potassium
These determinations were performed on a Perkin-
Elmer flame photometer. The standard curves employed for
extrapolation were those used at the laboratory where the
instrument was located.
25
V. carbon and Hydrogen
These analyses were run by Dr. Glenn Swinehart of
the Microcheiaical Laboratory of the California Institute
of Teclmology, Pasadena, California using standard micro-
combustion procedures. Determinations were made on the
undialysed lipid containing forms of all five extracts and
in addition a determination made on the dialysed, defatted
aliquot of preparation IV.
VI# Total Carbohydrate
A modification of the Sorensen and Haugaard method
(1933) was employed. Determinations were performed on the
unhydrolysed and hydrolysed M-antigens. Hydrolysis was
performed by reflux w ith 6N hydrochloric acid (200 weight
by volume against dry weight of M-rantigen used) for 18
hours at a temperature of 130®C maintained by an oil bath.
Two milliliter aliquots of the M-antigen, dissolved
in water, were placed in ice-cold tubes followed by 3 ml.
of the oreinol reagent. The tubes.were cooled and 10 ml.
of the sulfuric acid solution was added, the tubes re-
cooled and placed in a water bath at exactly 80®C for 2,
5 and 20 minutes without stirring. The tubes were then
placed in an ice-bath for five minutes, after which time
they were read in the Klett-Summerson colorimeter with a
green filter.
24
The same procedure was eix^loyed on a standard
which contained 90 milligrams of dextrose per 100 ml. of
water.
1. Oreinol One gram per 50 ml. of water, add
20 ml. of centrated sulfuric acid and an additional 50 ml.
of water.
2. Sulfuric acid Seven parts of concentrated
sulfuric acid to three parts of water.
VII. Lipid Determinations
Ten milliliters of M-antigen were evaporated to
dryness in a test tube and ten milliliters of di-ethyl
ether added and the mixture refluxed with a ’ ’cold finger**
condenser for 15 minutes. The ether was then removed by
decanting, placed in a tared aluminum evaporating dish and
evaporated at room temperature. . The aluminum evaporating
dish was then weighed. After weighing, the solubility of
the residue was tested with water, physiologic saline,
chloroform, absolute ethyl alcohol and benzene.
VIII. Enzyme Digestions
Six milliliters of M-antigen were evaporated to
dryness and the residue taken up with an equal amount of
water.
25
One milliliter aliquots in duplicate were used with
both pepsin and papain. One-tenth perücent solutions of
the enzymes were used; the pepsin solution having 2 drops
of IN hydrochloric acid added#
Equal amounts of the M-antigen and enzyme solu
tions were incubated at 40®G for twenty-four hours and
then heated for 30 minutes at 80®G in order to inactivate
the enzymes. The M-antigen was thermostable under such
treatment.
A control of one milliliter aliquot of Mrantigen
plus one milliliter of water was incubated with the test
solutions in order to rule out loss of potency due to pro
longed standing at elevated temperatures. A second control
consisted of 2 drops of IN hydrochloric acid added to 1 ml.
of water and 1 ml. of M-antigen. This to eliminate the
possibility of acid breakdown of the M-antigen. Lastly,
a control was set up using heat inactivated enzyme and
M-fraction.
The activity of the M-fraction was then tested by
use of the hemagglutination inhibition test after pH ad
justment on the pepsin tube and the corresponding control.
Breakdown of the antigen was also tested chromatographic-
26
IX. Filter Paper Eleetrophoresis
Filter paper was used as a support for the buffer
and the protein solution. Aluminum bars were used to con
duct heat away from the paper strips. Plastic sheeting
served as an insulator for the bottom bar and thus kept the
buffer saturated paper strip away from it. A thick lucite
strip of the same dimensions as the aluminum bar served to
insulate the upper bar from the paper strips as well as
serving as a repository (through a hole bored in the
lucite and lined with a lucite filler) for the sample until
its absorption into the paper.
A system of cross-pieces moved by tightening of
bolts over a spring applied pressure to the bars in order
to prevent accumulation of too much liquid in the system.
The apparatus was, first equilibrated for a period
of one to two hours in order to attain proper voltage and
wetness of paper and thus prevent protein movement due to
liquid flow into the paper.
I All electrophoretic runs were made using a
I potential gradient.of 5 volts/cm, A veronal buffer, 0.
at pH 8.6 was used. In order to prevent electrolytic
changes in the buffer, the carbon electrodes were placed
in vessels containing saturated KCl and. connected to the
buffer baths by U-tube bridges filled with buffer and
27
plugged with cotton at both ends.
Runs were made in duplicate with strips being re
moved at the end of four and eight hours. After removal
from the bars the strips were dried in a hot air oven at
100®C and then dipped into a solution of 0.1 per cent
ninhydrin in butanol.
Color development was obtained by drying in the
hot air oven.
In addition to the procedure described above, runs
were also performed using two strips of paper, one on top
of the other. At the end of the appropriate period of
time the top strip was developed as indicated above, and
the corresponding area on the bottom strip was cut out and
eluted with 1-2 ml. of physiologic saline.
The saline extract was then tested for immunologic
activity using the hemagglutination inhibition reaction.
In both types of runs approximately three milli
grams of M-antigen was applied to each bar.
%. Filter paper Chromatography of Acid Hydrolysates
Ten milliliters of M-antigen were evaporated to
dryness in a test tube and 2 ml. of 6N hydrochloric acid
were added and refluxed for 18 hours with a ’ ’cold finger”
condenser at a temperature of 130®C maintained in an oil
bath.
28
The acid was removed by blowing a stream of air
into the tube overnight* The residue was taken up with
0.5 ml. of water and its pH checked for neutrality. In
every instance the pH was approximately 6.6-7.
Both ascending and descending chromatograms were
employed at the start of the experiments but the majority
of the work was performed using ascending technic due to
the more precise separation afforded.
The solvents employed were saturated n-butyl al
cohol made acidic with acetic acid (6 parts n-butyl al
cohol, 3 parts water and one part acetic acid) and sat
urated phenol.
The chromatograms were run for approximately 16-18
hours each. When the saturated butyl alcohol was used as
the solvent, 0.1 per cent ninhydrin was incorporated thus
obviating the spraying step to develop the color. In such
cases the strips were merely dried in the hot air oven at
100®G. In the case of phenol as the solvent the strips,
after hot air drying, were sprayed with a 0,1 per cent
^ /
ninhydrin solution made up in the saturated n-butyl al
cohol solvent.
Runs were also made using the two dimensional
method with the same solvents. First the runs were made
using phenol as the solvent and after completion of the
29
run the solvent was permitted to air dry and then the sheet
was turned at a 90° angle and run in the n-butyl alcohol
with the ninhydrin incorporated in the solvent.
Reference amino acids were made up in water in a
concentration of 0.01 per cent.
Whatman No. 1 paper was used In all the determina
tions •
Results
The carbon, hydrogen and nitrogen values are pre
sented in Tables IV and V. Table IV gives the values
obtained for carbon, hydrogen and nitrogen on undialysed,
lipid containing samples without the ash content of the
j samples taken into account for the calculations. When the
I sodium determinations on an undialysed sample gave values
of approximately 10 per cent, new values were obtained on
a dialysed sample which indicated a sodium concentration
of approximately 2.3 per cent average for all samples. On ,
the basis of these results, the carbon, hydrogen and
nitrogen values were re-determined on a sample of prepara
tion IV which was dialysed and defatted. Table V presents
these values exactly as determined experimentally for
preparation IX. and recalculated.for all others on the basis
of ash content.
30
TABLE IV
ELEMENT AND COMPONENT ANALYSIS OP M-ANTIGEN
ON undialysed; ' lipid CONTAINING SAMPLES
Fraction I II Ill IV V S - T*
Carbon 44.7^ 45.03# 45.74# 45,57# 45.21# 56.61#
Hydrogen 6.31^ 7.81# 7.1^ 7.33# 7.34# 7.74#
Nitrogen 10.31# 10.46# 10.68# 10.78# 10.90# 8.45#
Phosphorus 0.43# 3.^ 2.9#
1.2#
0.68# 2.44#
Sodium 2.3^ 2.29# 2,41# 2.30# 2.34# -
potassium 0.2C# 0.5€^ 0.45# 0.50# 0.45#
Iron 0.00# 0.00# o . o q # O.OC# 0.00#
i
Total ash 13.10# 10.4# 11.20# 11.10# 11.40#
(
Total car
bohydrate
2.17# 2.30# 2.34# 2.23# 2.21#
Lipid All approximately 20-30# -
Reducing
sugar
—
-
-
-
— 17.40#
Glucosamine — - - — - 8.56#
^SGhwarzweiss and Toms elk ( 1949)
TABIE V
31
PERCENTAGE COMPOSITION OP M-ANTIGEN AS CALCULATED
ON THE BASIS OF ASH FREE MATERIAL
sample Carbon Hydrogen nitrogen Ash
I 51.50^ 7.81# 11.81# -
II 50.40# 8.71# 11.6C# -
III 51.50# 8.01# 12.02# -
IV 51.20# 8.20# 12.10# -
V 51.02# 3.20# 12.30# -
IV, dialysed, 51.00^
defatted, uneor-
reoted for ash
7.16# 12.50# 6.60^
IV, corrected
for ash
54.60# 7.67# 13.40# — '
32
Using these new values it can be seen that the
element analysis fits in closely with that expected for
the composition of a typical protein*
In general, the entire chemical analysis of the
extracts reported here differ from that reported by
Schwarzweiss and Tomscik (1949)* These workers have re
ported values of 06*61 per cent for carbon and 8.45 per
cent for nitrogen.
The total carbohydrate deteimination (Table IV)
indicated that on both the hydrolysed and unhydrolys ed
fractions the average value was approximately 2.2 per cent.
They have reported a value expressed as reducing sugar of
17.4 per cent.
Nowhere in their report is there any indication of
a lipid centent to the antigen. Table IV shows a value for
these preparations of approximately 20-30 per cent. Of the
solvents tested for the solubility of the ether extract
residue, all were effective except the water and physio
logic saline.
Enzymatic digestion with the use of proteinases
(pepsin and papain) resulted in complete loss of immuno
logic activity as is shown in Table VI. Further filter
paper chromatograms of the enzymatic hydrolysates revealed
several spots, identified as amino acids.
33
TABLE VI
EFFECT OP ENZYIATIC HYDROLYSIS OF M-ANTIGEN AS DETER
MINED BY THE HEMAGGLUTINATION INHIBITION REACTION
Material Immunologic Activity
M-antigen plus papain
M-antigen plus pepsin plus HGl
M-antigen plus water
M-antigen plus water plus HCl
M-antigen plus heat inactivated
papain
Negative
Negative
1:700,000
1:700,000
1:700,000
34
Filter paper ciiromatography of the acid hydrolysate
(Table VII) served as further evidence that the fractions
were of a protein nature. All five fractions showed the
same number of amino acids, as well as the same amino
acids. In all, nine amino acids were identified from
their approximate Rf values as compared to that of refer
ence amino acids.
Two dimensional chromatograms were plotted by
using the Rf values of phenol and n-butyl alcohol as the
ordinates. Figure 3 shows this plot and indicates the
identity of the amino acids in the hydrolysate of the M-
antigen as lysine, glutamic acid, serine, glycine, threo
nine, alanine, aspartic acid, leucine and isoleucine.
The iron determinations (Table IV) were performed
in an effort to assign a reason for the yellow-brown color
of the M-antigen. The zero value obtained ruled out iron
or iron containing compounds as an explanation.
The inconsistencies of the phosphorus values
(Table IV) permit no comparison with the 2.44 per cent
value obtained by Schwarzweiss and Tomscik (1949).
The filter paper electrophoretic determinations
showed that the M-antigen extract was heterogeneous in
that four components were discernible; two migrating to
wards the positive pole (negative* charge) and two migrating
35
TABLE VII
FILTER PAPER €HROMATGGRAPHY OF M-AHTIGEM HYDROLYSATES
WITH REGARD TO REFERENCE AMINO ACIDS
Reference Area Rf values
Amino Acid/M-antigen Butyl Alcohol Phenol
Lysine 0.05 0.11
M-1 0.04 0.10
Glutamic 0.12 0.28
M-2 0.10 0.29
Serine 0.16 0.37
M-3 0.14 0.36
Glycine 0,19 0.40
M-4 0.18 0.42
Threonine 0.22 0.49
M-5 0.25 0.51
Alanine 0.32 0.60
M-6 0.33 0.63
Aspartic 0.46 0.84
M-7 0.44 0.86
Leucine 0.63 0.87
M—8 0.63 0.89
Isoleucine 0.69 0.91
M-9 0.64 0.90
36
DIMENSIOmL GHROMTGOBAMS OP M-ANTIGEN
ACID HYDROLYSATES
leucine
re
H
na
ne
0 N o . 6 1 0 S - * 'I iiv e r s it 3 il2 o k s t o r e f 3 s A n g e te A * 3 # 6
Phenol Rf
.8 *9
FIGURE 3
37
towards the negative pole (positive charge)♦
Testing of the eluted portions revealed that the
immunologic activity was present in both components which
were negatively charged and also in the slower moving
component which was positively charged.
Serologically the eluate could be serially (two
fold) diluted to the eighth tube for all components and
still show immunologic activity in the hemagglutination
inhibition reaction. The approximate dilution of the M-
antigen in this tube was 1:700,000.
CHâPTER V
IMMUNOLOGY AND SMOLOGY
Adsorption Experiments
It had been reasoned that the minimum amount of M-
antigen which must be present in order to effect agglu
tination of a sensitized particle was 160 gamma per 0*1 ml.
of particles In their appropriate density. The average
titer of the various M-antigen extracts was approximately
0.004 gamma per 0.1 ml. of infectious mononucleosis serum
containing four hemagglutinin units (Table III). If the
highest titer serum to be encountered was taken at ap
proximately 8000 units, then one-tenth of a milliliter of
sensitized particles should contain 80 gamma of M-antigen
in order to react with the antibodies. However, this was
based on the supposition that multiple proportion reactions
occur and there is no evidence for this occurring. There
fore, twice the amount predicted was utilized or 160 gamma
of M-antigen per 0.1 ml. of particles.
Adsorption was attempted using the “batch" method
whereby the particles were suspended in a particular
liquid, i.e., water, saline or buffer, the density of the
suspension being approximately that of a 2 per cent sus
pension of sheep erythrocytes.
39
The alcohol from 1 ml. of the M-antigen extract
was evaporated and the residue taken up with 3 ml. of the
particle suspension. This was in keeping with the reason
ing outlined above as regards the minimum amount of M-
antigen which had to be present in order to effect agglu
tination.
This suspension was then incubated at 37®C for two
hours followed by incubation at 0-5®€ overnight. After
icebox incubation the particles were centrifuged and the
supernatant tested for the presence of protein with the
ninhydrin reaction and Immunologic activity with the hem
agglutination inhibition reaction.
If the supernatant was negative on both tests an
aliquot of the packed particles was removed and tested for
the presence of protein after washing three times. Those
particles showing a protein reaction were then washed
three times and resuspended in their appropriate liquid.
This suspension constituted the “antigen" which was used
in the agglutination reaction, as the substitute for the
sheep erythrocyte suspension in testing infectious mono
nucleosis sera.
A. Dowex-2 Colloidal Particles
The anion exchange resin was gently boiled for two
hours with twenty times its volume of IN HaOH* After
40
cooling, the mixture was filtered through a Buchner funnel.
The residue was then washed with distilled water
until the filtrate was near neutrality. This, in general,
took from 10-15 wasMiigs. The pH of the particles sus
pended in water was 7.5. The particles were also suspended
in a buffer at pH 5.5.
The anion exchange res in was also activated by
treatment with IN hydrochloric acid by boiling for two hours
and then washing with distilled water until the filtrate
was neutral. The pH of the particles suspended in water
was 6.5. The particles were also suspended in a buffer
solution at pH 5.5.
B. Dowex-1 colloidal Particles
The cation exchange resin wasactivated by treat
ment with IN hydrochloric acid in a similar, manner as was
the anion exchange resin dis cussed, above. A water sus
pension had a pH of 6.0; a buffer suspension of pH 5.5 was
also used.
C. Bentonite
Bentonite was gently boiled with IN hydrochloric
acid for one hour.. It was found that filtering was not
possible as the particles soon clogged the pores of the
filter paper. . The mixture was therefore centrifuged for
41
thirty minutes at 2000 RPM# the supernatant removed, dis
tilled water added, mixed, and the process repeated until
the pH of the supernatant was near neutrality.
The washed and treated particles were suspended in
a buffer solution of pH 8.0 and one at pH 5.0 by mixing
for three minutes in a Waring blendor.
D. Kaolin
Kaolin was treated as described above for bentonite
with the exception that the particles after treatment with
acid could be separated by filtration and the excess acid
removed by washing with distilled water.
! E. Alumina Adsorption (Fisher)
i
I Fisher’s alumina adsorption, mesh 80-200, was
I ground with a mortar and pestle until of a very find
I powdery consistency. This, without any further treatment,
was made up in a suspension of water and one of physiologic
saline.
F. Norite and carbon Black
Both of these were used without any further treat
ment other than preparing the suspension by mixing in a
Waring blendor using distilled water as the liquid phase.
G. Ferric Hydroxide
42
Colloidal ferric hydroxide was prepared by adding
0*3 grams of solid ferric chloride to 200 ml# of boiling
water# The resultant colloidal ferric hydroxide was dia-
lysed against two changes of distilled water for forty-
eight hours in order to remove the chloride ions#
This dialysed colloidal suspension constituted the
carrier for the adsorption of the M-antigen# It was found
that if longer periods of dialysis were used some aggrega-
I tion of the particles occurred and these larger particles
I were also used as a vehicle#
I Results !
I I
i All of the particles used with the exception of the!
! I
; bentonite adsorbed the M-antigen# This was. shown by the
I
negative immunologic activity of the supernatants after
centrifugation of the treated particles and by the neg-
I ative ninhydrin reaction of. the supernatants#
Using the Dowex-2 colloidal anion exchange parti
cles activated with hydroxyl groups and suspended in a
buffer at pH 5#5 and sensitized with M-antigen, the
standard sheep cell agglutination reaction was set up with j
the particles as the antigen instead of the sheep erythro- ;
cytes# No agglutinati on occurred#
Addition of sheep cells, after the sensitized
particles had been removed by centrifugation, gave no
45
hemagglutination of the erythrocytes* This indicated that
the antibody had combined with the M-antigen present on
the Dowex particle*
As a control on the possibility that the anti
bodies had adsorbed onto the particles themselves, serum
was tested for sheep erythrocyte hemagglutinins after
treatment with a suspension of unsensitized particles made
up in a buffer of pH 5*6* The hemagglutinins for sheep
erythrocytes were not removed by the unsensitized Bowex
particles* |
I
Similar results, i*e., no agglutination but in- j
activation of antibody, were obtained when normal human ■
serum was used as the diluent instead of saline and also
when 0*1 per cent bovine albumin was used as the diluent. |
I
Identical results were obtained with all the other |
particles which adsorbed the M-antigen* j
Absorption Experiments with M-antigen and j
I
Sera Containing Heterophile Antibodies j
I Methods, and Materials |
I I
i All of the sera which have been labeled as in- !
! . !
j factious mononucleosis were obtained from cases in the Los i
I Angeles area diagnosed as such on the basis of clinical
I symptoms, as well as hematologic and serologic findings*
44
All other sera showing heterophile antibodies were obtained
from patients clinically diagnosed as having infectious
diseases, blood dyscrasias or liver dysfunctions *
A rapid method of adsorption was employed whereby
for the infectious mononucleosis sera 0.3 ml. of M-antigen
in saline was added to 0.1 ml. of the undiluted serum and
' the mixture shaken mechanically for five minutes. For all
I
i other sera 0.1 ml. of M-antigen was used with 0.1 ml. of
i
undiluted serum. Duplicate runs were made using only
I saline and serum as controls in order to rule out loss of
j antibody due to shaking.
I After the absorption, period, 0.1 ml. of the mix
ture was added to tube number 1 of the standard sheep cell
aggluticnaition protocol (Table VIII) and dilutions made
from that point as was regularly done. In reading the
titer of the infectious mononucleosis sera the end point
was taken as two tubes higher due to the dilution of the
serum with the M-antigen.
Results
Within the confines of the infectious mononucleosis
serum series, co]iç)lete or nearly complete absorption of
the heterophile antibody occurred when using the M-antigen
as the absorbing agent (Table DC).
45
TABLE VIII
PROTOCOL FOR STANDARD SHEEP CELL ASGLUTINÂTION REACTION
Tubes Saline ml. Serum ml. Sheep rbc^ ml
1 0.4 0.1 of absorbed 0.1
2 0.25 0.25 of tube 1 0.1
3 0.25 0.25 of tube 2 0.1 (3)
4 0.25 0.25 of tube 3 0.1
5,6..n^ 0.25 0.25 of previous tube 0.1
n 0.25
—
0.1
1 — 1^ sheep erythrocytes in saline.
2 -- corpuscle caatrol.
(5) -- Tubes are centrifuged for three minutes at
1500 RPM and then read directly for presence
of agglutination by lightly tapping the
bottom of the tube.
46l
TABIE IX
HEmGGLUTimTlOH TITERS OP IKPECTIOTJS MONGHUCIEOSIS SERA
BEPmE AHD AFTER ABSORPTION WITH M-ANTIGEN
Serum
Before
Hemagglutinin Titer
Absorption After Absorption
1 1 7168 0
2
1 3584 0
3 1 3584 0
4 1 3584 0
1 5 1 3584 0
6 1 3584 0
7 1 1792 0
8 1 1792 0
9 1 1792 0 1
10 1 1792 0 '
1 11 1 1792 0 I
i 12
1 1792 1:56 I
j 13 1 1792 1:56 1
' 14 1 1792 1:56
! 15
1 1792
1:28 I
j 16 1 896 0
17 1 896 0
18 1 896 0
19 1 896 0
20 1 896 0
21 1 896 0
22
1 896 0
23 1 896 0
24 1 896 0
25 1 896 0 1
26 1 896 0 1
27 1 896 0
28 1 896 0
29 1 896 0
30 1 896 0 i
31
1 896 0 1
32 1 896 0 i
33
1 896
8 1
34 1 896 0
35
1 896 0
36
1 896 1:56
47
TABLE DC (continued)
HEMAGGLDTINATION TITERS OF IHPEGTIOUS MOHOTOGLEOSlS SERA
BEFORE AND AFTER ABSORPTION WITH M-ANTIGEN
Serum
Hemagglutinin Titer
Before Absorption After Absorption
i 37 1 896 1:28
; 38 1 896 1:28
i 39 1 896 1:28
40 1 896 1:28
1 41 1 896 1:28
42 1 896 1:28
43 1 448 0
44 1 448 0
45 1 443 0
46 1 448 0
47 1 443 0
48 1 448 0
49 1 448 1:56
50 1 448 1:28
51 1 448 1:28
52 1 448 1:28
53 1 224 0
54 1 224 0
55 1 224 0
56 1 224 0
57 1 224 0
58 1 224 0
59 1 224 G
60 1 224 0
61 1 224 0
62 1 224 0
63 1 224 0
64 1 224 0
65 1 224 0
66 1 224 0
67 1 224 1:28
63 1 224 1:28
69 1 224 1:28
70 1 224 1:28
71 1 224 1:28
72 1 112 0
48"
TABIE IX (continued)
HEmGGLIJTINATION TITERS OP INPECTI0ÜS MOHONUGLEOSIS SERA
BEFORE AND AFTER ABSORPTION WITH M-ANTIGEN
Serum
Hemagglutinin Titer
Before Absorption After Absorption
75 1:112 0
74 1:112 0
i 75 1:112 0
i 76 1:112 0
1 77 1:112 0
1 78 1:112 0
1 79 1:112 0
80 1:112 1:28
1 81 1:112 1:28
82 1:56 0
85 1:56 0
84 1:56 0
85 1:56 0
86 1:56 0
87 1:56 0
88 1:56 0
89 1:56 0
90 1:56 0
49
All other serums tested for the presence of hetero
phile antibodies, and in particular the presence of anti-
mononucleosis heterophile antibodies, indicated, at least
within the range of the experiments reported here, that
the anti-mononucleosis heterophile antibody was unique for
infectious mononucleosis (Table X).
50
TABLE X
HSMAGGLOTINATIGN TITERS GP SERA SHOWING HETEROPHIIE ANTI-
BODIES BEFORE AND AFTER ABSORPTION WITH THE M-ANTIGEN
Hemagglutinin Titer
Disease
Before Absorption After Absorption
Mumps 1:56 1:56
Mumps 1:14 1:14
Mumps 1:28 1:28
Mumps 1:448 1:448
Mumps 1:56 1:56
Mumps 1:14 1:14
Typhoid 1:28
l;28
Typhoid 1:7
Typhoid 1:28 1:28
Typhoid 1:7 —
Typhoid 1:14 1:14
Typhoid 1:14 1:14
Typhoid 1:14 1:14
Typhoid 1:28 1:28
Typhoid 1:7 —
Typhoid 1:14 1:14
Measles 1:56 1:56
Measles 1:112 1:112
Measles 1:224 1:224
Measles
1:56 1:56
Measles 1:56 1:56
Measles 1:14 1:14
Measles 1:14 1:14
Measles 1:7
-
Measles 1:7
-
Measles 1:7
-
Measles 1:14
Polio 1:112 1:112
polio 1:14 1:14
Polio 1:7 -
polio 1:7
-
Syphilis 1:7 -
Syphilis 1:14 1:14
Syphilis 1:28 1:28
Brucellosis 1:7 -
Brucellosis 1:14 1:14
51
TAB BE X (continued)
HEMAGGLUTIiSATIOH TITERS OP SERA SHOWIHG HETIROPHIIE ANTI
BODIES BEFORE AND AFTER ABSORPTION WITH THE M-ANTIGEN
Disease
Hemagglutinin Titer
Before Absorption After Absorption
Weil-Pelix Positive 1:14 1:14
Weil-Felix Positive 1:7
-
Weil-Pelix Positive 1:14 1:14
Weil-Felix Positive 1:28 1:28
Weil-Pelix Positive 1:14 1:14
Weil-Pelix Positive 1:14 1:14
Weil-Pelix Positive 1:28 1:28
Weil-Pelix Positive 1:14 1:14
Hansens 1:448 1:448
Serum sickness 1:112 1:112
Serum sickness 1:224 1:224
Infectious hepatitis 1:28 1:28
Infectious hepatitis 1:14 1:14
Infectious hepatitis 1:14 1:14
Infectious hepatitis 1:28 1:28
Hodgkins 1:14 1:14
Hodgkins 1:28 1:28
Dooley*s anemia 1:14 1:14
Lymphatic leucemia 1:14 1:14
Lymphatic leucemia 1:14 1:14
Rheumatic arthritis 1:28 1:28
Rheumatic arthritis 1:14 1:14
CHâFTER VI
DISCUSSION
Using lyophilized bovine erythrocyte stroma as a
starting material an alcoholic extract had been prepared
which reacted immunologically with the heterophile anti
bodies present in infectious mononucleosis. That such a
material was present in bovine erythrocytes bad been in
dicated by the utilization of such cells as an absorbing
agent for the heterophile antibodies in infectious mono
nucleosis.
Inasmuch as the bovine erythrocytes also absorbed
the antibodies in serum sickness serum, the problem had
been coi3f)licated by the necessity of obtaining an immuno-
logically singular extract. The mononucleosis antigen ex
tract reported upon in these experiments was such a
material. This was shown by the negative results obtained
using this extract in the.hemagglutination inhibition re
action with serum sickness serum. . The extremes to which
the material was diluted and still manifested activity was
taken as further evidence of, the immunologic purity of the
extract.
The chemical analysis of the extract led to the
conclusion that the mononucleosis antigen was. of a protein
I 53
I nature and that the lipid which was present did not have j
i . I
I any function in the immunologic activity of the material, j
The protein nature of the active material was |
I shown by the elemental analysis data which corresponds to a
I
I typical protein,
I The enzymatic hydrolysis of the mononucleosis anti-
i
I gen was taken as further evidence of the protein nature of
j the material. By the use of proteinases (pepsin and papain]
complete loss of immunologic activity resulted.
Acid hydrolysis revealed that nine amino acids
were present and that all of the fractions extracted had
the same amino acids. This showed the reproducability of
the product arrived at by the extraction procedures
employed.
It was felt that a polysaccharide was not involved
in the immunologic activity of the antigen since total
carbohydrate determinations indicated a value of approx
imately 2 per cent. Inasmuch as potency levels of the
mononucleosis antigen had been determined on.the basis of
dry weight of starting material, expressed as a dilution
factor; 2 per cent of the rather small amount of material
(0,056 mg. ) would have led to an activity expression never
before encountered in immunology.
The lipid content of approximately. ,20-30 per cent
64
which was removed from the mononucleosis antigen by mild
reflux with diethyl ether was of interest from several
aspects. First, the ease with which it was removed by
ether reflux from the protein made it very unlikely that
the lipid was intimately bound to the protein to form a
lipoprotein. Such complexes are not known to be readily
I broken apart. Secondly, it was of interest merely by its
I presence. When one considers the rather elaborate pro-
j cedure involved in obtaining the M-antigen, all of which
; utilized lipid solvents under conditions favoring extrac-
I
1 tion, it was rather surprising to find the lipid still
I
I present,
j Schwarzweiss and Tomscik ( 1949 ) have reported that
; the mononucleosis antigen as extracted by their method
, gave indications upon chemical characterization of being
a polysaccharide with glucose or glucosamine as the deter
minant portion, Enzymatic digestion with both pepsin and
trypsin revealed no loss of immunologic activity.
In an effort to reconcile these widely divergent
results with those reported in this paper, recourse was
made to an examination of where the two preparations
differed both in starting material and procedure for ex
traction. Basically, the only over-all difference lay in
the preparation of the stroma. However, it was rather
55
difficult to accept the possibility that lysing the eryth
rocytes with carbon dioxide saturated water and lyophiliza-
tion could make such a radical difference. This was a
remote possibility especially since the only reason carbon
dioxide saturated water was used as the lysing agent in
I the first place was due to its being a mild acid and thus
I
I lowered the pH to the approximate isoelectric point of the
I stroma. This insured maximum precipitation of the stroma,
I As further evidence that such a procedure probably had not
brought about a major change in the chemical composition
of the stroma, was the fact that in both procedures 10 |
I
per cent hydrochloric acid had been used to finally adjust j
the pH to 5,5, !
I I
I On the basis of the above discussion it was found
that no conclusion could be drawn as to the divergent
I results and that the problem could only be solved by
duplication by other workers. It should be recorded that
a search of the literature revealed no confirmation of the
results published by Schwarzweiss and Tomscik (1949).
The concept of the adsorption experiments, while
not original was fully applicable. Presented with a
soluble immunologic fraction, desirous of utilizing it in
a diagnostic test, it seemed highly feasible to attempt
adsorption onto a particle which would serve as the
r-------------------- ------------------------- -------------------
I " 56
I visible indicator system of serologic reaction. This
seemed even more attractive in view of the observation
that the material was a protein. There are many particles
such as alumina cream, kaolin and bentonite which have been;
used in protein chemistry for adsorption. With the intro- j
duetion of the ion exchange resins, it was felt that i
perhaps it was now possible to achieve what had not been j
successful to any degree in the past, |
That adsorption occurred with these particles whilej
agglutination did not occur was not anticipated and still j
has remained unexplainable. However, this in itself is a |
j I
major problem for immunologists, since the dynamics of j
1 I
j agglutination remain to be explained, 1
It was reasonably certain that the antibodies had |
j reacted with the mononucleosis antigen present on the j
I
I particles in that the serum would no longer hemagglutinate |
sheep erythrocytes. Therefore, it can be concluded that
I
the active site of the mononucleosis antigen was not in- !
volved in the adsorption process. The. control experiment j
ruled out the possibility of the antibody by itself ad- !
sorbing onto the particle, I
The absorption experiments of the M-antigen with j
infectious mononucleosis sera and other sera showing
heterophile antibodies indicated that the anti-mononucleosis
57
heterophile antibody was unique for infectious mono
nucleosis*
Accepting the validity of the observations that
the heterophile response in infectious mononucleosis was
unique, it was proposed that the Davidsohn differential
test be revised so that the mononucleosis antigen be
utilized as the absorbing agent instead of the guinea pig
kidney macerate* This was done for the following reasons*
There are two types of heterophile antibodies which will
absorb out with the guinea pig kidney macerate; the Porss-
man antibody and the serum sickness antibody* The kidney
macerate is not a single immunologic entity whereas the
M-antigen appeared to be more specific*
Any appreciable loss in titer would be diagnostic
from the serologic sense since the antibody seems to be
restricted to infecti.ous mononucleosis* No longer should
it be necessary to engage in empirical discussions as to
what constitutes a d^-^S^ostic titer before and after ab
sorption*
Several sera having a sheep cell titer of 1:56 were
included in these experiments* These sera were obtained
from patients clinically diagnosed as having infectious
mononucleosis* According to Davidsohn (1958) this was not
diagnostic for infectious mononucleosis, only titers of
58
1:112 or higher being considered* Since these sera showed
a loss in titer upon absorption with the mononucleosis
antigen, it was felt that serologically, the clinical
diagnosis had been confirmed*
The residual titers of the infectious mononucleosis
sera absorbed with M-antigen were presumed to be due to
the presence of other heterophile antibodies* Simmonde
and Markowitz (1952) have indicated that some infectious
mononucleosis sera may contain, in addition to the anti-
mononucleosis antibody, some anti-serum sickness anti
bodies*
CHAPTER VII
SUMMARY
A chemical analysis of a heterophile fraction
isolated frcaa bovine erythrocyte stroma was presented*
The material reacts immunologically with the heterophile
antibodies present in infectious mononucleosis*
Chemically, the material was a lipid-protein mix
ture with the immunologic activity present in the protein
portion*
Serologically, it had been indicated that the
heterophile antibody reacting with this material was
unique for infectious mononucleosis*
On the basis of the above it had been proposed
that the M-antigen be substituted as the absorbing agent
in place of the existiiQg guinea pig kidney in the David
sohn differential test*
BIBLIOGRAPHY
BIBLIOGRAPHY
Bailey, G. H*, and Raffel. S., Hemolytic antibodies for >
sheep and ox erythrocytes in infectious mononucleosis*
Jour* Clin* Invest*, 14:228, 1935*
Boyd, William C*, Fundamentals of Immunology, New York:
Interscience Publishers Inc*, 1947* 503 pp*
Carpenter, G*, Kahler, J*, and Reilly, E* B*, Elevated
titers of serum heterophile antibodies in three
instances of monocytic leukemia* Am* J* Med* Sci*,
220:195, 1950*
Davidsohn, I*, Test for infectious mononucleosis* Am. J*
Clin* Path*, Technical Supplement 8:56, 1938*
Davidsohn, I*, and Walker, P., The nature of the hetero
phile antibodies in infectious mononucleosis * Am* J*
Clin* Path*, 5:455, 1935*
Eaton, M* D*, Murphy, W* D*, and Hanford, V*, Heterogenetic
antibodies in acute hepatitis. J* Exper* Med*,
79:539, 1944*
Goldman, R., Pishkin, B*, and Peterson, E*, The value of
the heterophile antibody reaction in the lymphomatous
diseases* J* Lab, end Clin* Med*, 35:681, 1950*
Kabat, E* A*, and Mayer, M* M*, Experimental Immunochem
istry, Springfield: Charles 0* Thomas, 1948, 567 pp*
Kennedy, R*, The quantitative determination of iron in
tissue, J* Biol* Chem*, 74:385, 1927*
Leibowitz, S*, Heterophile antibody in normal adults and
in patients with virus hepatitis* Am. J* Clin* Path*,
21:201, 1951*
Paul, J* R*, and Bunnel, W* W*, The presence of hetero
phile antibody in infectious mononucleosis* Am* J*
Med* Sci*, 183:90, 1932.
Schultz, L* E*, Heterophile antibody titer in diseases
other than infectious mononucleosis*Arch* Int* Med*,
81:328, 1948*
62
Schwarzweiss, H*, and Tomcslk, J*, Beef erythrocyte ex
tracts reacting with hemagglutinins in infectious
mononucleosis and in horse serum sickness* Proc* Soc*
Exptl* Biol* and Med*, 72:693, 1949*
Simmonds, W*, and Markowitz, A*, Heterophile antibodies
preset in infectious mononucleosis. In press, 1962*
Sorensen, T*, and Haugaard, P*, Uber die Anwendbarkeit der
Orcinreaktion zur Bestimming der Art and Menge von
Kohlenhydrolgruppen in Biweisstoff en, Biochem*
Ztschr*, 260:247, 1933*
Southam, C*, Goldsmith, Y*, and Bruchenal, J*, Heterophile
antibodies and antigens in neoplastic diseases.
Cancer, 4:1036, 1951*
Stuart, C* A., Pulton, M*, Ash, R*, and Gregory, K*, The
relations between certain heterophile antibodies and
antigens. J* Inf, Dis., 59:65, 1936*
Stuart, G* A*, Griffin, A*, Wheeler, J., and Batey, S.,
A thermostable antigen in beef cells. Proc, soc*
Exptl* Biol* and Med*, 34:212, 1936*
Tomcsik, J*, and Schwarzweiss, H*, Isolation of the hetero
genetic mononucleosis antigen from the stroma of beef
erythrocytes * Proc *. Soc *. Bxptl* Biol* and Med *,
69:558, 1948*
Wat s on, J *, Johnson. P., Kahn, J*, and S tone, P *, Sub-
clinical inf ectious. mononucle osis with hepatitis*
Arch* Int* Med*, 88:618, 1951*
Werkheiser, W* G*, Research Report, Dept* of Biochem*,
Doheny Library, Ü* So* Calif*, 1948*
U n lve rs N y o fS eu th ern California Library
UMI Number: DP23896
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
D k eem aiion RtiblisMng
UMI DP23896
Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
Microform Edition © ProQuest LLC.
All rights reserved. This work is protected against
unauthorized copying under Title 17, United States Code
ProQuest LLC.
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, Ml 48106- 1346

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Studies on the cytology of bacteria and yeasts by means of ultraviolet photolysis

PDF

Studies on the bacterial oxidation of saturated fatty acids and their derivatives

PDF

Studies on the aerobic oxidation of fatty acids by bacteria

PDF

Nude mouse grown human nephroblastomas

PDF

Studies of frog retinal ganglion cell responses to temporal, spatial and chromatic stimuli

PDF

Studies of two naturally occurring compounds which effect release of acetylcholine from synaptosomes

PDF

The role of microbes in the formation of the tubeworm fouling community in Fish Harbor, Los Angeles Harbor, California

PDF

An analysis of the need and opportunity for the continuing liberal education of doctors of medicine

PDF

Public health in a retrenchment era: a choice about investing with people

PDF

The 'Widmar' (1981) decision and the status of religious liberty on the public college and university campus: Antecedents, effects, and prospects

A qualitative and quantitative study of the retinal ganglion cells of the rabbit (<italic>Oryctolagus cuniculus</italic>)

PDF

A qualitative and quantitative study of the retinal ganglion cells of the rabbit (Oryctolagus cuniculus)

PDF

Out of many...: A social history of the homosexual rights movement as originated and continued in Los Angeles, California

PDF

An analysis of traditional birth attendant (TBA) programs in selected third world countries

PDF

The innervation of the teeth and periodontium of the rat

PDF

Studies on cholesterol metabolism in the rat

PDF

Numerical analysis of ecological survey data

PDF

Certain factors influencing the fermentation of glucose by Lactobacillus beijerinckii

PDF

A study of Japanese students at the University of Southern California, 1946-1980: vocational impact of American academic experience on Japanese students after returning to Japan

PDF

The role of the sensorimotor cortical system in skill acquisition and motor learning: a behavioral study

PDF

An evaluation of an innovative master's degree program for administrators of ambulatory health care centers

Asset Metadata

Creator Markowitz, Abraham (author)

Core Title Chemical and immunological studies on a heterophile antigen extracted from bovine erythrocyte stroma

School Graduate School

Degree Doctor of Philosophy

Degree Program Bacteriology

Degree Conferral Date 1952-07

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag health and environmental sciences,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c30-262213

Unique identifier UC11227603

Identifier DP23896.pdf (filename),usctheses-c30-262213 (legacy record id)

Legacy Identifier DP23896.pdf

Dmrecord 262213

Document Type Dissertation

Rights Markowitz, Abraham

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA