Immunologic Properties of Anti-Rabbit Globulin Sera
This dissertation has been 63-2547 microfilmed exactly as received
ROGUL, Marvin, 1932- IMMUNOLOGIC PROPERTIES OF ANTI-RABBIT GLjOBUUN CHICKEN SERA.
The Ohio State University, Ph.D., 1962 Bacteriology
University Microfilms, Inc., Ann Arbor, Michigan IMMUNOLOGIC PROPERTIES OP ANT I-RABBIT
GLOBULIN CHICKEN SERA
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
MARVIN ROGUL, A .B ., H .S,
•a*#***#**
Tho Ohio State University 1962
Approved by
L d v iser Department of Microbiology ACOOWLEDGMEHTS
The author wishes to express sincere appreciation to
Dr. II. C. Dodd for his help and thoughtfulness throughout this investigation.
The author also wishes to thank Drs, If. J. Bigley,
C. Cohen, H, S. Rheins, and Mrs. V. B. Geyer for their aid.
Gratitude is also expressed to Ilessrs. P. H, Aldenderfer,
0. Pogue, C. F. Thayer, J. S. Widder, R. J. Zahransky, and
Hiss Joan Shillis for their assistance. CONTENTS
Page
INTRODUCTION...... 1
LITERATURE R E V IE W ...... 3
MATERIALS AND M ETH O D S...... 18
RESULTS ...... 31
DISCUSSION...... 83
SUMMARY...... 93
BIBLIOGRAPHY...... 95
AUTOBIOGRAPHY...... 100
ill TABLES
Table Page
1 Electrophoretic Analysis of Rabbit Serum Preparations Used in Immunizing Chickens ...... 32
2 Titration of Normal and Imnune Chicken Sera with Isosensitized Group A Rabbit Erythrocytes in 0.85 Per Cent Sodium C hloride ...... 35
3 Normal and Immune Chicken Sera Adsorbed with Rabbit Red Cells Titrated with Isosensitized Group A Rabbit Brythrocytos in O.85 P®r Cent Sodium C h l o r i d e ...... 36
4 Titration of Normal and Immune Chicken Sera with Isosensitized Group A Rabbit Erythrocytes in 8.0 Per Cent Sodium C hloride ...... 37
5 Normal and Immune Chicken Sera Adsorbed with Rabbit Red Cells Titrated with Isosensitized Group A Rabbit Erythrocytes in 8,0 Per Cent Sodium C hloride ...... 39
6 Titration of Normal and Immune Chicken Sera with Sensitized Sheep Erythrocytes in 0.85 Per Cent Sodium C hloride ...... 40
7 Titration of Normal and Immune Chicken Sera with Normal Sheep Erythrocytes in O.85 Per Cent Sodium C h l o r i d e ...... 41
8 Normal and Immune Chicken Sera Adsorbed with Rabbit Erythrocytes Titrated with Sensitized Sheep Erythrocytes in O.85 Per Cent Sodium Chloride . . . 43
9 Titration of Normal and Immune Chicken Sera with Sensitized Sheep Erythrocytes in 8.0 Per Cent Sodium C hloride ...... 44
10 Normal and Immune Chicken Sera Adsorbed with Rabbit Erythrocytes T itrated with Sensitized Sheep Erythrocytes in 8.0 Per Cent Sodium Chloride. . . . 45
iv TABLES— (Continued)
Page
11 T itration of Normal and Imnune Chicken Sera with. Rabbit Serum Cohn Fraction II (Gamma Globulin) Coupled to Human E r y th ro c y te s...... 48
12 T itration of Normal and Immune Chicken Sera with Rabbit Serum Cohn Fraction III (Beta Globulin) Coupled to Human E r y th ro c y te s ...... 49
13 T itration of Normal and Immune Chicken Sera with Rabbit Serum Cohn Fraction IV (Alpha Globulin) C oupled to Human E ry th ro c y te s ...... 50
14 T itration of Normal and Immune Chicken Sera with Rabbit Serum Cohn Fraction IV-1 (Alpha Globulin) C oupled to Human E ry th ro c y te s...... 51
15 T itration of Normal and Immune Chicken Sera with Rabbit Serum Cohn Fraction IV-4 (Alpha Globulin) C oupled to Human E r y t h r o c y t...... e s 52
16 T itration of Normal and Immune Chicken Sera with Rabbit Serum Cohn Fraction V (Albumin) Coupled to Human E r y th ro c y te s ...... 53
17 T itration of Normal and Immune Chicken Sera with Chicken Egg Albumin Coupled to Human Erythrocytes . 54
18 Composite of Titrations of Normal and Immune Chicken Sera with Rabbit Serum Cohn Fractions C oupled to Human E ry th ro c y te s...... 55
19 Electrophoretic Analysis of Commercially Prepared Rabbit Serum Cohn Fractions ...... 57
20 The Hemagglutination of Sensitized Erythrocytes by Anti-Na„+Al Chicken Sera After Adsorption with Cohn Rabbit Fraction II (Gamma Globulin) ...... 66
21 The Hemagglutination of Sensitized Erythrocytes by Anti-NRS Chicken Sera A fter Adsorption with Cohn Rabbit Fraction II (Gamma Globulin) ...... 67
22 The Hemagglutination of Sensitized Erythrocytes by Anti-NH.+F Chicken Sera After Adsorption with Cohn Rabbit Fraction II (Gamma Globulin) ...... 68
v TABLES— (Continued)
Page
23 The Hemagglutination of Sensitized Erythrocytes hy Anti-HRS+F Chicken Sera After Adsorption with Cohn Rabbit Fraction II (Gamma Globulin) ...... 69
24 The Hemagglutination of Sensitized Erythrocytes by Anti-Na«+Al Chicken Sera After Adsorption with Cohn Rabbit Fraction III (Beta Globulin) ...... • 70
25 The Hemagglutination of Sensitized Erythrocytes by Anti-HRS Chicken Sera After Adsorption with Cohn Rabbit Fraction III (Beta Globulin) ...... 71
26 The Hemagglutination of Sensitized Erythrocytes by Anti-NH.+F Chicken Sera After Adsorption with Cohn Rabbit Fraction III (Beta Globulin) ...... 72
27 The Hemagglutination of Sensitized Erythrocytes by Anti-HRS+F Chicken Sera After Adsorption with Cohn R ab b it F r a c tio n I I I (B eta G lo b u lin ) ...... 73
28 The Hemagglutination of Sensitized Erythrocytes by Anti-Nap+Al Chicken Sera After Adsorption with Cohn Rabbit Fraction IV (Alpha Globulin) ...... 74
29 The Hemagglutination of Sensitized Erythrocytes by Anti-HRS Chicken Sera After Adsorption with Cohn Rabbit Fraction IV (Alpha Globulin) ...... 75
30 The Hemagglutination of Sensitized Erythrocytes by Anti-HH.+F Chicken Sera After Adsorption with Cohn Rabbit Fraction IV (Alpha Globulin) ...... 76
31 The Hemagglutination of Sensitized Erythrocytes by Anti-HRS+F Chicken Sera After Adsorption with Cohn Rabbit Fraction IV (Alpha Globulin) ...... 77
32 The Hemagglutination of Sensitized Erythrocytes by Anti-Na-+Al Chicken Sera After Adsorption with Cohn Rabbit Fraction V (Albumin) ...... 79
33 The Hemagglutination of Sensitized Erythrocytes by Anti-HRS Chicken Sera After Adsorption with Cohn Rabbit Fraction V (Albumin) ...... 80
34 The Hemagglutination of Sensitised Erythrocytes by Anti-HH.+F Chicken Sera After Adsorption with Cohn Rabbit Fraction V (Albumin) ...... 81 vi TABLES— (Continued)
T a b le P age
35 The Hemagglutination of Sensitized Erythrocytes hy Anti-HRS+F Chicken Sera A fter Adsorption with Cohn Rabbit Fraction V (Album in) ...... 82
vii FIGURES
Figure Page
1 Electrophoretic Patterns Representative of Rabbit Serum Components Used to Immunize C hicks...... 33
2 Electrophoretic Patterns of Rabbit Serum Cohn Fractions at Different Dilutions ...... 58
3 Total nitrogen Precipitated From 0*5 ml of Chicken Antisera by Adding Various Amounts of Rabbit Serum Cohn Fraction I I ...... 60
4 Total Nitrogen Precipitated From 0*5 ml Chicken Antisera by Adding Various Amounts of Rabbit Serum Cohn Fraction I I ...... 6 l
5 Total Nitrogen Precipitated From 0*5 nil of Chicken Antisera by Adding Various Amounts of Rabbit Serum Cohn Fraction I I I ...... 62
6 Total Nitrogen Precipitated From 0*5 nil of Chicken Antisera by Adding Various Amounts of Rabbit Serum Cohn Fraction IV ...... 63
7 Total Nitrogen Precipitated From 0*5 ml of Chicken Antisera by Adding Various Amounts of Rabbit Serum Cohn Fraction V ...... 64
viii INTROOTCTION
Anti sera specific for human globulin are commonly used today for the detection of incomplete antibody on the cells and in the sera of individuals with so called "immunologic” diseases which are accompanied by either iso- or auto-antibodies*
Quite often rabbits are employed as experimental models simulating these human maladies and antiglobulin sera equivalent to
Coombs' sera must be produced in other species*
Many attempts to determine the best method for producing antisera to human globulins and the specificity of these sera have been recorded* However, few attempts have been made to determine the sensitivities and specificities of methods used to detect rabbit antibodies on rabbit erythrooytes*
Ho one procedure is conpletely adequate to ascertain the properties of antiglobulin sera and to this end a variety of methods were used in this study to express antibody activity qualitatively and quantitatively*
Chiokens were immunized with rabbit serum globulins and serum and the antiglobulin activity tested in a number of ways* Essentially, the antiglobulin sera were tested for their antiglobulin capacity with sensitized red oells in the usual 0.85 per oent sodium chloride and with 8*0 per cent HaCl as diluents since the latter is known to enhance precipitating antibody in chicken serum*
1 2
The specificity of these sera for Cohn fractions of rabbit
serum was also examined by their capacity to hemagglutinate rabbit and human red oells to which these fractions had been coupled by bis diazotized benzidine.
As with anti-human globulin sera, quantitative analyses of precipitates of rabbit serum fractions and the various antiglobulin chicken sera revealed a sim ilar lack of specificity between the various rabbit serum globulins. However, supernatants from antisera adsorbed with various fractions and tested with cells sensitized with rabbit antibody indicated the globulin classification of these anti b o d ie s . LITERATURE REVIEW
The use of antiglobulin sera to detect the presence of anti
body was first employed by Uoresohi (1908). The technique utilized
nonagglutinating dilutions of specific anti—bacterial and anti-red
blood cell sera reacted against their specific antigens. The sensi
tized cells were washed and then exposed to an antiglobulin serum,
hence causing agglutination to occur.
Many years later, Coombs, ilourant and Race (1945)» unaware
of Lloreschi's work, applied the same principle in demonstrating in
complete Rh antibodies.
Although Lloreschi, Mourant, and Race deserve recognition for
illum inating this procedure, the name Coombs’ test has been used by many workers as a synonym for the antiglobulin test.
A quantitative modification of the antiglobulin test has
demonstrated that the amount of sensitizing Rh antibody in erythro blastosis and of auto-antibody in acquired hemolytic anemia on erythrocytes could be correlated to the severity of erythroblastosis fetalis and the blood dyscrasia (Weiner and Gordon, 1953)*
Coombs and Uourant (1947) immunized rabbits with various preparations of human serum and its globulins and found that many were satisfactory, but alum precipitated human serum caused the pro duction of antiserum which best deteoted incomplete Rh antibodies on human erythrocytes. These workers then immunized rabbits with Cohn
3 fractions of human serum and utilized these same fractions to inhibit the anti-Rh globulin test. They found that anti-3) (Rho) antibody was mainly present in the gamma globulin fraction of human serum, in spite of the fact that Rh antibodies were elicited and partially neutralized by highly purified preparations of other serum proteins*
Repeating the same technique, Dacie (1951) found that the antiglobulin agglutination of human erythrocytes sensitized with in complete anti-X) (Rho) or the "nonspecific warm antibodies" of acquired hemolytic anemia was inhibited by small concentrations of gamma globulin, suggesting that these incomplete antibodies were gamma globulin. However, the antiglobulin agglutination of erythrocytes coated with normal cold antibody, with the cold antibody of acquired hemolytic anemias, and with the incomplete anti-group A antibody was only inhibited by very high concentrations of gamma globulin. It appears that these antibodies were not gamma globulin, and possibly some contaminant caused inhibition.
Rabbit antisera to human serum or globulins show a variety of specificities. For instance, these sera have been found to ag glutinate erythrocytes from patients with various types of hemolytic anemias and cells sensitized with anti-Rh antibodies, normal incomplete cold antibodies, or anti-group A human serum. Absorption with one type of sensitized erythrocyte made possible the preparation of anti globulin which no longer agglutinated the type used for absorption, but still agglutinated one or more other typos of sensitized cells
(Crawford and Mollison, 1951? Renton, 1952). 5
By differential absorption of Coombs' antisera, Cutbush et a l. (1955) showed that some human antibodies specific for human erythrocytes were gamma globulin and probably concentrated in the gamma—2 portion of the serum* On the other hand, some anti—human erythrocyte antibodies (notably, the cold antibodies) appeared to be in the alpha or beta portion of the serum and seemed to need another serum component for sensitization,
Dacie (1953) and co-wrrkers (Dacie, Crookston, and Christen son, 1957) found that certain cold antibodies would sensitize human erythrocytes without causing agglutination. The antiglobulin ag glutination of cells coated with these antibodies was not inhibited by gamma globulin and was dependent on the presence of C^, Cg, and
C^ components of complement. In contrast, antiglobulin agglutination of cells coated with various warm antibodies was inhibited by gamma globulin and independent of complement.
The purification and analysis of serum proteins by different methods has not alv/ays produced the same results. Imminoelectro- phoresis has revealed that in a human serum^anti-human serum system, the gamma globulin antigen is present in the gamma-2, gamma-1, beta, and alpha-2 globulin fractions of human serum. Moreover, this broad overlap of gamma globulin is in contrast to immune precipitation of starch gel diffusion systems, in which gamma globulin was well sepa rated from the beta and alpha portions. Although fractionation may be achieved by any one method, the antibodies and gamma globulin fractions may be revealed as complex mixtures by another. It is also 6 well known that the presence of specific antibodies in any electro phoretic fraction may vary depending on the species immunized, the antigen employed and the procedure of immunization (Williams and
Grabar, 1955? Porter, I960),
Recently, it has been shown that the injection of rabbits with bacteria coated with homologous rabbit antibody caused the pro duction of antisera containing isoprecipitins of various specificities which precipitated the sera of other rabbits. The authors postulated that antibodies with different specificities have different antigenic structures. By these methods, five rabbit gamma globulin antigens, which are of an hereditary character and seem to form three genetic systems, were obtained (Milgrom and Dubiski, 1957; Kelus, I960;
Dubiski, Dubiska, Skalba, and Kelus, 196l).
Similar results were obtained by Dray and Young (1958) when they immunized rabbits with normal rabbit sera plus paraffin oil type adjuvants. Isoprecipitins were found to serum antigens v/ith the electrophoretic m obilities of alpha, beta, and gamma globulins,
Adler (1956) reported that guinea pigs immunized with normal rabbit sera or rabbit globulin failed to yield antisera which would appreciably agglutinate sheop erythrocytes sensitized with sub- agglutinating amounts of anti-sheep erythrocyte rabbit serum. How ever, injection of guinea pigs v/ith Y/ashed immune aggregates to sheep cells and their homologous rabbit antibodies elicited production of potent antisera which caused hemagglutination of sensitized cells
(after proper absorptions of antisera with sheep cells). It was found also that the addition of normal rabbit sera to sensitized 7 sheep erythrocyte antigens retarded the immune response of the guinea pig and resulted in less potent antiglobulin serum#
Kelner and Hadel (1953a, 1953b) created experimental con ditions in rabbits which simulated human erythroblastosis fetalis#
Controlled matings resulted in fetuses with red blood cells which contained an antigenic factor not present in the mother rabbit.
Anti-rabbit globulin guinea pig serum was used to detect iso-iraimme antibodies prosent in the serum of the mother and in the serum and on the erythrocytes of the nev/born.
As an aid in differentiating rabbit red blood cell antigens,
Heard (1955) Has utilized anti-rabbit globulin goat serum to detect rabbit immune iso-antibodies on non-agglutinating sensitized cells#
Although chickens are used infrequently in experimental or practical immunological procedures, they have been found to be ex cellent antibody producers in response to small antigenic doses#
Chicken antisera have been used to differentiate and compare the antigenic specificities of normal adult, fetal and sickle cell hemo globins (Goodman and Campbell, 1953) and the nephlometrio quantitation of human serum gamma globulins and albumins (Goodman et a l .« 1957)*
LSunoz and Becker (1952) have utilized chicken antisera to study edestin, a protein which is insoluble in 0.85 per cent sodium chloride (lTaCl) but soluble in 8.0 per cent NaCl. Chickens have also proved to be effective producers of "coombs’ antisera11 used in the detection of anti-Rh antibodies (Jankovic, Simnonovic, and Lincoln,
1 9 5 8 ). 8
As early as 1903 Ewing and Strauss described the use of chicken antisera for medico-legal purposes. Later, Hektoen (1918) immunized chickens with large amounts of mammalian sera, injecting ten to twenty ml of sera intravenously and twenty to thirty ml intra abdominally. On the average, antibody titers reached their highest peak in nine to twelve days and thereafter declined.
Wolfe (1942) further investigated chicken antisera by in jecting chickens with very small quantities of sheep, bovine, and buffalo sera. By using the interfacial precipitin test technique, he found that the titer of chicken antisera increased in vitro on a g in g *
Wolfe and Bilks (1946) found that the iri vitro titers of chickens immunized with various serum proteins started to increase
11 hours after bleeding and peak titers were reaohed after five to eight days of in vitro aging. Titers determined within twenty-four hours were not indicative of the eventual final titers. Later,
Gengozian and Wolfe (1957) noted that storage of anti-bovine serum albumin (BSA) chicken serum for seven to twenty-one days at 4°C or
-20°C resulted in an increase in interfacial titers and quantitative determinations of precipitated nitrogen performed in a 1*8 per cent sodium chloride diluent. A decrease in total nitrogen precipitation occurred in aged antisera when quantitative tests were performed in
8.0 per cent llaCl. However, the nitrogen precipitated in the presence of 8,0 per cent NaCl was always greater than that precipitated in 1,8 per cent KaCl, regardless of the age of the antiserum. The addition of fresh normal chicken serum increased the amount of nitrogen 9 precipitated in 1*8 and 8.0 por cent NaCl, but did not change inter- facial titers. This demonstrated that there were no normally oc curring labile inhibitory factors in chicken antisera which diminished on aging. The authors suggested that the rise in quantitative nitrogen determinations elicited in 1*8 per cent NaCl diluents might be due to solubility factors resulting from aggregation of serum proteins and partial denaturation of proteins on aging. Also, the same non-specific material which coprecipitates with fresh antiserum in the quantitative nitrogen determination of tests carried out in
8.0 per cent NaCl may interfere with the reaction between antigen and antibody in the interfacial test. Aging may remove this substance.
Conversely, chickens which were immunized with human gamma globulin by Deutsch, Nichol, and Cohn (1949) did not exhibit a rise in antibody titer when the sera were aged in vitro from zero to nine days, whether estimated by quantitative nitrogen determinations or interfacial precipitation. However, a definite decrease in both alpha and gamma globulin was apparent in electrophoretic patterns of immune sera samples from which the antibody was removed by antigen absorption. According to these authors, quantitative precipitation assays established the amount of alpha globulin involved contributed approximately one-half of the total nitrogen found at the point of maximal precipitation.
It was in 1951 that Goodman, Wolfe, and Horton (1951) first found that increasing the concentration of the NaCl diluent to 8.0 per cent caused the maximum precipitation of antigen-antibody complexes employing bovine serum albumin and human gamma globulin as antigens 10
and their corresponding ohioken antisera. The greatest total pre
cipitation was found in high antigen excess. These antisera then
were fractionated and it was found that the euglobulin aliquot pro
duced a higher total nitrogen yield at 8.0 per cent NaCl than at 1.0
per cent NaCl, thus showing that increased precipitation reactions
in 8.0 per cent NaCl does not involve the coprecipitation of albumin
or pseudoglobulin portions of the antiserum. These authors con
jectured that 8*0 per cent NaCl might decrease the solubility of
antigen-antibody complexes and/or that the dissociation of complexes
may be greater in 1.0 per cent NaCl than in an 8.0 per cent m ilieu.
Goodman and Wolfe (1952) demonstrated that the antisera of
chickens, owls and pheasants immunized with bovine serum albumin
yielded increased antibody precipitation in diluent concentrations up to 12.0 per cent NaCl. Loss of recoverable antibody in low sodium
chloride concentrations could be reversed by raising the salt con
centration, and maximal antibody precipitation in 8.0 and 12.0 per
cent NaCl solutions could be partially dissolved by resuspension in
1.0 per cent NaCl.
In order to examine the effects of various salts and con
centrations on chicken serology, Goodman, Wolfe, and Goldberg (1954) utilized the following Hofmeister series of sodium salts,
SCN< I < Br < NO- < Cl < SO. 3 4
as diluents for anti-BSA and anti-human gamma globulin chicken sera
and their homologous antigens. (From left to right is an illustration
of the ability of these anions to salt proteins out of solution. The 11
reverse is the ability of these anions to bind proteins.) With any
particular ionic species differences in the amounts of specifio pre
cipitation were produced by varying the ionic strength, and differ
ences were noted using any one ionic strength of different ions. At
low ionio strengths (up to 0,26) precipitation was greater in the
iodide medium than in any other ionic media. When the ionic strength
of the iodide medium was increased, precipitation diminished. In hibition was more striking in SCN media and thus suggests that a
certain amount of anion binding by chicken antibody-antigen com plexes increase precipitation, but further binding decreases pre cipitation. With anions such as Cl and S0^, precipitation increased with each increase of ionic strength, and maximum precipitation with chicken antisera systems in the various sodium salts occurred at about 1.5 ionic strengths of Cl and S0^. Hence, these results sug gest that a salting out of soluble antibody-antigen aggregates is an important condition for obtaining complete precipitation with chicken antisera.
In an attempt to determine the specific reactants of chicken antieera, Goodman and Ramsey (1957) immunized chickens with human serum albumin, human gamma globulin, or orosornucoid. The resulting antisera were then absorbed in 8.0 per cent NaCl or 13.0 per cent
NaCl with the equivalence zone amount of antigen. The absorbed anti sera were examined by means of electrophoretic patterns and exhibited a marked reduction in gamma globulin but not alpha globulin. The serum gamma globulin fractions of immunized chickens were also sub jected to the same methods and were found to give comparable results. 12
The alpha globulin portions of the same antisera showed no copre—
cipitation in systems employing immune chicken gamma globulin.
Various chicken gamma globulin fractions were stable at 8.0 and
12.0 per cent NaCl but some showed instability in 1.0 per cent NaCl.
In certain systems these latter sometimes yielded coprecipitation of non-specific gamma globulin in 1.0 per cent NaCl but not in the
8.0 or 12.0 per cent solutions.
To elucidate further the mechanisms involved in chicken antibody-antigen reactions, Banovitz, Singer, and 7/olfe (1959) 0®“ ployed bovine serum albumin antigen and its homologous chicken anti- serum or ammonium sulfate precipitated immune globulin. The complexes of these reactants were made soluble and then studied in an electro phoretic Tiselius cell. The results revealed two well-defined antigen-antibody complex peaks, whereas using rabbit antisera with the same specificity results in only one peak. By differentially precipitating the BSA—anti-BSA chicken serum in 1.0 and 8.0 per cent NaCl it was possible to fractionate these two peaks partially and it was then demonstrated that precipitation in 8*0 per cent NaCl was a specific reaction. It is possible that these two peaks were the result of univalent and divalent antibodies; however, these authors were of the opinion that all the antibodies were probably divalent but varied in their binding abilities.
In a more extensive publication Banovitz and Wolfe (1959) attempted to repeat the work of Deutsch, ITichol, and Cohn (1949) by using human gamma globulin and bovine serum albumin and the entire globulin fraction or gamma globulin from the respective homologous 13 chicken antisera* Electrophoretic examination of absorbed and un absorbed antisera indicated that in essence only the gamma globulin component of chicken antisora contributes to antigen—antibody com plexes. It was not possible to demonstrate alpha globulin in re dissolved human gamma globulin—an ti—human gamma globulin chicken serum aggregates, and these workers could not substantiate the con tention that alpha globulin contributed up to 50 per cent of the protein of specific precipitates (Deutsch, Nichol, and Cohn, 1949).
Using agar diffusion and ultracentrifugation techniques
Llakinodan, Gengosian, and Canning (1960) attempted to determine the mechanism or mechanisms which were involved in the effects of aging antiserum and increasing the salt concentration for serological re actions. It was found that a normal serum protein would copre cipitate with BSA—anti-BSA chicken serum aggregates in 1.5 M NaCl regardless of the zones of precipitin reaction or the age of the antiserun. This protein was shown to be a macroglobulin with the mobility and salting-out properties of a beta or gamma globulin. In
0.15 II NaCl, however, coprecipitation occurred with aged antiserum but not with fresh antiserum. These authors also noted the formation of a flocculating or gelatinous material associated with aging of the antisera.
Recently, Orlans, Rose, and Ilarrack (1961) have studied fowl antibodies to rabbit gamma globulin, bovine serum albumin, and the epsilon toxin of Cl. welchii. Specific precipitates wore redissolved either in antigen excess or in solutions of lower salt concentration than those in which they had been formed. These were used to measure 14
the sedimentation and diffusion coefficients and the electrophoretic
m otilities of the soluble complexes of fowl antibody with antigen.
The fowl antiserum to any one antigen was found to contain two types
of homologous antibodies having the same electrophoretic mobility,
but giving two distinct bands of precipitation with rabbit antiserum
prepared against chicken serum globulin* The values obtained for the
molecular weight of soluble complexes indicate that ono type of
chicken antibody has a molecular weight of about 600,000 and the other
180,000. The data also suggested that the latter antibody combined
with only one molecule of antigen in antigen excess. Both types of
antibodies were precipitated by antigenin 0*9 and 8,0 per cent NaCl.
The largest precipitates formed at higher salt concentrations con
tained more of the low molecular weight antibody, together with
another component that was neither antigen nor antibody. It is pos
sible that the macroglobulin found by Makinodan et a l. (i960) may be
this latter component or the heavy antibody.
The study of the effect of physical factors on the serology
of chicken sera has been mainly concerned with the effects of varied
salt concentrations on the precipitin reaction. As a departure from
this line, Gengozian and Wolfe (1956) noted that the dilution of
anti-bovine serum albumin in 8.0 per cent NaCl prior to combination
with BSA resulted in decreased antibody recovery when the two were
combined. This diraunition was also apparent when the volume of the undiluted reacting system was increased. Ammonium sulfate fractions
of this serum yielded similar results after dilution. When normal
serum (stored for three months at -20°C) was used as the volume 15 diluent, greater total amounts of nitrogen were precipitated per ml.
This suggests that some of the antibody population of chicken anti
sera are salt labile and that perhaps low grade antibody may have a high degree of dissociation and undergo desorption in large volumes of 8.0 per cent NaCl*
Wolfe, Mueller, and Neese (1959) have found that after in itially incubating fowl antiserum and its antigen, larger amounts of precipitate were obtained by centrifugation at 22°C than at 4°C.
Precipitation was maximal after three hours incubation at 37°C and immediate warm centrifugation, in contrast to additional incubations
(cold or warm) and/or cold centrifugation.
The antibody tite rs of male and female chickens immunized with bovine serum and bovine serum albumin has illustrated that there is essentially no difference in antigenic response due to sex and that chickens become immunologically mature at five weeks of age
(Wolfe and Dilks, 1948; Wolfe, M ueller, Neese, and Tempelis, 1957).
When chickens were immunized with two different amounts of bovine serum albumin (40 mgs/kg body weight and 200 mgs/kg body weight) the peak antibody titer was attained earlier with the smaller amount of antigen, but maxitmm antibody production occurred with the larger amount of antigen. The subcutaneous route elicited greater antibody content in sera than the intravenous method when large amounts of antigen were used. A single injection of antigen yielded high titered sera within five to eight days, but whether small or large, when single or triple doses of inocula were used, the antibody peak was greatest from nine to thirteen days after the initial injection. The 16 antibody content was very low three to four weeks later, but per sisted in some cases for two months after a m ultiple series of in- jeotions (Wolfe and D ilks, 1946j Brown and Wolfe, 1954)*
Schmidt and Wolfe (1953) estimated the antibody tite rs of ohickens individually immunized with one of either six mammalian proteins, two plant proteins or type one pneumococcal polysaccharide*
In general, animal proteins resulted in higher antibody tite rs than plant proteins although thyroglobulin, beef hemoglobin and the poly saccharide could only be detected qualitatively by flocculation t e s t s *
When bovine serum albumin and human gamma globulin were in jected sinultaneously, the chicken antibody tite rs to both antigens were lower than their respective controls, which were injected with only one antigen. The anti—human gamma globulin antibody content reached a peak before the BSA and its maximum tite r was comparable to its control. However, the antibody response to BSA was reduced.
These results suggest that the human gamma globulin which has a high molecular weight may have a crowding out effect on the lower weighing
BSA. The simultaneous injection of BSA and bovine hemoglobin shows no detectable antibody response to hemoglobin in the experimental or control animals, but response to BSA was considerably reduced in chickens receiving both as compared to the chickens injected only with BSA. The authors postulate that although BSA and bovine hemo globin are approximately the same weight, the bovine hemoglobin may compete with the BSA for antibody producing reactive sites (Abramoff and Wolfe, 1956). 17
Anaphylaxis of chickens immunized with BSA was hest demon
strated when the animals were challenged six to eight days after
sensitization and severity increased with increase in the content
of serum antibody. Anaphylaxis was best avoided when the birds were
challenged before the fourth day or after the fifteenth day following
sensitization.
Ali (1959) lias found that the naturally occurring rabbit
hemagglutinin in normal chicken serum is best demonstrated in 0.85
per cent ITaCl and only weakly aotive in an 8.0 per cent ITaCl solution.
In contrast, the hemagglutinin in anti-rabbit globulin chicken serum was best demonstrated in O.85 per cent UaCl as well as in 8.0 per
cent ITaCl and anti-rabbit globulin sera absorbed with normal rabbit
cells still showed high titers of hemagglutinin when titrated in 8.0
per cent ITaCl solution. This seemed to indicate that antibody pro
duced in response to rabbit serum globulin is specific for a globulin
on normal rabbit red blood cells and its maxi mm agglutination occurs
only in the presence of high salt concentration. MATERIALS AND METHODS
Preparation of Rabbit Globulin and Serum Fractions
Normal Rabbit Sera
The blood of at least three rabbits was obtained by cardiac puncture and placed in a 37°C incubator for three hours* The sera were decanted from the clots, pooled and oentrifuged at 1,500 rpm for five minutes to remove any remaining cells and then stored at —20°C.
Ammonium Sulfate Precipitated Globulin
One-half volume saturated ammonium sulfate ( ) was slowly added to the serum with constant stirring. The pH was adjusted to 7*8 with 1*0 N sodium hydroxide. After incubation at room temper ature for one hour, the suspension was centrifuged at 7,000 rpm for five minutes. The supernatant was discarded and the sediment was dissolved in 0.9 per cent sodium chloride (NaCl) to the original volume of the serum. The process of adding (RH^^SO^, adjusting pH, incubation and centrifugation was repeated. The precipitate was then washed two times with a one—third saturated (HH^gSO^ solution using one-half the original volume of serum. After redissolving the pre cipitate in 0.9 per cent ITaCl to the original volume of serum, the o globulin solution was dialyzed against 0.9 per cent ITaCl at 4 C for twenty-four hours (Cushing and Campbell, 1957)*
18 19 Alum Adsorbed Rabbit Globulin
One ml of rabbit serum was placed into each of a series of centrifuge tubes. Wine ml of a 20*0 per cent sodium sulfate solution were added gradually with shaking. The tubes were then incubated in a 37°C water bath for three hours with frequent shaking, after which the tubes were centrifuged at 12,000 rpm for ten minutes. The super natants were decanted and physiological saline was added to make the volume up to 1*0 ml and to redissolve the globulin.
Five ml of this globulin solution were diluted with 16.0 ml of distilled water in a 50*0 ml centrifuge tube. To this solution, 18.0 ml of a 10*0 per cent solution of potassium alum in distilled water were added. The pH was adjusted to 6*5 with 5 N sodium hydroxide to precipitate the globulin and alum. This was centrifuged at 1,500 rpm for five minutes. The supernatant fluid was aspirated and 40*0 ml of saline containing merthiolate at a dilution of l/lOOO were added. The precipitate was resuspended in this solution, centrifuged at 1,500 rpm for five minutes, and the supernatant fluid aspirated.
The same process was again repeated. Twenty ml of merthiolate saline solution were added to the precipitate, mixed, and resuspended for injection (modification of Proom, 1943)•
Preparation of Immune Chicken Sera
Anti-Wormal Rabbit Sera
Each chicken was injected intraperitoneally with 0*5 ml of pooled rabbit Bera on two consecutive days and then with 1*0 ml each day for four days. 20
Anti-Normal Rabbit Sera Enulsified with Freund's Adjuvant
An enulsion consisting of 2*5 ml normal rabbit sera, 2*3 ml
Falba and 2*5 ml of mineral oil containing 1*0 mg per ml killed
Hycobacterium tuberculosis var. hominis was injected in equally
divided doses into each thigh muscle of a chicken. Falba and the
mycobacterial suspension are termed Freund's adjuvant and each is
added in an amount equal to the antigen.
Anti-(UH^gSO^ Precipitated Rabbit Globulin Enulsified with Freund’s A djuvant
An emulsion consisting of 2.5 ml (KH^J^SO^ precipitated
globulin, 2,5 ml Falba, and 2.5 ml of the oil and bacteria suspension was injected in equally divided doses into each thigh naiscle of a
c h ic k e n .
Anti-Sodium Sulfate Precipitated Rabbit Globulin Adsorbed on Alum
Each chicken was injected with 5*0 ml of this globulin sus pension into each thigh muscle. The total of 10,0 ml represented the protein of 2.5 ml of the original globulin suspension.
Chickens and Treatment of Antisera
Adult white leghorn chickens were obtained from the Ohio State
University Poultry Science Department.
In the preliminary studies one chicken was used in the course of each immunization procedure. After a single course of immunization,
5.0 ml of blood was obtained by cardiac puncture from each chicken at 21 various intervals up to twenty-five days. The "blood was allowed to clot and the serum was decanted and centrifuged to remove all cells*
The antiserum to each antigenic preparation was stored at -20°C,
Twelve days after the last serum was stored, all the sera were thawed and heat inactivated at $6°C for thirty minutes. These antisera were adsorbed with ra"b"bit erythrocytes and then titrated against sensitized erythrocytes, the titers were recorded and a decision was made as to the "best time to exsanguinate the fowl in order to obtain highest potency antisera. The chickens were refreshed with a single in jection of the same antigenic preparation (l.O ml of normal rabbit sera in the repeatedly injected chicken) and exsanguinated by bleeding from the heart seven to nine days later. These antisera are desig n a te d :
Designation Immunizing Preparation
Anti-HH^+F precipitated globulin with Freund’s adjuvant
Anti-ITa^+Al lJa^SO^ precipitated globulin with alum adjuvant.
Anti-NRS+F Normal rabbit sera with Freund's a d ju v a n t
Anti-NRS Normal rabbit sera
Norm c h ic k None
These antisera and normal chicken serum were stored at —20°C for at least twelve days. They were all heat inactivated before use.
In the subsequent production of antisera each chicken was im munized with the proper antigen as before and three to five days later reinjected in the same manner (with the exception of the 22
chickens receiving normal rabbit sera each day for six days). Seven
to nine days after the last injeotion, the chickens were fasted for
24 hours and then exsanguinated by intracardiao puncture and the re
sulting antisera were stored at -20°C for a minimum of twelve days
and heat inactivated before use. These antisera were designated in a manner similar to the aforementioned antisera. These sera were used primarily for the quantitative precipitation tests.
Anti-Sheep Red Blood Cell Rabbit Sera
A pooled Bample of rabbit antisera to sheep red blood cells was obtained from the stock used in the serology course in the Depart ment of Ilicrobiology.
Iso-Immune Rabbit Sera - Anti-Group A Erythrocytes
Anti-group A erythrocyte iso-immune rabbit sera were obtained from Dr. Carl Cohen of the B attelle Uemorial Institute.
ITon-Agglutinating Sensitization of Sheep Erythrocytes
One-tenth ml of an anti-sheep erythrocyte rabbit sera sample was doubly diluted in phosphate buffered 0.85 per cent sodium chloride of pH 7.4 in agglutination tubes. To each tube was added 0.1 ml of a
2.0 per cent suspension of sheep erythrocytes in phosphate buffered saline. The tubes were shaken and incubated at 37°C for thirty minutes, centrifuged at 1,500 rpm for thirty seconds and read for ag glutination. The dilution in the last tube to demonstrate a positive hemagglutination was designated as one hemagglutinating unit. The next higher dilution was termed an one-half hemagglutinating unit. 23
Equal volumes of a 2.0 per cent sheep red blood cell sus
pension and an one-half hemagglutinating unit dilution were mixed
in a 50 ml oentrifuge tube* The mixture was agitated to insure
homogeneity and then placed in a 37°C water bath for thirty minutes.
The mixture was then removed and centrifuged at 1,500 rpm in a
clinical centrifuge. The supernatant was discarded and the sedimented
erythrocytes were resuspended in one hundred volumes of chilled
phosphate buffered saline. The cell suspension was reoentrifuged,
the supernatant aspirated, and the cells were washed again in cold
phosphate buffered saline. The wash fluid was again discarded and
the cells were once again reconstituted to their original 2.0 per
cent volume by the addition of phosphate buffered saline. The cells were dispersed in the phosphate buffered saline and lightly centri
fuged for the purpose of allowing any agglutinated aggregates to
settle to the bottom. Only the superior portions of the cell sus pensions were used in the experiments.
The same method was used for the non-agglutinating sensiti
zation of sheep red blood cells to be used in 8.0 per cent sodium
chloride (NaCi) solutions, except the cells were finally resuspended
in 8.0 per cent NaCi of pH 7*1*
Non-Agglutinating Sensitization of Rabbit Group A Erythrocytes
Nine volumes of 4*0 per cent rabbit erythrocytes in phosphate buffered saline were mixed with 1.0 volume of 0.33 psr cent Bacto— trypsin in phosphate buffered saline. This mixture was incubated at
37°C for fifteen minutes, washed and centrifuged twice in phosphate 24
'buffered saline, and then restored to its original 4*0 per cent volume with phosphate buffered saline*
A 4*0 per cent suspension of normal erythrocytes was also prepared from the same rabbit*
Two series of doubling dilutions of anti-A erythrocyte rabbit iso—immune serum were made in 0,05 ml amounts in phosphate buffered
saline in agglutination tubes. Into each tube of one series was added 0*05 ml of a 4*0 per cent trypsinized rabbit erythrocyte sus pension. Into the second series, the same procedure was followed with normal erythrocytes. The tite r of iso-immune serum with the trypsinized cells was compared to non-trypsinized cells and the highest dilution exhibiting a 4 plus reaction with trypsinized cells and negative reaction with normal cells was termed the sensitizing d ilu t i o n *
A 4*0 per cent suspension of rabbit group A erythrocytes was mixed with an equal volume of a sensitizing dilution of anti—group A rabbit serum in a 50 nil centrifuge tube, incubated at 37°C for 30 minutes, centrifuged, the supernatant aspirated, and the cells re- suspended in 50 volumes of chilled phosphate buffered saline. The cells were recentrifuged and washed again. The erythrocytes were finally reconstituted to the original suspension volume with phos phate buffered saline*
The same method was used for the non-agglutinating sensiti zation of rabbit group A erythrocytes to be used in 8*0 per cent sodium chloride, except the cells were finally resuspended in 8*0 per cent NaCi of pH 7*1* 25
Titration of Chicken Antisera to Rabbit Globulins and Sera with Sensitized Sheep and Rabbit Red Blood Cells
Doubling dilutions of normal and immune chicken sera were
made in phosphate buffered O.85 per cent NaCi pH 7»4 (phosphate buf
fered saline) and in an 8*0 per cent NaCi solution of pH 7*1» To
each tube was added an equal amount of sensitized 2*0 per cent sheep
erythrocytes, or 4*0 per cent rabbit red blood cells. The equal
amounts were 0*05 sil of rabbit cell suspension and 0*1 ml sheep cell
suspension. The tubes were then incubated at 37°C for thirty minutes,
centrifuged for thirty seconds at 1,500 rpm, and read for hemag
glutination.
As a control, the same procedures were carried out with normal sheep and rabbit red blood cell suspensions.
The same procedures were employed with normal and immune
chicken sera which were adsorbed with rabbit erythrocytes. These
sera were adsorbed with equal amounts of washed, packed, and pooled normal rabbit erythrocytes. This mixture was incubated at room temperature, with frequent agitation for thirty minutes, centrifuged
at 1,500 rpm, and the adsorbed serum was then removed.
Coupling of Rabbit Serum Fractions to Rabbit and Human Red Blood Cells
Cohn fractions of rabbit serum proteins were obtained from
Pentex Incorporated, Kankakee, Illinois. These included fractions II
(gamma), III (beta), IV (alpha), IV-1, IV-4 (alpha sub-fractions 1 and 4) and V (albumin). Chicken egg albumin was used as a control. 26
Six-tenths ml of a serum fraction solution containing 0*01 gm
of the fraction per ml was added to a centrifuge tube containing
0,15 ini of washed and packed rabbit erythrooytes. To this was added
1,5 ml o f a 1x15 dilution of bis-diazotized benzidine. The contents
of the tube were mixed and allowed to incubate at room temperature
for ten minutes. The tube was centrifuged, the supernatant decanted,
and the cells were resuspended in 10,5 ml of 1,0 per cent chicken
serum in phosphate buffered saline pH 7*4* (The chicken serum was previously adsorbed with an equal volume of rabbit erythrocytes.)
The tube was again centrifuged, the supernatant aspirated, and the cells resuspended in 7»5 ml of the 1,0 per cent chicken serum in phosphate buffered saline.
Doubling dilutions of normal and immune chicken sera were made in 0.1 ml amounts in phosphate buffered saline in round bottom agglutination tubes. One-tenth ml of the protein coupled erythrocyte
suspension was added to each tube. The tubes were shaken and allowed to incubate for one to two hours at room temperature. The tube bottoms were then observed for agglutination patterns graded as +
(positive) or - (negative) hemagglutination. The cells were then centrifuged at 1,500 rpm for one minute, and the hemagglutination again recorded.
This procedure proved unsatisfactory and the same technique was repeated, but human group 0 Eh positive erythrocytes were sub
stituted for rabbit erythrocytes and a phosphate buffer solution of pH 7»3 (no sodium chloride) replaced the phosphate buffered saline. 27
The method used was sim ilar to that described by Stavitsky and
Arquilla (1955).
Nitrogen Quantitation of Immune Precipitates
Cohn rabbit serum fractions II, III, IV, and V were used in the precipitation experiments. These serum fractions were diluted in 0.9 per cent sodium chloride solution. Various amounts of the solution were placed in micro—Kjeldahl digestion flasks containing two glass beads. One-half ml sulfuric acid was added to each flask.
The water waB evaporated gradually by boiling with constant agitation over a free flame until dense white fumes filled the flask and the residue had charred. The tubes were removed from the microburners and allowed to cool. Three to four drops of 30.0 per cent hydrogen peroxide were added directly to the solution. The tubes were then heated until the fluid became water clear and the heating was con tinued at a gently boiling temperature for five minutes. The material was then transferred to a 50*0 ml volumetric flask containing 0.8 ml of a 40.0 per cent sodium hydroxide solution and diluted to 25.0 ml with distilled water. The contents of the volumetric flask were mixed, cooled, and then 12.0 ml of the modified Nessler’s reagent
(Polin-Wu modification) were added and at once the contents were diluted to 50.0 ml with distilled water and mixed well. Quantities ranging from 0.1 and 1.0 ml volumes of standard solutions containing
0*01 and 0.1 mg of nitrogen, respectively, wore mixed in a 50.0 ml flask with the aforementioned reagents and treated in a sim ilar manner. These mixtures were all read in the Beckman KU spectro- 28 photometer at a wavo length of 48O m illi microns. The nitrogen determinations were calculated from a graph of the standards and also by simple proportions of optical density#
It should he noted that phosphates inhibit Hesslerization, no hydrogen peroxide should he placed in the blank, and that the ratio of sulfuric acid to the neutralizing sodium hydroxide solution is very critical#
Biuret Determinations
The same amounts of rabbit serum fractions used in the micro-
Kjeldahl te3t were each made up to one ml with phosphate buffered saline. To each sample, 1#5 ml of the biuret reagent were added#
The solution was mixed, incubated at 37°C for thirty minutes, and read at a wave length of 555 millimicrons in a Beckman DCJ spectro photometer (Kabat and liayer, 1961)#
For each protein antigen, a graph was prepared consisting of an abscissa which represented micrograms nitrogen as determined by the micro- Kjeldahl and an ordinate representing the optical density of the biuret for the same protein sample#
ITitrogen Determinations of Immune P r e c i p i ta t e s
Doubling dilutions of Cohn rabbit serum fractions were pre pared in 0,5 ml quantities in phosphate buffered saline in 12#0 ml conical graduated centrifuge tubes. To each tube was added 0.5 ml of normal or immune chicken sera. These sera were pooled aliquots of antisera ranging in age from three months to two years. Prior to 29 their use, they were heat inactivated and centrifuged at 8,000 rpm to clear them of debris. The reactant3 were then incubated at 37°C for three hours with frequent shaking and stored overnight in a 7 °0 refrigerator.
The tubes were then placed in a 4—6°C refrigerated centrifuge and centrifuged at 1,800 rpm for one hour. The supernatants were then decanted and stored at -20°C, The precipitates were suspended in ml of chilled 0,9 pe:r cent sodium chloride, centrifuged at
1,800 rpm for one hour, and the supernatants discarded. Two to three drops of 0,5 M Bodium hydroxide were added to each tube and gently heated until the precipitates were dissolved. To each tube, 1,5 ml of the biuret reagent were introduced, and the solution was increased to a volume of 2,5 ml by the addition of distilled water. All the tubes were incubated at 37°C for thirty minutes and then reqd in a
Beckman XRJ spectrophotometer at wavo length 555 m illim icrons. The readings were compared with the graphs made for each antigen and re corded as the total nitrogen of the reactants.
Determination of Excess Antigen and Antibody of Supernatants
Two drops of each supernatant were placed into two aggluti nation tubes. Three drops of anti-normal rabbit sera chicken sera were introduced into one tube to detect excess antigen, and appropriate amounts of antigen were placed in the second tube to detect excess antibody. The tubes were placed in a 37°C water bath for three hours and then overnight in a 7°C refrigerator. The next morning the con tents were read for precipitation and the reactions recorded. 30
Titration of Supernatants with Sensitized Red Blood Cells
The supernatants of each precipitation reaction were separated into two aliquots. Each supernatant of one group was adsorbed with an equal amount of washed and packed sheep red blood cells. Two drops of the adsorbed supernatants were doubly diluted in phosphate buf fered O.85 per cent sodium chloride of pH 7«4 (phosphate buffered saline). To each tube was added two drops of a 2.0 per cent sensitized sheep erythrocyte suspension. These reactants were incubated at 37 °0 for thirty minutes, centrifuged at 1,500 rpm for thirty seconds, and the intensity of agglutination recorded*
The second group of supernatants were each adsorbed with an equal volume of washed and packed rabbit erythrocytes. One drop of the adsorbed antisera was doubly diluted in phosphate buffered saline.
To each dilutic.. was added one drop of a 4*0 per cent solution of sensitized rabbit group A erythrocytes. These reactants were incubated at 37°C for thirty minutes, centrifuged at 1,500 rpm for thirty seconds, and the agglutination intensity read and recorded*
Electrophoretic Analysis
Electrophoretic analyses were performed on rabbit sera and globulin preparations used for immunization and on the Cohn fractions.
All samples were analyzed by the paper strip electrophoretic method using a Spineo analytrol. RESULTS
Several methods have been suggested for producing high
potency mammalian antisera to human globulin and serum antigens for
the purpose of detecting human antibodies on human erythrocytes.
Since the use of chickens anti sera to detect rabbit globulins is
relatively unexplored, it was decided that those methods used to
produce antiserum to human globulin would be utilized in producing
chicken antisera to rabbit 3erum preparations.
Intrinsic to this problem is the fact that chicken precipi
tating antibodies develop an increased titer on aging in vitro and that chicken antisera precipitate antigens more efficiently in 8,0
per cent sodium chloride (ITaCl) solutions than in 0,85 por cent
ITaCl s o l u tio n s .
Therefore, it was decided that the capacity of these antisera
to hemagglutinate sensitized cells would be observed after at least twelve days of aging and in different salt solutions.
The antigenic preparations were rabbit serum and ammonium
sulfate and sodium sulfate precipitated globulins which were quali tatively and quantitatively examined by the paper strip electro phoretic method. From the data in Table 1 and Figure 1 it is evident
that the ammonium sulfate and sodium sulfate precipitated antigens
consisted mainly of gamma globulins and some beta globulins. In one
case of sodium sulfate precipitation a small amount of globulin (7*0
31 32 per cent) was found in the alpha globulin region, but it is most likely that this was the trailing edge of the beta globulin.
TABLE 1
ELECTROPHORETIC ANALYSIS OP RABBIT SERUM PREPARATIONS USED IN IMMUNIZING CHICKENS
P e r C ent Composition* Serum Treatment Gamma B e ta Alpha Albumin
Normal Rabbit Sera 1 9 .3 1 3 .1 1 4 .2 5 3 .4
Ammonium S u lf a te 85*0 1 5 .0 — —
Sodium Sulfate 8 0 .0 2 0 .0 — —
■ * Average of five different analyses.
These antigens were then used as immunizing agents in the following manners The ammonium sulfate precipitated rabbit globulin suspension was incorporated into Freund*s adjuvant and introduced intram uscularly into the thighs of a chicken; the resulting antiserum was designated anti-NH^+F. The sodium sulfate precipitated rabbit globulin sus pension was adsorbed on to potassium alum and injected intranuscularly into the thighs of a chicken; the resulting antiserum was designated anti—Nag+Al. Normal rabbit serum was em ulsified with Freund*s adjuvant and inoculated into the thigh muscles of a chicken; the re sulting antiserum was designated anti—NRS+F. Normal rabbit serum was injected intraperitoneally in chickens for six days and the re sulting antiserum was designated anti-NRS. Normal chicken serum was designated norm chick. I. ELECTROPHORETIC PATTERNS REPRESENTATIVE OF RABBIT SERUM COMPONENTS USED TO IMMUNIZE CHICKS
Normal Rabbit Serum Na2S04 Precipitated Globulins (NH^g SO4 Precipitated Globulins 34
The chickens were hied at various intervals and the serum
samples were stored for at least twelve days. The sera were then
heat inactivated^ adsorbed with rabbit red blood cells and titrated
against rabbit erythrocytes sensitized with iso-immune antibody and
sheep cells sensitized with rabbit hemolysin. In general it was
found that the highest antibody titers for both systems (dilutions
of approximately 1/1024) were obtained between seven and eleven days
after the last injection of antigen. Titers of sera obtained fifteen
to seventeen days later had diminished and by the twenty-firBt and
twenty-fifth days the chicken antisera did not demonstrate any anti
globulin activity. Bach chicken was then refreshed with one in
jection of the antigen used previously and exsanguinated seven to
nine days later#
The results of chicken antisera titrated against isosensi
tized rabbit red blood cells in O.85 per cent sodium chloride (NaCi)
are listed in Table 2. It can be seen in the controls containing unsensitized rabbit erythrocytes that the titers of hemagglutinins
for normal rabbit erythrocytes were so high that the antiglobulin
activity had been masked. After adsorption with equal volumes of
packed normal rabbit erythrocytes, the antiglobulin antibodies were
readily demonstrable as illustrated in Table 3»
Hemagglutinins for normal rabbit erythrocytes were also
demonstrable in unadsorbed chicken antisera diluted in 8#0 per cent
NaCi (Table 4). Nonetheless, a high degree of antiglobulin activity
was demonstrable in anti—Na^+Al serum and anti-NRS serum before ad
sorption as compared to controls. A comparison of the titers of 35
TABLE 2
TITRATION OP NORMAL AND IMMUME CHICKEN SERA WITH ISOSENSI TIZED OROUP A RABBIT ERYTHROCYTES IN 0 .8 5 PER CENT SODIUM CHLORIDE
Reciprocal of Chicken Serum D ilution Serum______16 32 64 128 2 5 6 512 1024 2048 4096 8 1 9 2 1 6,384 C Norm Chick 4 4 4 4 3 1 —
Anti-NHH+F 4 4 4 3 II — — — ” --
A n ti Na2+Al 4444432 + — Anti-NRS 4 4 4 2 1 + - - - — — — Anti-NRS+F 4 4 4 4 3 2 1 — —
T itration of Normal and Immume Chicken Sera with Normal Group A Rabbit Erythrocytes in O.95 p e r cent Sodium Chloride Norm Chick 4 4 4 3 + — — — — — —
Anti-NHij+F 4 4 3 2 + — — — — — —
Anti-Na2+Al 4 4 4 4 3 + — — — — — -
Anti-NRS 4 4 3 1 1 — — — — — — Anti-NRS+F 4 4 4 2 1 — — — — — — 36
TABLE 3 NORMAL AND IMMUNE CHICKEN SERA ADSORBED WITH RABBIT RED CELLS TITRATED WITH ISOSENSITIZED GROUP A RABBIT ERYTHROCYTES IN 0.83 PER CENT SODIUM CHLORIDE
Reciprocal of Dilution S erum______16 32 64 128 256 512 1024 2048 4096 8192 16,384 C Norm Chick 2 1 1 + — — —
Anti-NH^+F 3 2 II + — — — — — — Anti-Nag+Al 4 4 4 4 4 3 2 + — — --
A nti-N R S 4 4 2 1 1 — — —
Anti-NRS+F 4444311 + — —
Normal and Immune Chicken Sera Adsorbed with Rabbit Red Cells Titrated with Normal Group A Rabbit Erthrocytes in O .85 Per Cent Sodium C h lo rid e Norm C h ick 1 + — Anti-NH^+F — — — — — — —
A n ti-N a 2+Al + — — Anti-NRS — — — — — — --
Anti-NRS+F 3 2 — — — — — 37
TABLE 4
TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH ISOSENSI TIZED QROUP A RABBIT ERYTHROCYTES IN 8 .0 PER CENT SODIUM CHLORIDE
Reciprocal of Dilution S erum______2 4 8 16 32 64 128 256 512 1024 2048 4096 8192 C
Norm Chick 4 2 2 11 + — — — — — — — —
Anti-NH4+F 2 11111+ — — — — — — —
A n ti-N a 2+Al 4443322 2 2 2 2 1 1 — A nti-N R S 4 4 4 3 3 2 1 1 1 — — — — — Anti-NRS+F 4 4 4 3 3 3 2 1 + — — — -- --
T itration of Normal and Immune Chicken Sera with Normal Group A Rabbit Erythrocytes in 8.0 Per Cent Sodium Chloride Norm Chick 2 4 11 + + — — — — — — — — Anti-NHjj+F 2 11 1 --- — — — — — — — — Anti-Na2+Al 4 3 4 4 2 1 1 1 + — -- — -- Anti-NRS 3 3 3 2 1 1 + — — — — — — —
Anti-NRS+F 4 3 3 3 2 1 38
hemagglutinins for normal rabbit red cells in diluents of 0.85 and
8*0 per cent NaCi (Tables 2 and 4) indicates that perhaps the titers
were slightly less in 8*0 per cent NaCi, with the exception of anti-
Na^+Al which remained the same when compared to agglutination in
0*85 per cent NaCi*
Table 5 illustrates that the antiglobulin titers of chicken
antisera adsorbed with rabbit erythrocytes were all depressed in
8*0 per cent NaCi with the exception of anti-Na^+Al, which appeared
to have greater agglutinability than when titrated in a 0*85 per cent
NaCi menstrum (Table 3)» Agglutinins for normal rabbit red blood
cells were adsorbed out of most of the sera with the exception of
anti-Na^+Al.
Antiglobulin specificity was also demonstrable in chicken antisera tested with sensitized sheep erythrocytes (anti-sheep erythro cyte rabbit sera) in 0,85 P©1* cent NaCi (Table 6), This was especially noticeable in anti-Nag+Al and anti—IIRS+F when compared to the hemagglutinin titers for normal sheep red cells. The anti-sheep
cell and antiglobulin titers of the other antisera closely approxi mated each other. Closer inspection indicated that the sheep erythro cyte agglutinin tite r was greatest in those antisera with higher antiglobulin titers. To verify this, the eayeriment was repeated and
Table 7 shows the results of five samples, each consisting of a pool of seven chicken antisera, tested with normal sheep red cells. The data emphasize that immunizing chickens with rabbit globulins in creased the sheep erythrocyte agglutinin tite r. A sim ilar phenomenon 39
TABUS 5
NORMAL AND IMMUNE CHICKEN SERA ADSORBED WITH RABBIT RED CELLS TITRATED WITH ISOSENSITIZED OROUP A RABBIT ERYTHROCYTES IN 8 .0 PER CENT SODIUM CHLORIDE
Serum______2 H 8 16 32 64 128 256 512 1024 2048 4096 8192 C Norm C h ick + 1 + — — - — — —
Anti-NH^+F +21 I + - — Anti-NA2+Al 3332222 2 1 1 1 1 1 Anti-NRS 111 + --- — — — — Anti-NRS+F 11111 + — — — —
Normal and Immune Chicken Sera Adsorbed with Rabbit Red Cells T itrated with Normal Qroup A Rabbit Erythrocytes In 8.0 per cent Sodium C h lo rid e Norm Chick + - - — — — — — — —
Anti-NHij+F - - - — — — — — — —
A n ti-N a 2+Al 3 2 2 1 + — — — — — Anti-NRS - - + — — —
A nti-N R S+F TABLE 6
TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH SENSITIZED SHEEP ERYTHROCYTES IN 0.85 PER CENT SODIUM CHLORIDE
Reciprocal of Chicken Serum Dilution Serum 2 4 8 16 32 64 128 256 512 1024 2048 4096 8192 16,384 C
Norm Chick + 2 1 + —
Anti-NH4+F 4 1 1 1 — —
Anti-Na2+Al 4 4 4 4 4 4 3 2 1 1 1 1 1 1 —
Anti-NRS 4 4 4 4 3 3 2 1 — __
Anti-NRS+F 4 4 4 4 4 3 2 2 2 1 1 1 — ——
Titration of Normal and Immune Chicken Sera with Normal Sheep Erythrocytes in0.85 Per Cent Sodium Chloride
Nona Chick 3 1 1 + — — —
Anti-NHij+F 4 3 2 —
Anti-Na2+Al 4 4 4 4 2 1 1
Anti-NRS 4 4 4 3 1 1 —
Anti-NRS+F 4 4 4 4 2 1 — 41
TABLE 7
TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH NORMAL SHEEP ERYTHROCYTES IN 0 .8 5 PER CENT SODIUM CHLORIDE
Reciprocal of Dilution Serum______2 4 8 16 32 64 128 256 512 C Norm Chick 2 1 Anti-NH^+F 2^44443 3 1 1
A n ti-N a 2+A l 4 4 4 3 1 1
A nti-N R S 4 4 3 2 1 1 ----- —
A nti-N R S+F 4 4 4 4 2 1 1 42 has heen reported "by Friedman and Yeatman (195&) "who found that chickens immunized with rabbit kidney produced antibody with an affinity for sheep red blood cells*
The chicken antisera were then adsorbed with rabbit erythro cytes and tested with sensitized sheep cells in 0*85 per cent UaCl
(Table 8). Comparison with Table 6 demonstrates that the anti- globulin titers appeared to be slightly less and no sheep cell ag glutinins were observed at a dilution of 1 to 16. However, all anti sera except anti—NH^+F demonstrated definite antiglobulin antibodies in titers exceeding 1 to 16. It is conceivable that rabbit red blood cells may have adsorbed the sheep red blood cell agglurxnins, but the question was not pursued*
Table 9 illustrates that sheep red cell and antiglobulin titers were not easily differentiated in 8*0 per cent NaCl and all tite rs were less when compared to those obtained in O.85 per cent
ITaCI (Table 6).
Adsorption of the chicken sera with rabbit red cells and titration with sensitized and normal sheep erythrocytes in 8*0 per cent HaCl (Table 10) showed that almost all hemagglutinins for both sensitized and normal sheep cells were removed, except from anti —
Ua^+Al. Even with this serum, no distinct antiglobulin specificity was demonstrable because of the sim ilarity of the titers for normal and sensitized sheep cells*
To summarize, the following can be stated for both systems of erythrocytes sensitized with rabbit antibodies: (l) It is de sirable to use adsorbod antiglobulin sera because agglutinins for 43
TABLE 8
NORMAL AND IMMUNE CHICKEN SERA ADSORBED WITH RABBIT ERYTHRO CYTES TITRATED WITH SENSITIZED SHEEP ERYTHROCYTES IN O.85 PER CENT SODIUM CHLORIDE
Reciprocal of Chicken Serum D ilution Serum______16 32 64 128 256 512 1024 2048 4096 8 1 9 216 ,3 8 4 C
Norm Chick — — —
A nti-N H 4+F — — -- — — — — — —
A n ti-N a 2 +A l 4 4 3 2 2 1 — — — — —
Anti-NRS 3 1 1 1 — — — — — — — —
Anti-NRS+F 3 4 2 2 2 2 1 +
Normal and Immune Chicken Sera Adsorbed with Rabbit Erythrocytes Titrated with Normal Sheep Enthrocytes in O.8 5 Per Cent Sodium Chloride Norm Chick — — — — —
Anti-NH^+F — — — — — -- — — — — — —
A n ti-N a 2 +A l — — -- Anti-NRS — — —
A n ti-N R S + F 44
TABLE 9 TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH SENSITIZED SHEEP ERYTHROCYTES IN 8 .0 PER CENT SODIUM CHLORIDE
Reciprocal of Chicken Serum D ilution Serum 2 4 8 16 32 64 128 256 512 1024 2048 4096 C
Norm C hick
Anti-NH^+F 1 1 - rH + 1 i i i i Anti-Na2+Al 3 3 2 1 1 i i
A nti-N RS 3 1 1 1 1 Anti-NRS+F 3 2 2 1 1 + — - - —
T itration of Normal and Immune Chicken Sera with Normal Sheep Erythrocytes I n 8 .0 P er Cent Sodium Chloride Norm C hick A nti-N H ^+F 1 + -
A n ti-N a 2 +Al 2 1 1 1 +
A nti-N RS 1 1 + A nti-N RS+F 2 2 1 1 1 45
TABLE 10
NORMAL AND IMMUNE CHICKEN SERA ADSORBED WITH RABBIT ERYTHRO CYTES TITRATED WITH SENSITIZED SHEEP ERYTHROCYTES IN 8 .0 PER CENT SODIUM CHLORIDE
Serum 2 4 8 16 32 64 128 256 512 1024 2048 4096 C Norm C hick
Anti-NH4+F
A n ti-N a 2 +Al 3 111+— — — ......
Anti-NRS
Anti-NRS+F
Normal and Immune Chicken Sera Adsorbed with Rabbit Erythrocytes Titrated with Normal Sheep Erythrocytes in 8.0 Per Cent Sodium Chloride
Norm C hick
Anti-NH4+F
Anti-Nag+Al 3 2 1 1 1 — — — —
Anti-NRS
Anti-NRS+F 46 normal rabbit erythrocytes mask antiglobulin activity and immuni zation of chickens with rabbit globulins increased sheep erythrocyte agglutinins, (2) The use of 8*0 per cent NaCl is undesirable for antiglobulin hemagglutination demonstrations except, perhaps when using antiserun to sodium sulfate precipitated globulins adsorbed on alum , (3) The use of 8,0 per cent salt solution causederythrocytes to crenate and the cells also tended to cling together in a !,stringyM formation. However, this was quite different from agglutination.
In order to determine the ability of the various chicken antisera to detect rabbit serum protein components, various fractions were coupled to red blood cells by diazotization. These altered cells were then titrated v/ith the anti-rabbit globulin chicken sera*
Two major difficulties were immediately encountered. The first was the frequent lysis of rabbit erythrocytes after the coupling process was completed. This was corrected by using a phos phate buffered solution of pH 7»3 which did not contain sodium chloride. The second problem was the failure to detect anti-rabbit globulin antibodies in the chicken sera when certain rabbit serum fractions were coupled to rabbit erythrocytes. Such antibodies were detectable when rabbit serum Cohn fraction II (gamma globulin) and
Cohn fraction IV-4 (alpha globulin) were coupled to rabbit erythro cytes. No reactions were observed with Cohn fraction III (beta globulin), Cohn fraction IV (alpha) or Cohn fraction IV-1 (alpha).
Curiously, flocculation was observed at the interface when these latter three fractions were layered v/ith chicken antisera. Thus, precipitin type reactions were obtained which were not demonstrable 47
"by hemagglutination* In contrast, when all the above rabhit serum
fractions were coupled to human erythrocytes, each was detectable by antiglobulin hemagglutination*
Tables 11 through 17 illustrate the diverse specificities
of the chicken antisera for the various rabbit serum proteins coupled to human erythrocytes. These proteins were contained in rabbit serum
Cohn fractions II, III, IV-1, IV-4» and V (electrophoretic analysis listed in Table 19) • Tests with normal chicken serum and the various chicken antisera tested with egg albumin coupled to human erythrocytes were used as controls*
Table 18 is a composite of antibody titers found in Tables 11 through 17. The latter tables were included to demonstrate the in tensity of hemagglutination at each dilution, but comparison is easier using a composite of titers* A serum dilution of 1 to 2048 was the limiting dilution in this procedure*
Examination of the hemagglutination patterns show that each antiserum with the exception of anti-HH^+F contained antibodies specific for a wide range of serum proteins. The titers for fractions
II, III, and IV in anti-IIH^+F serum are probably significantly high to indicate specificity when compared to the control reactions with egg albumin and normal serum, but they are also significantly lower than the titers of the other antisera. Apparently, this serum con tains little antibody specific for the other three fractions.
As can be seen in Table 18, when the cells were centrifuged the resulting titers approximated those obtained by hemagglutination patterns. However, the titers of anti-KRS and anti-HRS+F sera were 48
TABLE 11
TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH RABBIT SERUM COHN FRACTION I I (QAMMA GLOBULIN) COUPIED TO HU MAN ERYTHROCYTES
Hemagglutination Patterns
Reciprocal of Dilution Serum 24 81632 64 128 256 512 1024 2048 C Norm C hick +
Anti-NH4+F + + + + + + i i + + A n ti-N a 2+Al + + + + + + + + +
Anti-NRS + + + + + + + + + + +
Anti-NRS+F + + + + + + + + + + +
Agglutination After Centrifugation
Norm C hick 3 1+
Anti-NHij+F 3 3 2 2 11 1 +
A n ti-N a 2+Al 4 4 4 4 4 4 2 2 3 2 2 —
Anti-NRS 12 1111 + ■
Anti-NRS+F 4 2 2 2 2 2 1 1 1 1 1 — 49
TABLE 12
TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH RABBIT SERUM COHN FRACTION I I I (BETA GLOBULIN) COUPIED TO HU MAN ERYTHROCYTES
Hemagglutination Patterns
Reciprocal of Dilution Serum 2 4 8 16 32 64 128 256 512 1024 2048 C Norm Chick + +
Antl-NHjj+F + + + + + + — —
A nti-N a2+Al + + + + + + + + + + + —
Anti-NRS + + + + + + + +
ANTI-NRS+F + + + + + + + + + + + —
Agglutination after Centrifugation
Norm Chick 3 +
Anti-NH^+F 3 2 2 1 1 11 —
A nti-N a2+Al 2 2 2 2 2 2 11 1 11 —
Anti-NRS - 1+
Anti-NRS+F 2 2 1 1 1 ! + — 50
TABLE 13
TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH RABBIT SERUM COHN FRACTION IV (ALPHA GLOBULIN) COUPUED TO HUMAN ERYTHROCYTES
Hemagglutination Patterns
Reciprocal of Dilution Serum______2 4 8 16 32 64 128 256 512 1024 2048 C Norm Chick + +
Anti-NH4+F + + + + + + +
A nti-N a2+Al + + + + + + + + + + +
Anti-NRS + + + + + + + + Antl-NRS+F + + + + + + + + + + +
Agglutination after Centrifugation
Norm Chick 1 1 — -- — —
Antl-NH4+F 4 2 2 1 1 1 +
A nti-N a2+Al 342222 2 1 1
Anti-NRS +
Anti-NRS+F 3 1 1 1 1 + — — -- 51
TABLE 14
TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH RABBIT SERUM COHN FRACTION IV -1 (ALPHA GLOBULIN) COUPLED TO HUMAN ERYTHROCYTES
Hemagglutination Patterns
Reciprocal of Dilution Serum 2 4 8 16 32 64 128 256 512 1024 2048 C
Norm Chick + — — — — —
Anti-NH/j+F + + + — — — — —
A ntl-N a2+Al + + + + + + + + +
A n ti—NRS + + + + + —— —- — —- —— —- ——
Anti-NRS+F + + + + + + + + +
Agglutination after Centrifugation
Norm Chick 1 + + — — — — —
Anti-NHii+P 4 2 1 1 1 — — — —
Anti-Na2+Al 3 3 2 1 2 1 1 -- —
Anti-NRS 1 + — — — —
Anti-NRS+F 2 2 1 i l l — — — 52
TABLE 15 TITRATION OF NORMAL AND IMMUNE CHICKEN SERA WITH RABBIT SERUM COHN FRACTION IV -4 (ALPHA GLOBULIN) DOUPIZD TO HUMAN ERYTHROCYTES
Hemmaglutlnation Pattern
Reciprocal of Dilution Serum 24 816 32 64 128 256 512 1024 2048 C
Norm Chick +
Anti-NH4+F + + + +
A nti-N a2+Al + + + + + + + +
Anti-NRS + + + + + + + + + + + + + + 1 Anti-NRS+F + + + + + + + + 1 Agglutination after Centrifugation
Norm Chick 2+
Anti-NH4+F 3 22 1
A nti-N a2+Al 4 4 3 3 3 2 11 1
Anti-NRS 211 1 1 1 --
Anti-NRS+F 211 2 2 111 53
TABLE 16
TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH RABBIT SERUM COHN FRACTION V (ALBUMIN) COUPIED TO HUMAN ERYTHROCYTES
Hemagglutination Patterns
Reciprocal of Dilution Serum 24 816 32 64 128 256 512 1024 2048 C Norm Chick +
Antl-NHi|+P + + +
A n ti-N a 2+Al + + + + + + + + —
Anti-NRS + + + + + + + + — —- — —— Anti-NRS+F + + + + + + + + + + Agglutination after Centrifugation
Norm C hick 1
Antl-NHij+F 4 22 1 1 +
A n ti-N a 2+Al 4 4 3 3 3 3 3 2 2 1 1
Anti-NRS 3 2 2 1 2 1 1 11 +
Anti-NRS+F 4 3 3 3 3 2 2 2 2 1 1 — 54
TABLE 17
TITRATION OP NORMAL AND IMMUNE CHICKEN SERA WITH CHICKEN EGG ALBUMIN COUPLED TO HUMAN ERYTHROCYTES
Hemagglutination Patterns
Serum 2 4 8 16 32 64 128 256 512 1024 2048 C Norm Chick + — — — — — Anti-NH4+F + + — — — — —
A n ti-N a 2+Al + + + +— — — Anti-NRS — — — — — —
Anti-NRS+F + — — — — —
Agglutination after Centrifugation
Norm Chick 1 — — — — —
Anti-NH^+F 3 2 1 — — — — — —
A n ti-N a 2+Al 4 4 1 1 1 + ------
Anti-NRS — — — — -- —
Anti-NRS+F 1 1 — ------— — — - - TABLE 13
COMPOSITE OF TITRATIONS OF NORMAL AND IMMUNE CHICKEN SERA WITH RABBIT SERUM COHN FRACTIONS COUPLED TO HUMAN ERYTHROCYTES
Hemagglutination Patterns* Serum Egg** Fraction fraction fraction Fraction Fraction Fraction Albumin II III IV IV-1 IV-4 V Norm Chick 2 2 4 4 2 2 2
Anti-NHij+F 4 64 64 128 8 16 8
Antl-Nag+Al 16 2048 2048 2048 512 236 256
Anti-NRS — 2048 256 256 32 2048 256
Anti-NRS+F 2 2048 2048 2048 512 2048 1024
Agglutination after Centrifugation*
Norm Chick 2 4 2 4 2 2 2
Anti-NHij+F 8 128 128 64 32 16 32
Antl-Na^+Al 32 2048 2048 512 128 512 2048
Anti-NRS — 64 4 — 2 64 512
Antl-NRS+F 4 2048 64 32 64 256 2048 Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. **Control. 56 erratic with this method. Red cells to which fraction IV had heen coupled were not agglutinated in anti—HRS after centrifugation and titers of this serum with other fractions, except fraction V, were low. Titers of anti-HRS+F with rabbit serum Cohn fractions III, IV,
IV-1, and IV-4 were lower after centrifugation. Because of the be havior of anti—MRS and anti—KRS+F it was concluded that hemaggluti nation patterns provided a more reliable and sensitive method than centrifugation.
In order to determine the sensitivities of these antisera, quantitative nitrogen determinations were performed on precipitates obtained by mixing constant amounts of chicken antisera with varied dilutions of rabbit serum Cohn fractions. Electrophoretic analysis
(Table 19 and Figure 2) showed that each Cohn fraction contained mixtures of globulins and albumin. Thus, no precipitate could be considered specific in terms of a specific antibody reacting with a
3ingle antigen, especially since the antigens used to immunize the chickens were not employed to precipitate the antisera. Nevertheless, high dilutions of Cohn fractions would dilute out components origin ally present in small amounts and consequently emphasize that com ponent present in the highest concentration. By using rabbit serum
Cohn fractions II, III, IV, and V to cover the electrophoretic range from gamma globulin to albumin in conjunction with the dilutions it was thought that certain conclusions could be inferred from the pre cipitin curves. The supernatants resulting after precipitation were tested with erythrocytes sensitized with rabbit antibody and an 57
TABLE 19
ELECTROPHORETIC ANALYSIS OP COMMERCIALLY PREPARED RABBIT SERUM COHN FRACTIONS
Per Cent Composition Cohn Fraction Oamma Beta Alpha Albumin
I I (gamma) 8 2 .2 13-5 4.3 —
I I I (b e ta ) 32.2 26.8 33 .2 7 .8
IV (a lp h a ) 6 .6 9-9 20.5 6 3 .0
V (albumin) 2 .5 3-9 4 .3 89*3 IV-1(alpha) 10 .3 11.6 38.8 39-3 IV-4(alpha) 6 .5 9-2 10.7 73-6 Fig. 2. ELECTROPHORETIC PATTERNS OF RABBIT SERUM COHN FRACTIONS AT DIFFERENT DILUTIONS
FRACTION IT FRACTION M s s
FRACTION m FRACTION 3L
— I/IO DILUTION OF STOCK SOLUTION. — -1 /5 0 DILUTION OF STOCK SOLUTION. 59 indication of the globulin classification of these antibodies was obtained by hemagglutination inhibition.
On thawing the chicken sera prior to the precipitin tests it was noted that floccules occurred which became soluble after incu bation at 37°C and reappeared in the 3erum blank of each chicken serum after refrigerated centrifugation. It was concluded that this was the demonstration of a cryoglobulin* but the amount was so small that it fell below the sensitivity of the biuret test and was un measurable. Precipitin curves were plotted for normal chicken serum, but any precipitates which occurred (cryoglobulin) fell below the sensitivity of the biuret test.
The total amounts of nitrogen precipitated when various dilutions of rabbit serum Cohn fractions in 0.5 ml amounts were mixed with 0.5 ml of chicken antisera are plotted in Figures 3 through 7*
In Figures 3 and 4 (Fraction II) the first peaks were probably due to the reaction of rabbit gamma globulin with the antibody in the various chicken antisera* Up to 90»0 micrograms of antigen, the anti-Hag+Al chicken serum was the most sensitive antiglobulin serum for rabbit serum Cohn fraction II. In greater amounts of antigen anti—URS+F and anti-ERS caused the greatest nitrogen precipitation, whereas anti-NH.+F and anti-Ha0+Al were depressed, 4 2 As antigen concentrations increased it was expected that minor components of the fraction would become serologically demon strable by increased nitrogen precipitation with anti-KRS and anti—
1JRS+F, because these antisera were produced in response to the largest array of serum proteins. Micrograms total N Precipitated 240 240 160- 120 0 4 80 t 0 , Fig. 3. 3. Fig. irgas nie N de t 0. m Antisera ml .5 0 to added N Antigen Micrograms 50 HCE ATSR B ADN VROS MUT OF AMOUNTS VARIOUS AODING BY ANTISERA CHICKEN OA NTOE PEIIAE FO 05 OF l 0.5m FROM PRECIPITATED NITROGEN TOTAL 100 ABT EU CH FATO H FRACTION COHN SERUM RABBIT 150 250 0 0 2 0 350 400 425 2 4 0 0 4 0 5 3 00 3 ni—NS F Sera F — Anti + NRS NA - Anti i ^F Sera F I^ N ti- n A ni- R Sera NRS - Anti 2 +A Sera Al + Micrograms total N Precipitated 0 0 2 0 4 2 160- 120 0 4 - 0 8 Fig. - - ti 0 4. 4. irgas nie N de t 05m Antisera ml 0.5 to added N Antigen Micrograms HCE ATSR B ADN VROS MUT OF AMOUNTS VARIOUS ADDING BY ANTISERA CHICKEN 10 OA NTOE PEIIAE FO 05m OF ml 0.5 FROM PRECIPITATED NITROGEN TOTAL 20 ABT EU CH FATO H FRACTION COHN SERUM RABBIT 30 50 080 60 704 0 niNS Sera Anti-NRS iNRS+F Sera F + S R ti-N n A H Anti-N iNAg+AI Sera I A + g A ti-N n A 4 Sera F 0 9 100 Micrograms total N Precipitated Fig.5. Fig.5. 300 0 0 2 250 5 ABT EU CH FATO HI H FRACTION COHN SERUM RABBIT - 350 O - / lOO-i 150 - - HCE ATSR B ADN VROS MUT OF AMOUNTS VARIOUS ADDING BY ANTISERA CHICKEN 0 0 10 0 20 0 30 0 450 400 350 300 250 200 150 100 50 OA NTOE PEIIAE FO 05 OF l 0.5m FROM PRECIPITATED NITROGEN TOTAL irgas nie N de t 05 l Antisera ml 0.5 to added N AntigenMicrograms ni- R Sera NRS Anti- niNU Sera Anti-NhUF t- +F Sera F S+ R nti-N A Sera Al + NA2 Anti-
500
T 9 Micrograms total N Precipitated TOTAL 6 a i F - 0 8 2 240-r* 160 + 40 80 4 CIKN NIEA Y DIG AIU AONS OF AMOUNTS VARIOUS ADDING BY ANTISERA CHICKEN ' 0 0 10 0 20 0 30 400 350 300 250 200 150 100 50 irgas nie N de t 05m Antisera ml 0.5 to added N Antigen Micrograms trogen e g o r it n V ABT EU CH FRACTION32 COHN SERUM RABBIT RCPTTD RM . m OF ml 0.5 FROM PRECIPITATED t niNS Sera Anti-NRS niN4 Sera Anti-NH4F Anti NA - niNS+F Sera F + Anti-NRS 2 + l Sera Al+ 450 Micrograms total N Precipitated 7. Fig. 200 - 0 4 2 280 160 120 - 0 4 - 0 8 - - HCE ATSR B ADN VROS MUT OF AMOUNTS VARIOUS ADDING BY ANTISERA CHICKEN irgas nie N de t 05m Antisera ml 0.5 to added N Antigen Micrograms 0 0 10 0 20 0 45 2 55 625 575 525 475 300 250 200 150 100 50 OA NTOE PEIIAE FO 05 l OF ml 0.5 FROM PRECIPITATED NITROGEN TOTAL V ABT EU CH FATO 2 FRACTION COHN SERUM RABBIT ni- R F Sera F NRS+ Anti - NH Anti - ni- R Sera NRS Anti - Anti - NA Anti - 4 2 + Al Sera Sera Al + Sera F 65
When Tables 20 through 23 (fraction II) were compared with
Figures 3 and 4, in the case of anti-Nag+Al, a correlation was noted between the amount of antigen which initially depressed the total nitrogen precipitated and the inhibition of antiglobulin hemaggluti nation. This was not so readily apparent with the other antisera.
When rabbit serum Cohn fraction III was precipitated by the various antisera (Tables 24 through 27), the anti-Nag+Al chicken serum precipitated the greatest amounts of nitrogen up to 40*0 miorograms of antigen nitrogen (observed in Figure 5)* At greater amounts of antigen more nitrogen is precipitated respectively by anti-KRS+F> anti-NRS > anti-Ka^+Al ^ anti-NH^+F.
Comparison of Tables 24 through 27 with Figure 5 illustrated that antiglobulin hemagglutination was sometimes inhibited after the initial retardation of precipitated nitrogen by Cohn fraction III.
The precipitation of nitrogen by the addition of rabbit serum
Cohn fraction IV is plotted in Figure 6. In the presence of small amounts of antigen the greatest amounts of nitrogen precipitated were
Caused respectively by anti-KRS > anti—NRS+F > an ti—Na^+Al > and anti—
1TH^+F. In larger amounts of antigen it is anti—HES+F > anti-Nag+Al > anti-KRS > anti-IIH^+F. Very little depression of antiglobulin hemag glutination occurred (Tables 28 through 3l) when this fraction was added to the chicken antisera. The slight inhibition which was ob served was probably due to beta and gamma globulin contaminants.
This became more apparent when the percentages of these components in fraction IV were noted in Table 19• TABLE 20
THE HEMAGGLUTINATION OF SENSITIZED ERYTHROCYTES BY ANTI-Nag+Al CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION I I (GAMMA GLOBULIN)
Total N Hemagglutina tion* Fraction II Precipitated Contents of Supematent Rabbit Iso-immune Anti-Sheep RBC Micro gm N Microgm N Antigen Antibody Antibody Rabbit Antibody
389.50 24.5 + — — 2 __ 444.75 28.5 + ------—-- 2 2 2 .3 8 44.0 + a m w 2 — a* w 111.19 80.5 + 2 — 55.59 9 1 .0 + — 2 — 27.79 115.5 + — 2 — 13.90 1 2 3 .0 + -- 8 2 6.95 8O.5 trace — 16 2 3.^7 60.5 — 64 8 1.74 34.0 — + 64 16 0 .8 6 23.5 -- + 64 16 Serum Blank — — + 64 16 Serum BlankReacted w ith Unsensitized Cells 2 1------Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 21
THE HEMAGGLUTINATION OF SENSITIZED ERYTHROCYTES BY ANTI-NRS CHICKEN SERA AFTER ADSORP TION WITH COHN RABBIT FRACTION I I (GAMMA GLOBULIN)
Total N Hemagglutination* Fraction II Precipitated Contents of Supernatent Rabbit Iso-immune Anti-Sheep RBC Micro gm N Micro gm N Antigen Antibody Antibody Rabbit Antibody
889.50 2 7 2 .2 + 8 W .75 217-8 + trace 8 — 2 2 2 .3 8 173.0 + + 8 — 111.19 98.5 + + 8 — 55.59 5 8 .0 + + 16 ■ — 27.79 46.0 + + 32 — 13.90 46.0 + + 64 2 6.95 54.0 + + 128 4 3.47 48.5 trace + 128 16 1.74 18.5 — + 64 64 0 .8 6 1 6 .0 — + 256 64 Serum Blank —— + 512 64 Serum Blank Reacted with Unsensitized Cells 8 * Reciprocal of Highest Dilution Demonstrating a PosMve Agglutination. TABLE 22
THE HEMAGGLUTINATION OF SENSITIZED ERYTHROCYTES BY ANTI-NHii+F CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION I I (GAMMA GLOBULIN)
Total N Hemagglutination* Fraction II Precipitated Contents of Supematent Rabbit Iso-immune Anti-Sheep RBC Micro gm N Micro gm N Antigen Antibody Antibody Rabbit Antibody
889.50 54.0 + MW 16 4 444.75 49.0 + — 16 8 222.38 48.5 + -- 16 8 111.19 43.0 + — 16 8 55.59 54.0 + — 16 8 27*79 6 3 .0 + — 32 8 13.90 5 6 .0 + -- 64 32 6.95 48.0 trace — 64 128 3.^7 43.0 » + 256 256 1.7* 28.5 -- + 256 64 0 .8 6 19.0 -- + 256 128 Serum Blank —-- + 256 128 Serum Blank Reacted with Unsensitized Cells 16 16 7------Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 23
THE HEMAGGLUTINATION OF SENSITIZED ERYTHROCYTES BY ANTI-NRS+F CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION I I (GAMMA GLOBULIN)
Total N Hemagglutination* Fraction II Precipitated Contents o f Supematent Rabbit Iso-immune Anti-Sheep RBC Micro gm N Micro gm N Antigen Antibody Antibody Rabbit Antibody
889.50 2 1 9 .8 + 4 444.75 1 9 2 .0 + + 4 2 2 2 2 .3 8 104 + + 4 ------111.19 85 + + 2 2 55.59 73 + + 8 4 27.79 62 + + 16 2 13.90 58 + + 32 8 6.95 43.5 — + 32 8 3.^7 22.5 — + 32 8 1.74 3 1 .0 — + 32 32 0 .8 6 1 8 .0 -- + 64 64 Serum Blank ** « — + 64 64 Serum BlankReacted with Unsensitized Cells + 4 4
Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-Na2 +Al CHICKEN SERA APTER ADSORPTION WITH COHN RABBIT FRACTION I I I (BETA GLOBULIN)
Total N Hemagglutination* Fraction III Precipitated Contents of Supematent Rabbit Iso-immune Anti-Sheep RBC Micro gm N Micro gm N Antigen Antibody Antibody Rabbit Antibody
862.00 56.5 + 2 4 431.00 139.5 + — 2 2 215.50 102.5 + 4 4 107.75 102.5 + — 4 4 53.68 109.5 + — 8 16 26.94 112.0 + — 32 64 13.47 88.5 + — 32 64 6.73 58.5 trace + 128 128 3.37 37.0 — + 128 128 1.68 15.0 -- + 64 128 0.84 9.0 -- + 128 256 Serum Blank —— + 128 256 Serum Blank Reacted with Unsensitized Cells 2 16
' i t 1 - - - - T _ . . _ - Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 25
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-NRS CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION I I I (BETA GLOBULIN)
T o tal N Hemagglutination* Fraction III Precipitated C ontents of Supematent Rabbit Iso-immune Anti-Sheep RBC Micro gm N Micro gm N A ntigen Antibody Antibody Rabbit Antibody
8 6 2 .0 0 274.0 + 2 431.00 331.0 + tra c e 2 — 215.50 235.5 + + 4 — 107.75 I6 5 .O + + 4 — 53.88 90.5 + + 4 2 26.94 68.5 + + 8 16 13.47 6 1 .0 + + 16 32 6.73 44.0 + + 64 32 3.37 31.0 tra c e + 64 64 1.68 1 5 .0 -- + 128 64 0.84 4.0 -- + 256 64 Serum Blank — — + 256 64 Serum Blank Reacted with Unsensitized C ells 2 f------Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 26
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-NH^+F CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION I I I (BETA GLOBULIN)
T otal N Hemagglutination* Fraction III Precipitated Contents of Supematent Rabbit Iso-immune Anti-Sheep RBC Micro gm N Micro gm N Antigen Antibody Antibody Rabbit Antibody
862.00 68.5 + 8 431*00 97.0 + — 8 — 215*50 80.5 + — 8 — 107*75 58.5 + — 8 — 53.88 53.0 + — 8 26.94 53*0 + — 32 — 13.47 24.5 + — 64 4 6.73 17.0 + trace 64 16 3.37 9.0 + 64 64 1.68 — + 128 64 0.84 —— + 128 64 Serum Blank —— + 256 128 Serum Blank Reacted with Unsensitized C e lls 8 — I ------Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABI£ 27
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-NRS+F CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION I I I (BETA GLOBULIN)
Total N Hemagglutination* Fraction III Precipitated C o n ten ts of S u p e m a te n t Rabbit Iso-immune Anti-Sheep RBC M icro gm N M icro gm N Antigen Antibody Antibody Rabbit Antibody
062.00 347.0 + 8 431.00 337.0 + 8 4 215.50 2 6 7 .0 + 16 2 107.75 178.0 + tr a c e 8 2 53.88 134.5 + + 8 8 26.94 8 1 .0 + + 16 16 13.47 46.5 + + 16 16 6.73 2 0 .0 -- + 32 32 3.37 9.0 — + 64 128 1 .6 8 3.0 + 64 32 0.84 -- + 128 64 Serum Blank — + 256 256 Serum Blank Reacted with Unsensitized C e lls 8 4 # — • — ■ — 1 — ■ . — . Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 28
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-Na2+Al CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION IV (ALPHA GLOBULIN)
T o ta l N Hemagglutination* Fraction IV Precipitated Contents of Supematent Rabbit Iso-immune Anti-Sheep RBC M icro gm N M icro gm N A ntigen A ntibody A ntibody Rabbit Antibody
833-00 120.0 + 32 256 416.50 148.0 + —. 32 64 208.25 118.0 + -- 64 128 104.13 6 8 .5 + 128 128 52.06 68.5 + + 128 128 26.03 3 0 .0 + + 128 256 13.02 33.0 + + 128 128 6.51 24.5 + + 64 64 3.25 2 3 .0 + + 128 128 1.63 2 7 .0 + + 128 256 0 .8 1 2 3 .0 — + 128 256 Serum Blank — + 128 256 Serum Blank Reacted with Unsensitized C e lls — —
Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 29
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-NRS CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION IV (ALPHA GLOBULIN)
Total N Hemagglutination* Fraction IV Pi»eclpitated Contents of Supernatent Rabbit Iso-immune Anti-Sheep RBC M icro gm N Micro gm N Antigen Antibody A ntibody Rabbit Antibody
833.00 138.0 + 32 16 416.50 132.0 + — 16 32 208.25 1 1 6 .0 + — 16 128 104.13 164.0 + + 32 128 5 2 .0 6 232.0 + + 32 128 26.03 2 6 8 .0 + + 32 256 13.02 198.0 + + 64 128 6.51 118.0 —- + 64 512 3-25 6 1 .5 — + 128 128 1.63 44.0 — + 64 512 0.81 2 7 .0 — + 64 256 Serum Blank — -- + 128 256 Serum Blank Reacted with Unsensitized C e lls -- —
Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 36
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-NH4+F CHICKEN SERA AFTER ADSORPTION WITH COHN TABBIT FRACTION IV (ALPHA GLOBULIN)
T o ta l N Hemagglutination* F r a c tio n IV Precipitated Contents of Supematent Rabbit Iso-immune Anti-Sheep RBC M icro gm N M icro gm N Antigen Antibody Antibody Rabbit Antibody
833*00 68.5 + 4 8 416.50 55*0 + — 8 16 208.25 49*5 + — 32 32 104.13 42.0 + — 32 64 52.06 21.0 + -- 32 64 26.03 24.5 + 32 128 13*02 12.5 + ------32 64 6.51 6.0 + ------64 128 3*25 12.5 + ------64 128 1.63 6 .0 + + 64 128 0 .8 1 12.5 — + 128 128 Serum Blank —— + 64 128 Serum Blank Reacted with Unsensitized. Cells ■■ “ ¥------Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 31
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-NRS+F CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION IV (ALPHA GLOBULIN)
T o ta l N Hemagglutination* F r a c tio n IV Precipitated Contents of Supernatent Rabbit Iso-immune Anti-Sheep RBC M icro gm N M icro gm N A n tig en A ntibody Antibody Rabbit Antibody
8 3 3 .0 0 2 7 2.O + _ M 4 8 416.50 2 0 5 .0 + — 8 16 208.25 1 7 8 .0 + + 32 64 104.13 170.5 + + 32 128 5 2 .0 6 174.0 + + 32 256 26.03 2 2 8 .0 + + 64 512 13.02 1 8 2 .0 + + 64 256 6.51 1 2 6 .0 + + 32 512 3-25 70.5 + + 32 256 1.63 43.0 -- + 32 256 0.81 37.5 — + 64 256 Serum Blank —— + 64 512 Serum Blank Reacted with Unsensitized Cells —— — 1 1 Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. 78
As anticipated by the electrophoretic analysis of the anti gens used in immunizing the chickens, anti-Na.+Al and anti-NH.+F were 2 4 hardly capable of precipitating small amounts of Cohn fraction V
(Figure 7 and Tables 32 through 35)* On the other hand anti-HRS and anti-lIRS+F quite effectively precipitated fraction V and had similar curves.
The most clearly defined equivalence zones were exhibited by anti-Nag+Al (Tables 20 and 24 Contents of Supernatant) and anti-
NH^+F (Tables 22 and 26) when reacted with fractions II and III and anti-llH^+F reacted with fraction IV (Table 30), Since each Cohn fraction consisted of serum protein mixtures it was not surprising that no other definite equivalence zones were noted.
The complete inhibition of hemagglutination of sensitized rabbit and sheep erythrocytes occurred when the chicken antisera to rabbit serum preparations wore adsorbed with rabbit serum Cohn fractions II (Tables 20 through 23) and III (Tables 24 through 27)* liach less inhibition was observed with fraction IV (Tables 28 through 31) and fraction V (Tables 32 through 35) not neutralize hemagglutination at all.
These data suggest that the rabbit iso-immune anti-A erythro cyte antibodies and anti-sheep erythrocyte rabbit antibodies were predominantly gamma globulins and perhaps some of the antibodies were beta globulins. TABLE 32
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-Na2+Al CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION V (ALBUMIN)
T o ta l N Hemagglutination* F r a c tio n V Precipitated Contents of Supernatant Rabbit Iso-immune Anti-Sheep RBC M icro gm N Micro gm N Antigen A ntibody A ntibddy Rabbit Antibody
1206.00 95.0 + __ 64 128 603.00 48.5 + — 64 256 301.50 2 6 .0 + — 128 256 150.75 11.0 + — 64 256 75.38 15.0 + — 128 256 37.69 0.0 + — 128 256 18.84 11.0 + 64 256 9.42 11.0 + -- 128 256 4.71 6 .0 + — 128 512 2.36 6 .0 + — 128 128 1.18 1 8 .5 tr a c e — 128 128 Serum Blank ——— 64 128 Serum Blank Reacted with Unsenslzed Celia — — # Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABIE 33
THE HEMAGGLUTINATION OF SENSITIZED ERYTHROCYTES BY ANTI-NRS CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT HI ACTION V (ALBUMIN)
Total N Hemagglutina tion* F ra c tio n V Precipitated Contents o f Supernatent Rabbit Iso-immune Anti-Sheep RBC M icro gm N M icro gm N Antigen Antibody Antibody Rabbit Antibody
1206.00 72.0 + 64 256 6 0 3 .0 0 5 8 .0 + — 64 256 301.50 38.5 + — 64 256 150.75 9 7 .5 + — 64 256 75.38 1 5 1 .0 + — 64 256 37.69 2 8 2 .0 + — 128 512 18.84 273.0 + — 128 256 9-42 2 0 9 .0 + — 64 256 4.71 139.0 —— 64 512 2 .3 6 7 2 .5 —— 64 256 1 .1 8 39-0 — tra c e 128 256 Serum Blank — — + 128 , 2 5 6 1 Serum Blank Reacted with Unaensltized Cells —
Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 3^
THE HEMAOOLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-NHh+P CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION V (ALBUMIN)
T o ta l N Hemagglutination* F ra c tio n V Precipitated Contents of Supernatant Rabbit Iso-inmune Anti-Sheep RBC Micro gm N Micro gm N Antigen Antibody A ntibody Rabbit Antibody
1 2 0 6 .0 0 3 9 .0 + _ 32 64 6 0 3 .0 0 3 8 .0 + — 64 128 301.50 1 5 .0 + — 128 128 150.75 1 8 .0 + — 128 128 75*38 1 2 .0 + — 128 256 37*69 2 6 .0 + 128 128 18.84 1 5 .0 + ---- 64 256 9 .4 2 15*0 + ---- 128 256 4.7 1 1 5 .0 + ---- 256 256 2 .3 6 1 9 .0 + ---- 128 512 1 .1 8 2 1 .0 tr a c e ---- 256 512 Serum Blank —— ---- 256 256 Serum Blank Reacted with Unsensitized C e lls 2 2 U — — ------■■■ — 1 " — - — Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. TABLE 35
THE HEMAGGLUTINATION OP SENSITIZED ERYTHROCYTES BY ANTI-NRS+F CHICKEN SERA AFTER ADSORPTION WITH COHN RABBIT FRACTION V (ALBUMIN)
T o ta l N Hemagglut in a t ion* F ra c tio n V Precipitated C o n ten ts of Supernatant Rabbit Iso-immune Anti-Sheep RBC M icro gm N M icro gm N A ntigen Antibody Antibody Rabbit Antibody
1206.00 71.0 + 32 64 603.00 48.5 + ... 64 256 301.50 64.5 + ---- 64 128 150.75 7 1 .0 + -- 64 256 75.38 128.0 + -- 128 512 37.69 1 165.5 + + 128 256 18.84 2 5 2 .0 + + 64 128 9.42 171.5 + 128 512 4.71 112.0 ■ ■ (HP + 128 512 2.36 6 5 .O ---- + 64 256 1 .1 8 39.0 ---- + 128 256 Serum Blank ---- + 128 256
Serum Blank Reacted with Unsensitized Cells — -- f ------Reciprocal of Highest Dilution Demonstrating a Positive Agglutination. DISCUSSION
The forecoing results show that chicken anti sera to rabbit serum or its globulin fractions could bo successfully used as anti- globulin sera for the detection of rabbit antibodies on sensitized erythrocytes. However, the efficiency of each antiserum to detect rabbit globulins on erythrocytes varied considerably under different tost conditions.
Various protein fractions of rabbit serum prepared by the
Cohn procedure were electrophorotically analyzed. Hemagglutination tests employing these fractions artificially coupled to erythrocytes and quantitative precipitation of these fractions showed that chicken antisera contained antibodies specific for at least several protein fractions. The data also provided information on the probable protein identity of rabbit red cell iso-antibodies and sheep cell hem olysin.
In addition, several interesting topics such as the antigenic relationship of some rabbit globulin to an antigen in sheep erythro cytes} the failure of certain rabbit serum fractions to couple with rabbit erythrocytes so as to be demonstrable by antiglobulin sera} and the production of immune chicken hemagglutinins for rabbit erythrocytes which were best demonstrated in 8.0 per cent ITaCl, will be discussed.
83 84 Inherent tc any discussion of antibody specificity is the necessity to consider the limitations of the tests employed, the physicochemical differences of antibodies with the same specificity, and the properties of antigens used for either immunization or serological testing.
After comparison of the electrophoretic analyses of the im munizing agents to analyses of the various Cohn fractions used for precipitation with chicken antisera, it was not surprising that all the antisera were capable of precipitating fractions II and III, and it was anticipated that antisera to rabbit serum (anti-IIRS and anti-
IKtSt?) would precipitate large amounts of rabbit serum Cohn fractions
IV and V, while antisera to rabbit globulins precipitated by ammonium sulfate (anti-UH^+P) and by sodium sulfate (anti-Ua^+Al) would cause little precipitation of these fractions until large amount of antigen were added. Various dilutions of the antigens were used so that the component present in the greatest concentration would be emphasized in the highest dilution. The data obtained from hemagglutination of rabbit serum Cohn fractions coupled to erythrocytos closely corrobo rated the results of the precipitin tests, with the exception of anti— lTag+Al. This antiserum was produced in response to m ainly gamma and beta rabbit serum globulins and yet, in the hemagglutination tests it demonstrated specificity for all the various rabbit serum fractions attached to erythrocytes, even those which primarily consisted of alpha globulin and albumin.
Similarly, Coombs and Ilourant (1947) found that an albumin fraction (Cohn) of human serum caused the production of rabbit 85
antibody which strongly agglutinated Rh positive cells sensitized
with incomplete human antibody. This albumin fraction, was not
capable of neutralizing the Coombs' test, but purified gamma globulin
fractions did cause inhibition. Williams and Crabar (1955) clarified
this inconsistency by discovering that gamma globulin molecules with
the same antigenic specificity differ from each other in molecular
weight, sokubility characteristics and electrophoretic mobilities.
Moreover, during the separation of serum fractions, it appeared that
small amounts of gamna globulin complexed with other globulins or
albumin, and migrated with higher electrophoretic mobility rates than
normal globulins. In view of these facts it is conceivable that
complexes of albumin or alpha globulin could migrate with the slower
moving gamma and beta globulins. It has also been demonstrated (Cohn,
Wetter, and Deutsch, 1949) that the use of antigens containing minute
amounts of highly antigenic impurities resulted in the production of
antisera with highly distorted serological specificities.
One possible explanation for the unexpectedly wide spectrum
of specificity of anti-ITag+Al for various serum fractions was that the
sodium sulfate precipitated globulins may have contained smaller
quantities of highly antigenic complexes of alpha globulin and albumin
v/hich were not electrophoretically detectable.
Differences in the sensitivities of hemagglutinating and pre
cipitating tests may also account for discrepancies in specificity*
The graphically illustrated nitrogen precipitation curves exhibit
various peaks, depressions and broadened areas for each antiserum.
Electrophoretic examination of the Cohn fractions suggest that the 86
initial peaks were probably due to interaction of antibodies with
a specificity for that antigenic component which was present in the
highest concentration. Many workers (Munoz and Becker, 1952; Grabar,
19505 Cohn, Deutsch, and Wetter, 1950; Cohn, Wetter, and Deutsch,
1949) consider these irregularities to he due to the depression of
nitrogen precipitation hy excess antigen, new antigen—antibody com
plexes as antigenic impurities increase in concentration, and the
insolubility of antigens in high concentration. Even antigens which
appeared homogeneous by ultracentrifugal and electrophoretic studies were easily distinguished in the antigen excess zones of precipitation
t e s t s .
If only the initial portions of the precipitin curves for anti-Wag+Al serum with fractions II and III are considered, it is obvious that this antiserum precipitated greater amounts of nitrogen than the other antisera. However, precipitation of fractions IV and
V by this antiserum is comparatively poor until large amounts of antigen are added. This indicated that only in high concentrations of fractions IV and V could minor antigens (perhaps gamma and beta globulins) become serologically demonstrable by precipitation.
The hemagglutination test employing antigens artificially coupled to erythrocytes is many times more sensitive than the pre cipitation test. Yet, Stavitsky (1954) contends that this sensitivity is sometimes a hindrance to the determination of major specificities*
That the great sensitivity of the hemagglutination and hemagglutination inhibition reactions is not always advantageous is shown by the experiments on the hetero geneity of antigens and antisera. It is difficult to 87
rule out the participation in the over-all hemag glutination reaction of minor impurities in the antigens and antiserum. . * . it has been shown that a horse immunized to three times recrystal— lized egg albumin developed more antibody to the small amount of conalbumin impurity than to the major egg albumin component. • • • However, in general the heterogeneity of antigen-antibody must be studied by a variety of immunochemical tech niques including hemagglutination, absorption of the sera to remove heterologous antibodies, the Oudin method and modifications of the qualitative precipitin technique. • • •
Thus another theory exists for the unexpected specificity of anti—ITa^+Al. It is possible that the combination of a highly sen sitive test and the unique ability of this antiserum to detect minute quantities of gamma and beta globulins in fractions IV and V may have caused the hemagglutination of these fractions attached to erythro c y te s .
The experiments of Richter and Haurowitz (i 9 6 0 ) p ro v id e another possibility for the seeming disparity between hemagglutination and precipitin tests. During attempts to determine how long antibody synthesis to proteins occurred in rabbits, these workers discovered that antibody present in the circulation three or more weeks after antigen injection no longer was demonstrable by precipitation, but was detectable for several months by specific agglutination of antigen coated red blood cells. It is generally conceded that larger amounts of antibody are necessary to cause precipitation than are needed for agglutination and because of physicochemical differences antibodies with the same specificity are not always demonstrable by the same method. The failure of certain rabbit serum fractions to attach to rabbit erythrocytes by diazotization in such a way so as to be 88 demonstrable in antiglobulin tests may also provide misinformation concerning the specificities of antiglobulin chicken sera. Therefore it was necessary to check antisera "by interfacial precipitation techniques, then diazotize serum fractions to human erythrocytes and then recheck the attachment with an antiglobulin serum of known specificity. Since fractions II and III (mainly gamma and beta globulins) completely inhibited hemagglutination of cells by anti globulin serum it was concluded that rabbit antibodies were pre dominantly gamma and perhaps some were beta globulin. In some anti sera, antiglobulin hemagglutinin adsorption could be correlated with the antigen excess zone follovring the initial precipitation curve peak.
Various investigators have employed a number of preparations to produce competent Coombs1 antiserum fo r th e d e te c tio n of human antibodies on erythrocytes. Coombs and Uourant (1947) compared a variety of antigens and found the highest antiglobulin hemagglutinin titers were produced by rabbits immunized with human serum adsorbed on alum. Proom (1943) also advised the use of this antigen to produce highly effective precipitating antisera.
In order to evaluate the sensitivities of anti-rabbit globulin chicken sera, the sera were rated by correlating (l) the data on pre cipitin curves obtained by the addition of amounts of antigen up to
100,0 micrograms of rabbit serum Cohn fraction II and up to 40.0 micrograrns of fraction III; (2) antiglobulin titers with antibody sensitized red cells; and ( 3 ) antibody titers obtained with Cohn fractions II, III, and IV attached to erythrocytes. 89
In this frame of reference anti-Nag+Al and anti-HRS+F were the best antiglobulin hemagglutinating and precipitating sera.
Anti-HRS was moderately effective and anti-NH^+F the least sensitive serum. In the presence of fractions IV and V anti-HRS and anti-HRS+F produced the largest amounts of precipitate. However, these fractions were not implicated in antibody detection.
It has been shown that high salt concentration (approximately
8.0 per cent ITaCl) is necessary for the maximum precipitation of antigen by chicken antisera (Goodman, Wolfe, and Horton, 1951? Good man, Wolfe, and Goldberg, 1954; Banovitz and Wolfe, 1959; Orlans,
Rose, and Uarrack, 1961). However, only in the case of anti-Ha^+Al was the antiglobulin hemagglutinin titer increased in a diluent of
8.0 per cent ITaCl. In all other antisera the antiglobulin titers were much lower than those found in the usual physiological saline.
It would seem that for most antiglobulin hemagglutinating tests utilizing chicken antisera O.85 per cent ITaCl would be the most desirable diluent.
It is not uncommon for animals immunized with serum from another species to produce antibodies with a specificity for some antigen on the erythrocyte of the donating 3pecies. For instance, rabbits immunized with human serum will produce antibodies to a globulin-like substance on normal human red blood cells (Stratton and Jones, 1955)*
Ali (1959) found that the hemagglutinin for normal rabbit red cells in chicken antiserurc to sodium sulfate precipitated serum globulins adsorbed on alum was maximally demonstrated in O.85 per 90 cent ITaCl as well as in 8.0 per cent ITaCl. Furthermore, after ad sorption with normal rabbit cells such antiglobulin serum still showed high titers of hemagglutinins when titrated in 8.0 per cent sodium chloride. The naturally occurring rabbit hemagglutinin in normal chicken serum was maximally demonstrated in 0.85 per cent
ITaCl and only weakly active in 8.0 per cent ITaCl solutions. The findings wore verified in tho present study. When chickens were immunised with various rabbit globulin and serum preparations the titers of hemagglutinins for normal rabbit erythrocytes were almost the same for immune and normal chicken sera tested in 0 .8 5 p e r c e n t
ITaCl. In the presence of o.C per cent ITaCl the titers of hemag glutinins in immune sera were quite varied and seemed to bo directly related to antiglobulin activity in O.Cfj per cent ITaCl. In other words it appears that the groater the antiglobulin capacity, tho higher the titer of hemagglutinins for normal rabbit red cells in
8.C per cent ITaCl. This suggests that rabbit serum globulin is capable of producing chicken antibodies which have an affinity for a globulin on rabbit erythrocytes and can only be demonstrated in solutions of high NaCl concentration.
In addition to this finding it was also demonstrated that immunisation of chickens with rabbit globulin and serum produced antibody with a specificity for sheep erythrocytes. In the study of
Friedman and Yeatman (lS?56) the immunisation of chickens with rabbit kidney produced an antibody which agglutinated sheep red blood cells.
By differential chemical extraction these workers determined that the antigen involved was not the classical Forssman antigen, but rather 91 another heterophilic type. Raffel ( 1 9 6 1 ) is of the opinion that the Forssman antigen is not a single entity in its various biological associations and the Leterogenetic relationship depends on similar rather than identical chemical structures. It is most probable that the heterophile antigens found in rabbit kidney and serum are the same entity and bear an antigenic relationship to an antigen in sheep erythrocytes,
"he high titers of normally occurring and immune hemagglutinins present a problem in the determinations of serological sensitivity and specificity by masking antiglobulin titers. For this reason, it is suggested that sera be adsorbed with erythrocytes prior to anti globulin hemagglutination tests.
During the course of immunisation it was noticed that those chickens which received six injections of rabbit serum intraperitone— ally often appeared listless and in poor health on the fifth or sixth day after the initial injection. According to Makinodan et a l.
(1 9 5 2) anaphylactic shock occurred six to eight days after sensiti zation and was best avoided if the chickens were challenged before the fourth day or after the fifteenth day. In accordance with these findings it seemed that chickens receiving six injections of rabbit serum suffered from protracted anaphylaxis. Mortality was highest in this group and it was decided that in the future the use of anti—
URS+F would be comparable to and more expedient than anti-IIRS.
Hektoen (1918) and Brown and Wolfe (1954) found that high titers of precipitating antibody wore usually found nine to thirteen 92 days after immunization of chickens with serum proteins, and there after titers decreased to a minimum at three weeks. Synthesis of anti globulin hemagglutinating antibodies was in close accord with, these findings for precipitating antibodies. In general the highest titers of antisera occurred seven to eleven days after the last injection of antigen and by three to four weeks antiglobulin hemag glutinin activity was no longer demonstrable* SUMMARY
1, Four chicken antise?a prepared by the injection of normal rabhit serum (anti-HRS), normal rabbit serum in Freund's adjuvant (anti-NRS+F), rabbit serum globulins precipitated by NagSO^ and adsorbed to alum (anti-rNag+Al), and globulins precipitated by (NH^gSO^ in Freund's adjuvant (anti—BH^+F) were examined for antiglobulin specificity and sensitivity* 2* The hemagglutinins for normal rabbit erythrojytes in normal and anti-rabbit globulin chicken sera were well demonstrated in 0*85 and 8*0 per cent NaClf however, the immune hemagglutinins were best expressed in 8*0 per oent NaCl* Immunization with rabbit globulins also increased the titers of hemagglutinins for normal sheep erythrocytes* 3* After adsorption with rabbit erythrocytes all the anti sera with the exception of anti-NH^+F were capable of detecting iso immune and anti—sheep erythrocyte rabbit antibodies on erythrocytes* Anti-NH^+F detected only iso-iramune rabbit antibodies. 4* In an attempt to determine the sp ecificities and sensi tivities of the chicken antisera, rabbit serum Cohn fractions II (gamma), III (beta), IT (alpha), and V (albumin) were employed in quantitative nitrogen precipitation tests and these same fractions plus fractions IV—1 (alpha) and IV—4 (alpha) were coupled to human erythrocytes to be used in hemagglutination tests* None of the 94 antisera showed a specificity for any one fraction and discrepancies were noted when the results of hoth procedures were compared* 5* The supernatants of the antisera precipitated with the -various Cohn fractions were titrated with antibody sensitized erythrocytes in order to determine the globulin classification of these rabbit antibodies*
6. Cohn fractions II and III effectively neutralized the
antiglobulin capacity of the antisera, indicating that rabbit iso
immune and anti-sheep erythrocyte antibodies are mainly gamma and perhaps beta globulin* 7* By correlating the data it was decided that anti-Hag+Al and anti-HES+F were the best chicken antisera for detecting rabbit antibody on erythrocytes. Anti-MRS was moderately effeotive and anti-HH^+F the least active antiglobulin serum* BIBLIOGRAPHY
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Stratton, F. and Jones, A. B. 1955* The Beactions Between Normal Human Bed Cells and Antiglobulin (Coombs) Serum* J* Im munol. 75*423* Wiener, A* S. and Gordon, E. B. 1953* Quantitative Test for Anti- body-Globulin Coating Human Blood Cells. J. Clin. Path. 23*429. Williams, Jr., C. A. and Grabar, P. 1955* Immunoelectrophoretic Studies on Serum Proteins. III. Human Gamma Globulin. J. Immunol. 74*404. Wolfe, H. B. 1952. Preoipitin Production in Chickens. I . Inter facial Titers as Affected by $iantity of Antigen Injected and Aging of Antisera. J. Immunol. 44*135* Wolfe, H. B. and Bilks, S. 1946. Precipitin Production in Chickens. II. Studies on the in vitro Bise of the Inter facial Titers and the Formation of Precipitins. J. Immunol. 52:1946. Wolfe, H. B. and Dilks, E. 1948. Precipitin Production in Chickens. III. The Variation in the Antibody Eesponse as Correlated with the Age of the Animal. J. Immunol. 58*245* Wolfe, H. B,, Mueller, A. P., and Neese, J. C. 1959* Physical Factors Affecting Maximum Precipitation of the BSA-Anti- BSA Fowl System. J. Immunol. 2:195* Wolfe, H. B., Mueller, A., Neese, J., and Templis, J. 1957. Precipitin Production in Chickens. XVI. The Belationship of Age to Antibody Production. J. Inmunol. 79*142. AUTOBIOGRAPHY
I, Marvin Rogul, was Born In St. Louis, Missouri, June 19* 1932. I received my secondary-school education in the public schools of St. Louis, Missouri, and my undergraduate training at the Uni versity of Missouri, which granted me the Bachelor of Arts degree in 1953* Prom the University of Missouri, I received the Master of Science degree in 1957 • In 1958 I enrolled in the Department of Microbiology in The Ohio State University. While completing the requirements for the degree of Doctor of Philosophy I served suc cessively as a graduate assistant in the Department of Microbiology and as a research assistant in The Ohio State University Research Foundation.
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