antibody heterogeneity and serological reactions · for measuring antibody activity, for example,...

BACERIOLOGICAL REVIEWS, June 1967, p. 157-174 Vol. 31, No. ICopyright © 1967 American Society for Mcrobiology Printed in U.S.A.

Antibody Heterogeneity and Serological ReactionsROBERT M. PIKE

Department of Microbiology, The University of Texas Southwestern Medical School, Dallas, Texas 75235

INTRODUCTION................................... 157COMPARISON OF yG AND eyM IMMUNOGLOBULINS................................... 158

Sequential Appearance in Serum................................... 158Factors hIfluencing Kind of Antibody Produced.................................. 159

Nature of the antigen ................................... 159Dosage of antigen................................... 159Other factors................................... 159

Heat Inactivation................................... 159Stability of Union with Antigen ............. ...................... 160Agglutination........................................... 161Precipitation................................... 161Complement Fixation ................................... 162Lysis........................................... 163Bactericidal Activity................................... 164Neutralization................................... 164Opsonization ................................... 164Hypersensitivity................................... 165Reactions Involving Rheumatoid Factor................................... 165

yA IMmUNOGLOBULiN................................... 166OUTLOOK................................... 167SUMMARY................................... 167LITERATURE CITED................................... 168

INRODUCIONThe antibody activities of serum are attributable

to a family of globulins which can be distinguishedby their chemical, physical, and antigenic proper-ties. The extreme heterogeneity of these immuno-globulins (35) with respect to electrophoreticmobility, molecular size, and structure has beenemphasized in a succession of reviews of whichseveral have appeared within the year (39, 44, 81).A standard system of classification and nomen-clature (27) has been recommended to avoid theconfusion which would inevitably result from adiverse terminology. As the criteria for recogniz-ing the different classes of immunoglobulins havebecome better defined, it has been increasinglyapparent that immunological reactivity is oftendependent upon the kind of antibody present.Although an antibody of one class can participatein several immunological reactions in compli-ance with Zinsser's unitarian theory (157), onekind of antibody may be much less efficient thananother or may fail completely to bring about agiven reaction. Our present purpose is to sum-marize recent information regarding the capacityof different immunoglobulins to elicit variousserological reactions. Although it is impossibleto separate completely the serological activity ofantibodies from other phases of antibody func-

tion, no attempt will be made to review the manybiological problems concerned with antibodyformation and distribution. The cellular origin ofimmunoglobulins (80, 152) and immunologicalmemory (142) are important related subjects notincluded in this discussion.The three major classes of immunoglobulins

known to have antibody activity are -yG (IgG),7S, usually mercaptoethanol-resistant; yM (IgM),19S, mercaptoethanol-sensitive; and yA (IgA)which may occur as 7 to 17S, depending on thedegree of polymerization (41). Two other immu-noglobulins have been identified: yD, which ac-counts for less than 1% of normal human serumimmunoglobulin (lSa), has not yet been shownto have serological activity; a globulin associatedwith human reagin has been designated yE (63).These five globulins have antigenically differentheavy chains. In addition, subclasses and allo-types have been described (39, 41). The -yG and-yM are most readily isolated and identified, andhave been most extensively studied in relation toimmunological properties. The first portion ofthis review will, therefore, be concerned with acomparison of the activities of these two immuno-globulins. Since the serological activity of a givenserum specimen may be influenced by the rela-tive concentrations of these two classes of anti-

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body, it also will be profitable to examine thecircumstances which may affect their occurrence.Finally, the more limited information regarding-yA will be summarized.

COMPARISON OF yG AND -yM IMMUNOGLOBULINS

Sequential Appearance in SerumAccording to the experience of most investi-

gators, the first antibody to be detected in theserum after antigenic stimulation is yM. Thisinitial antibody usually declines or disappearscompletely in several weeks to be replaced by yGwhich may persist for long periods of time. Sucha sequential synthesis has been studied most ex-tensively in the rabbit and has been reported in awide variety of species (Table 1) in response tosoluble and particulate antigens of diverse compo-sition. The tabulation includes not only instancesin which the sequence was observed after a singleprimary stimulus but also situations in which asecondary stimulus or hyperimmunization wasrequired to elicit detectable amounts of yG.Although this pattern appears to be a generalone, there are variations in the degree to which-yM is replaced by yG, depending on the kind ofantigen, the dosage, and the species. Of specialinterest is the fact that the early 'yM response inthe turtle (52) and the goldfish (144) is unusuallyprolonged as compared to that observed in mam-mals. There are some reports in which no -yG atall has been detected (10, 30, 79), but others haveobtained a yG response to the same antigens.The relation of the antibody sequence to thecellular aspects of the immune response and someof its implications have been reviewed by Uhr(142).There have been only a few opportunities to

observe the sequence of antibody appearance innaturally occurring infections. Murray et al.(84, 85) have shown that the antibody in primarytyphus is predominantly yM, whereas that in therecrudescent disease is 'yG. Serum from patientswith leptospirosis taken from the 2nd to the 5thweek after onset showed an increase in the propor-tion of yG as determined by agglutination ofsome serotypes but not of others during thistime interval (93). In heifers (109) and humanvolunteers (103) experimentally infected withBrucella, the early agglutinin was yM; oyG wasnot observed until about 2 weeks after infection.More recent observations, however, suggest

that the sequential synthesis of yM and yG maybe more apparent than real. Freeman and Stavit-sky (40), by means of radioimmunoelectrophore-sis, detected the simultaneous appearance of 'yGand -yM antibodies in rabbits after injection ofhuman serum albumin and bovine -y-globulin.

TABLE 1. Reports recording the early appearanceof yM followed by the appearance

of yG antibody activity

Species Antigen Reference

Rabbit

Mouse

Rat

Guinea pig

Cattle

Sheep

Man

Chicken

Turt le

FrogGoldfish

Serum proteins

Hemocyanin

HaptenesDiphtheria toxoidErythrocytes

Salmonella H antigenSalmonella 0 antigenLeptospiresMycobacteriaPoliovirusBacteriophage

SalmonellaActinophageSheep red cellsOvalbuminFerritinInfluenza virus

Bacterial flagella

Foot and mouth diseasevirus

BacteriophageArbovirus

Brucella

Foot and mouth diseasevirus

Diphtheria and tetanustoxoids

Salmonella H antigenBrucellaDiphtheria toxoid

Serum albuminBacteriophage

Hemocyanin and serumalbumin

BacteriophageBacteriophage

10, 11,15, 53

10, 11,30

911, 10611, 95,

12910, 1199, 147932913210, 11,

143

1406934418

1

24, 28,50

14313

109, 110,111

24

78

79, 141103106

17, 105112, 144

52

144144

Using a method which measured the antigen-binding capacity of rabbit antibodies for humanserum albumin, Osler et al. (89) found evengreater quantities of 'yG than yM within the firstweek after antigen injection. Although the syn-thesis of -yG and -yM may begin at about the

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same time, it is obvious that 'yM will appear topredominate in early sera if the methods usedfor measuring antibody activity, for example,agglutination, favor the detection of yM (40, 89).

Factors Influencing the Kind ofAntibody ProducedNature of the antigen. Protein antigens have

usually been found to stimulate a preponderanceof 'yG antibody after the early 'yM has decreasedor disappeared (10, 53). The same has been trueof viruses (18, 28, 50), bacteriophage (69, 112,143), and simple haptenes (9). On the other hand,the lipopolysaccharide somatic antigens of entero-bacteria stimulate yM antibodies predominantlyin man and the rabbit (10, 79, 86, 97, 99, 147),whereas in the guinea pig the antibodies toEscherichia coli polysaccharide were almost en-tirely yG (22).

There is evidence that the physical state of theantigen may be a factor. Ada et al. (1) comparedthe response of rats to the monomer and polymerforms of flagellin and noted a greater tendency ofthe latter to stimulate -yM. Lindqvist and Bauer(77) found that aggregated bovine serum albumininduced a consistently higher yM response inrabbits than did nonaggregated albumin. Rabbitsimmunized with thyroglobulin bound to acrylicresin particles showed a 20-fold increase in yM ascompared to rabbits immunizd with thyroglobu-lin alone (139). A marked yM response in rab-bits was obtained by the injection of particulatedeoxyribonucleic acid (DNA)-methylated bovineserum albumin complex (119). These observa-tions strongly suggest that particle size may in-fluence the kind of antibody produced.Dosage of antigen. There are indications that

the dosage of antigen or the intensity of anti-genic stimulation can influence the proportion of-yG and yM produced. It was first reported thatbacterial lipopolysaccharide antigen elicited yMantibodies only (10, 79), but, when largeramountsof antigen were injected over longer periods oftime, considerable amounts of yG were alsodemonstrable (97, 99, 107, 147). Only yM ag-glutinins for sheep erythrocytes were found aftera single injection, whereas both yG and yMappeared after two injections in rabbits (11).Svehag and Mandel (132) observed that a rela-tively small dose of poliovirus in rabbits elicitedonly yM, but, when a much larger dose was given,the initial -yM was followed by yG. It must berecognized, of course, that the greater the totalquantity of antibody, the greater the chance ofbeing able to detect a given proportion of a minorcomponent. The relative efficiency of a serologicalprocedure in detecting different classes of anti-body must also be considered (89, 99).

Other factors. A variety of other factors mayalso influence the kind of antibody produced.Treatment of mice with thorotrast prolonged the-yM response to Salmonella typhimurium, asmeasured by opsonic activity, and increased theyM response to sheep cells, while completelyinhibiting the production of yG (65). Endotoxininjection and X irradiation prolonged the yMresponse to bacteriophage (143). That differentspecies may respond differently to the same anti-gen was shown by Dixon et al. (30), in that rab-bits produced both yM and 'yG after intravenousinjection of hemocyanin, whereas rats producedyM only. There was a yG response in rats,however, when the antigen was mixed withFreund's adjuvant.

It is apparent from these examples that both'yG and yM antibodies can result, under theproper circumstances, from the injection of anyantigenic material so far studied, although oneor the other may. predominate in a particularsituation. There are several naturally occurringantibodies, however, which thus far have beenfound only as -yM globulins. The natural anti-bodies of the mouse for sheep erythrocytes (3,151), and the normal rabbit antibody for thepoliovirus (131) are in this category, although yGwith the same specificity can be produced byartificial immunization. At first, the only naturalantibodies of man detected by reactions withgram-negative bacteria were yM (34, 82), but,by indirect fluorescent-antibody staining, a majorportion of natural human antibody to Escherichiacoli and Neisseria gonorrhoeae was found to be'yG (26a, 26b). Differences in the sensitivities ofthe tests used probably accounts for the earlierfailures to detect natural yG (26b). The coldagglutinins which occur in patients with hemo-lytic anemia (42) and with pneumonia due toMycoplasma (71) have been identified as yMwith the exception of one instance in a patientwho had cold agglutinins of the yA class (6).Although the sheep cell antibody developing ininfectious mononucleosis, which is completelyabsorbed by boiled beef cells, is yM (71, 76, 91,94), antibody produced in rabbits by the injec-tion of boiled beef cells was found in both yMand -yG fractions (24a). The rheumatoid factorswhich are usually considered to be antibodies tonormal 7-globulin have the same physicochemi-3al and antigenic properties as other yM anti-bodies (73). There is increasing evidence, how-ever, that rheumatoid factor activity may also bepresent in -yG and -yA globulins (55).

Heat InactivationIt has been known for many years that anti-

bodies differ with respect to resistance to inacti-

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vation by heat, and that heat resistance tends toincrease with time after immunization (74). Kiddand Friedewald (68) described a naturally oc-curring antibody in rabbit serum which fixedcomplement with extracts of rabbit tissues, andfound that its activity was destroyed by heatingat 65 C for 30 min; specifically induced antibodieswere not affected by this temperature. Theydiscussed the significance of this antibody inrelation to "nonspecific" complement-fixationreactions observed by numerous other investi-gators, and noted that such reactions could fre-quently be avoided by heating the serum to adegree which would not affect specific antibodies.Natural sheep hemolysin and Wassermann reaginfound in normal rabbit serum showed a heatlability comparable to the antibodies for rabbittissues (68). Studying autoantibodies producedin rabbits by the injection of rat tissue, Ashersonand Drumonde (7, 8) found that macroglobulinantibody was destroyed by heating at 65 C for30 min, whereas the low molecular weight anti-body was stable at this temperature.The recognition of differences in heat inactiva-

tion of Brucella agglutinins has received practicalapplication in tests to distinguish nonspecificallyoccurring agglutinins from agglutinins resultingfrom infection (149). Two types of nonspecificagglutinins have been detected, both of whichare more susceptible to heat inactivation thanare the specific agglutinins (149). The nonspecificagglutinins resemble the early postvaccinal orpostinfection agglutinins in heat lability and insedimenting more rapidly on ultracentrifugationthan the low molecular weight agglutinins whichappear later (109-111).As long ago as 1904, it was observed that

agglutinins for bacterial flagella were more stableon heating than were agglutinins for somaticantigens (19). This observation was repeatedlyconfirmed over a period of 50 years without ade-quate explanation (98). When it was reportedthat somatic agglutinins tended to be yM whereasantiflagellar antibodies were predominantly yG(10), Pike and Schulze prepared antisera toseveral antigens and compared the heat stabilityof 7yG and 7M antibodies to each of these anti-gens (98). The 7G antibodies to the typhoidsomatic antigen withstood a temperature of 70 Cfor 30 min, whereas the yM antibody activity waspartially destroyed at 70C and completely in-activated at 75 C. The same relative suscepti-bilities to heat were shown for yG and 'yM anti-bodies to sheep cells, leptospires, bovine serumalbumin, and the flagellar antigens of the typhoidbacillus. It became apparent that the observeddifference in heat stability of somatic and flagellar

agglutinins was merely a reflection of the relativeheat stability of the predominant type of antibodyglobulin involved.This difference in heat lability of yG and yM

seems to be a consistent finding, since it has beenshown for-tetanus and diphtheria antitoxins pro-duced in sheep (78) and for rabbit antibodies forthe poliovirus (131). Locke and Segre (78) foundthat inactivation at 65 C for 1 hr provided a morereliable measure of macroglobulin antibody thandid treatment with mercaptoethanol. This differ-ence in heat resistance is compatible with thewell-known susceptibility of normal antibody toheat (68, 124) and the finding that most normalantibodies are yM (124, 131).

Stability of Union with AntigenThe firmness with which antibody binds to

antigen is a property often referred to as avidity.Less avid antibody dissociates from its union withantigen more readily than does antibody of highavidity. The literature pertaining to this subject issometimes difficult to interpret because of thevaried criteria used for measuring avidity. Theeffect which firmness of attachment may have onthe end result of an antigen-antibody reactiondepends on the kind of reaction involved. Whenlysis of erythrocytes or bacteria is the end result,an antibody which can more readily dissociatefrom antigen may produce serial injuries to cellsalthough only a limited amount of antibody ispresent. When neutralization of a toxin or a virusis the end result, dissociation would liberate toxinor infectious virus, and antibody activity wouldtherefore be less effective. The well-known factthat antitoxins produced in the early stages ofimmunization tend to have irregular neutralizingpower and are easily dissociated from toxin ondilution (102) would suggest that yM may be ingeneral less avid than yG. The greater bactericidaleffect of yM (107), as well as its inability to neu-tralize toxins and enzymes (106), would alsoimply low avidity.In some instances in which the avidity of yG

and yM antibody has been compared, the dataseem to support the suggestion that yM is lessavid. Robbins et al. (107) found that yM dis-sociated from S. typhimurium more readily inacid solution than did -yG. Furthermore, absorp-tion preferentially removed yG (58). In virus-antibody mixtures, Svehag (131) showed thatinfectious virus was released more readily from'yM in acid solution than from yG, indicating agreater avidity of 0G.

Other experiments have led to different conclu-sions. One method used to measure the stabilityof the union between antibody and erythrocytes

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has been to determine the degree of cell-to-celltransfer. Taliaferro (134) and Goodman andMasaitis (47) found that rabbit yG anti-sheephemolysin was transferred to a greater extentthan -yM, indicating a greater avidity for yM.Greenbury et al. (51) also concluded that 'yM ismore avid than -yG, since a given number ofhuman A cells fixed a considerably greater pro-portion of the yM antibody than of the yG. Bymeasuring the degree of dissociation of bacterio-phage from guinea pig antibodies, Finkelsteinand Uhr (38) found that yM obtained 1 weekafter immunization had a higher avidity than yGobtained 1 week later.Attempts to compare the avidity of yG and

,yM are complicated by the fact that the affinityof antibody increases with time after immuniza-tion (32, 53, 135), and the increase can occur inone molecular class of antibody (70, 128, 134).Studying changes in the quality of antibody dur-ing the course of immunization, Grey (53) foundthat, at the same stage of immunization, thedissociation rates of rabbit anti-bovine serumalbumin yG and yM were similar. Even if therewas a difference, insufficient yM was present toaccount for the observed change in bindingstrength with time. The avidity of both yG and7yM guinea pig antibodies for bacteriophage in-creased with time after immunization (38). Inthe light of these observations, any comparison ofthe avidity of different classes of antibody shouldtake into account the time factor as well as otherconsiderations discussed by Finkelstein and Uhr(38).

AgglutinationIt is apparent from many observations that

both yG and yM antibodies are capable of caus-ing agglutination, but to different degrees.Greenbury et al. (51) compared rabbit 131I-labeled -yG and yM antibodies for human erythro-cytes and found that over 100 times as much yGas yM was required to give 50% agglutination(Table 2). On a molecular basis, they calculatedthat 19,000 molecules of yG per cell were re-quired for 50% agglutination, whereas only 25molecules of yM per cell were required. Otherreports (57, 64, 77, 99, 107, 133) giving quantita-tive data show considerable variation (Table 2),but most agree that smaller amounts of yM than,yG are required for agglutination. One notableexception is the report of Heremans et al. (57)in which human yG and yM displayed approxi-mately the same agglutinating activity for Bru-cella. In a passive agglutination test, the sameauthors found that considerably more 'yM than7G antidiphtheria toxin was required for ag-

glutination. Other reports in which passive ag-glutination was employed to demonstrate humanantibodies to penicillin (146), and to demon-strate rabbit (53) and chicken (105) antibodiesto bovine serum albumin, indicate higher aggluti-nating activity for yM without giving quantita-tive comparisons. Although the diversity ofantigens, reaction end points, and methods ofassay used in these studies would be expected toresult in variations in the quantitative data, it isunlikely that this could account for all the differ-ences observed. An additional important variableis evident from the work of Osler et al. (89), whofound that yG rabbit anti-human serum albuminobtained from early bleedings after a single injec-tion possessed feeble if any hemagglutinatingactivity, in contrast to yG antibody from hyper-immune sera which showed higher specific ag-glutinating activity than yM from the same sera.Furthermore, there was considerable variationin the agglutinating properties of the immuno-globulins of different animals.

It is apparent from most of the work cited thatagglutination tends to favor the detection of yMantibody, particularly if early sera are examined.In comparing the agglutinating efficiency of thetwo immunoglobulins, it is important to selectan appropriate basis for comparison. To notethat one serum fraction has a higher agglutinintiter than another merely describes the distribu-tion of antibody activity in the sample of serum.Comparing agglutinating activity with some otherserological manifestation which itself may bevariable can give a distorted impression. Theabsolute amount of antibody expressed as theweight of antibody nitrogen or the molecularconcentration would, therefore, seem to be amore appropriate basis for comparison.

PrecipitationStudying rabbit antibodies to bovine serum

albumin, Benedict et al. (15) obtained evidencethat hemagglutination detected antibodies whichcould not be demonstrated by precipitation.Several investigators have reported little or noprecipitation with yM antibodies for serum al-bumin (53, 105, 123), although these antibodieswere readily detected by passive hemagglutina-tion. Both -yM and yG, however, gave precipitinlines in gels with Salmonella endotoxin (107, 147),with soluble rickettsial antigen (84), and withcoccidioidin (120). Mulholland et al. (83) re-ported quantitative precipitation results withS. enteritidis endotoxin and rabbit sera whichcontained predominantly 'yM antibody. It is wellknown that rheumatoid factor, a yM globulin,reacts with normal 7y-globulin to form a precipi-

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TABLE 2. Agglutinating activity of yG and -yM antibody

Agglutinating activity per mlSpecies Antigen Reference

-YG .... M

Rabbit Human erythrocytes 0.45-0.59 pig of pro- 0.00-0.0045 pAg of Greenbury et al. (51)teina proteina

Rabbit Human erythrocytes 0.05 ,g of Nb 0.004 pg of Nb Tada and Ishizaka (133)Human Human erythrocytes 0.01 prg of Nb 0.0004-0.0008 pg of Ishizaka et al. (64)

NbRabbit Salmonella typhi- 25-50 jpg of protein6 1-5 pig of proteins Robbins et al. (107)

muriumRabbit S. typhosa 1.44 ig of proteina 0.13 pug of proteina Pike et al. (99)Human Brucella 10.9 pug of protein6" 9.4 pg of protein6" Heremans et al. (57)Rabbit Human serum albu- 0.29 jug of protein" 0.15 pug of protein" Lindqvist and Bauer

min (77)Human Diphtheria toxin 1.6 pg of protein6" 7.5 pg of proteins Heremans et al. (57)

a Amount required for 50% agglutination.b Minimal amount required for agglutination."Based on total purified immunoglobulin.

tate (33, 56). Tada and Ishizaka (133) obtainedprecipitation with both yG and yM antibodies tohuman blood group A substance, but the floccu-lation time for 'yM was much slower, and inhibi-tion of precipitation by excess antigen was lessapparent with yM than with yG. The slow floc-culation time of 'yM is consistent with the ob-servation of Grey (53) that yM had a slowerassociation rate with antigen than yG, possiblydue to differences in molecular charge and size.The relative insolubility of the yM precipitate inantigen excess has also been observed with rabbitantibody to typhoid endotoxin (99) and withrabbit antibody to bovine serum albumin (77).In all three instances, yG precipitin curves showedtypical inhibition of precipitation in the region ofantigen excess.

In studying the precipitating and agglutinatingcapacities of yG and 'yM antibodies to typhoidsomatic antigen, it was found that yG was a muchmore effective precipitin than 'yM when agglutinintiter was used as the basis for comparison (99).When precipitin curves were determined for thetwo classes of antibody with varying amounts ofendotoxin, the ratio of antigen to antibody atequivalence was roughly the same in both curves.This suggests that the ability of yG and 'yM toprecipitate this antigen was of the same order ofmagnitude when compared on a weight basis.There is no clear explanation for the contrast-

ing agglutinating and precipitating capacities of7G and 'yM antibodies. The probability that theyM molecule has five (77) or more (60, 86a) com-

bining sites may account in part for its markedagglutinating ability, but one would expect ananalogous advantage to show up in precipitation.

Complement FixationPrior to 1960, it had often been observed that

the ability of various sera to fix complement wasvariable with respect to other serological proper-ties. For example, in virus infections complement-fixing activity of serum may appear later thanother demonstrable antibodies (136). In studyingthe antibody response of guinea pigs to foot-and-mouth disease virus vaccine, Graves et al. (50)found that neutralizing antibody was demon-strable in the serum within the first week afterinoculation, whereas complement fixation wasnot obtained until the 16th day. This delay in theappearance of complement-fixing activity wasexplained by showing that the early antibody waspredominantly yM. It was not until over 50% ofthe neutralizing activity was found in the yGfraction that complement-fixing activity couldbe detected. This provided a clear illustration ofthe superior complement-fixing capacity of yGantibody reacting with a viral antigen. A similarsituation has been described in guinea pigs inocu-lated with several arboviruses (13); the yM frac-tions of serum did not fix complement, and thiswas invariably associated with 7G antibody.Thus, a probable explanation is provided for theobserved delay in the appearance of complement-fixing antibodies in a number of virus diseases.The relation of complement fixation to the

classes of antibodies to a bacterial antigen hasbeen shown in brucellosis. Heremans et al. (57)found that both 7G and 'yM antibody obtainedfrom the serum of a patient with brucellosis fixedcomplement, but the 7G was markedly moreeffective than the yM. In comparing the ag-glutinating and complement-fixing activity of

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bovine sera and milk against Brucella, Andersonet al. (5) observed that failure to fix complementwas correlated with the presence of mercapto-ethanol-susceptible agglutinins. Sera and milkcontaining mercaptoethanol-resistant agglutininsfixed complement well. After inoculation ofcalves with live B. abortus, the complement-fixingtiter of the serum paralleled the titer of mercapto-ethanol-stable agglutinin. These observationsmay be significant in relation to the diagnosis ofchronic brucellosis in man, since Reddin et al.(103) have reported that the sera of patients withbacteriologically proven chronic brucellosis con-sistently contained mercaptoethanol-resistant ag-glutinins; in contrast, the sera of patients in whomthe only evidence of chronic infection was a lowagglutinin titer contained mercaptoethanol-susceptible agglutinins. These authors suggest are-evaluation of the complement-fixation test forsignificance in the diagnosis of brucellosis.A very striking example of the difference in

complement-fixing properties of yG and -yM wasprovided by the studies of Murray et al. (84, 85),comparing the serological reactions in primaryepidemic typhus with those in recrudescent typhus(Brill's disease). The complement-fixation reac-tion in primary typhus was usually negative unlessa relatively large amount of antigen was used (acharacteristic designated as high antigen require-ment), and inactivation of sera at 60 C instead of56 C caused a marked reduction in titer. In con-trast, complement fixation was readily obtainedwith sera from patients with recrudescent typhusby use of 1 unit of antigen, and the reactivity ofthe sera was not altered by inactivation at thehigher temperature. Furthermore, the Weil-Felix titers, which depend upon the agglutinationof Proteus, tended to be high in primary and lowin recrudescent typhus. These differences in sero-logical patterns were explained by showing thatthe predominant antibody in primary typhus isyM, whereas that of recrudescent typhus is yG.Thus, in addition to the relative heat lability andmarked agglutinating properties of yM, theweaker complement-fixing activity of this class ofantibody was shown in relation to the natural his-tory of an infectious disease in man. It is of inter-est to recall that Zinsser, a strong proponent of theunitarian theory, expended great effort to showthat typhus fever and Brill's disease, now knownto be recrudescent typhus (84), were due to thesame infectious agent (158).The fixation of complement in relation to the

type of antibody has been further elucidated bythe identification of two varieties of low molecularweight antibodies in guinea pigs which are desig-nated 7S 71 and 7S y2 according to their electro-

phoretic mobility (14). The 7S 72 fixes comple-ment (21) but does not attach to the skin to givepassive cutaneous anaphylaxis (PCA; 90),whereas the 7S 7l fails to fix complement butdoes give PCA. That this situation is not peculiarto the guinea pig is shown by the finding of simi-lar antibodies in mouse antihemocyanin serum(36) and in horse antihapten serum (70). The 7S7yl component of horse serum did not fix comple-ment, whereas the 7S y2 component did.

Resistance to inactivation by mercaptoethanol(54) has been widely used as a simple means ofdistinguishing yG from yM, although partialinactivation of chicken yG has been observed (16).It now appears, however, that, although some ofthe serological activity of 'yG 'may not be af-fected by mercaptoethanol, this compound de-stroys most of its complement-fixing ability (150)as well as other complement-requiring functions(127). Schur and Christian (122) suggest thatcomplement fixation is associated with thosedouble bonds which are more labile to mercaptanreduction. The complement-fixing titer of human7yG for Mycoplasma pneumoniae, however, wasnot reduced by treatment with mercaptoethanol(121).

LysisSince the lysis of erythrocytes by antibody is

dependent upon the presence of complement, itmight be predicted that the type of antibody moreeffective in fixing complement would also have thestronger lytic action. Apparently, the reverse istrue. In 1957, Stelos and Talmage (129) reportedthat 7M rabbit antibodies for sheep cells werefrom 50 to 100 times more efficient in causinghemolysis than were -yG antibodies when com-pared on the basis of combining power. On amolecular basis, it was estimated by Wigzell et al.(152) that mouse 7M was 100 to 1,000 times moreefficient than yG. A convincing demonstration ofthe greater lytic effectiveness of 7M has been pro-vided by Humphrey and Dourmashkin (60). Elec-tron microscopic examination of sheep cellslysed by antibody and complement revealed thesites of damage as holes in the cell membrane. Itwas calculated that two to three molecules of yMantibody were sufficient to make one hole,whereas about 1,000 times as many molecules of7yG were required for this effect. Observations byGoodman (46) and by Goodman and Masaitis(48) revealed a lack of correlation between theamount of complement fixed and the hemolyticactivity of serum. Since the antibody with thegreater molecular weight fixed less complementper hemolytic unit, it appeared that this antibodyutilized complement more efficiently in the hemo-

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lytic process than did the antibody of lower molec-ular weight.The difference in the hemolytic efficiency of

TG and TM has been apparent in the applicationof the plaque technique for detecting antibody forerythrocytes produced by single cells (67) and inthe adaptation of this technique for the detectionof cells producing antibodies to bacterial antigens(75). The parallel appearance of plaque-formingcells and TM serum antibodies indicated that onlyTyM-producing cells were being detected by thismethod (75, 117, 152). When anti-TyG serum isadded to the system, however, plaques appear inthe region of TG-producing cells (31, 130).

Bactericidal ActivityThe killing of gram-negative bacteria by anti-

body and complement not only provides a highlysensitive method for detecting antibody, but isalso presumably an important defensive mecha-nism. The bactericidal activity of normal humanserum for aerobic enteric bacteria was found toreside almost entirely in the TM fraction (82).Landy et al. (75) found over 99% of the bacterici-dal antibody for S. enteritidis in rabbit serum aftera single injection of endotoxin to be TM. Just re-

cently, the bactericidal effect of normal humanserum on some of the anaerobic, gram-negativebacteria indigenous to the oral cavity and intesti-nal tract also has been identified with the TMglobulin (34). Spitznagel (127), however, re-

ported that the normal guinea pig antibody re-

sponsible for the release of 32p from labeledEscherichia coli in the presence of complement isyG. If this is the same antibody as that which isbactericidal, normal antibody in man and guineapig must belong to different classes. Although thenormal bactericidal antibody has usually beenidentified as yM, yG antibody against gram-nega-tive organisms can be produced by hyperimmuni-zation (97, 147), and this also has bactericidalactivity. It appears to be less effective, however,than yM in relation to agglutinating capacity(147). Quantitative comparisons made by Robbinset al. (107) showed that on a weight basis 18 timesas much yG as yM was required to kill 50% ofa standard sample of S. typhimurium. The de-structive effect of yM antibody for both erythro-cytes and bacteria thus seems to be relativelymore efficient than that of TyG.

NeutralizationAntibodies have the capacity to neutralize

toxins, enzymes, and viruses without participa-tion of complement. Neutralizing activity hasbeen found in both yG and TyM antibodies forfoot-and-mouth disease virus (23, 24, 28), polio-

virus (12, 131, 132), influenza virus (12), severalarboviruses (13), and bacteriophage (143). As arule, virus-neutralizing power has been first de-tected in the TM, but, in sera collected later afterimmunization, yG becomes the predominant neu-tralizing antibody. A possible exception wasnoted in bovine serum in which the major portionof the neutralizing antibody for the virus of bovinediarrhea was TM (37). These studies show thedistribution of neutralizing activity in variousserum fractions, but offer no comparison of therelative efficiency of yM and TG on the basis ofequivalent concentrations of antibodies. Svehag,however, did show that normal yM antibody forpoliovirus formed weaker complexes with virusthan did immune yG antibody (131).Over the past 20 years, much has been written

concerning the heterogeneity of antitoxic anti-bodies, indicating variations in ability to precipi-tate toxin and to neutralize it (102). Much of thisinformation is difficult to correlate with theclasses of immunoglobulins as currently described.Of special interest is the recent report of Robbins(106), showing that yM diphtheria antitoxin pro-duced in rabbits did not inactivate toxin, al-though it did combine with toxin and causedstrong indirect hemagglutination. The yG wascapable of producing both agglutination and in-activation of the toxin. Likewise, yM combinedwith lysozyme but did not neutralize its enzymaticactivity, whereas TG did neutralize this enzyme.The reported ability of TM antidiphtheria toxoidto neutralize at 5 C but not at 23 C (11) should befurther explored.

Opsonization

Very few observations on the relative abilitiesof TG and yM to prepare antigenic particles forphagocytosis have been reported. The opsoniceffect of pig serum for S. typhimurium was associ-ated with a macroglobulin (66). Michael andRosen (82) found the yM fraction of normal hu-man serum more effective than TG in clearingS. typhosa from the blood of mice, but this merelyindicated the location of the antibody activityand did not constitute a comparison of specificactivity. Turner et al. (140) found that both yGand TM promoted phagocytosis of mouse typhoidbacilli, as determined indirectly by the clearanceof organisms from the peritoneal cavity afterinjection. Since this was the only means of anti-body assay used, no comparison of the relativeefficiency of the antibodies could be made. TheyG and yM antibodies in chick sera for goaterythrocytes showed parallel opsonizing andagglutinating titers (126). This suggests that yM

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is the more effective opsonin, since it is definitelythe more effective agglutinin.

Using removal of S. typhimurium from thecirculation of inoculated mice as a measure ofopsonic activity, Robbins et al. (107) concludedthat yM was 500 to 1,000 times as efficient as yGas an opsonin. On the contrary, Smith et al. (125)found that yG rabbit antibodies to P. mirabiliswere more effective than yM in promoting theingestion of these bacilli in a leukocyte-bacterialsystem without complement. These apparentlycontradictory observations were not based oncomparable experiments, however, since inges-tion of bacteria by phagocytes is only one ofseveral factors determining blood clearance.Rowley et al. (118) showed that immunity to

mouse typhoid could be transferred passively bymeans of phagocytic cells from immune mice. Inthis instance, at least, it was claimed that so-called cellular immunity was dependent on yMantibody adsorbed to cells, and that this antibodycould be eluted from the cells with urea. The possi-bility that this phenomenon might influence theoutcome of experiments involving phagocytosisshould be considered.

HypersensitivityThe varied reactons which are known to be

manifestations of hypersensitivity are so diversethat one would hardly expect the same kind ofantibody to be involved with all types. Generali-ties in this area are hazardous because of speciesdifferences in the antibodies involved. A discus-sion of the many aspects of this subject is avail-able (26) and this subject is beyond the scope ofthis review. Some recent observations on theparticipation of molecular types of antibody inallergic phenomena, however, seem to be perti-nent.Two varieties of 7S antibody have been recog-

nized in the guinea pig (14, 148). The 7S 'y2,which appears to be analogous to the -yG ofman, gives only weak (148) PCA or none at all(90). The 7S -yl antibody mediates both PCAand systemic anaphylaxis (90, 148). The simi-larities and differences between these 'yl anti-bodies and the anaphylactic antibodies of man,dog, rabbit, mouse, and rat, all of which migrateelectrophoretically faster than y2 globulins, havebeen extensively reviewed by Bloch (20a). PCAassociated antibody of the rat sediments betweenthe 75 and 19S components, is destroyed byheating at 56 C for 30 min, and is present in lowconcentration (20).

Osler et al. (89) found rabbit yM to be ineffec-tual in PCA, as did Lindqvist and Bauer (77).Human yM, according to Ovary et al. (91), didnot give PCA in the guinea pig.

Both 7S zyl and 7S y2 of the guinea pig canpassively sensitize to give the Arthus reaction(21). Furthermore, both wyG and -yM producedin the rabbit against human blood group A sub-stance induced reversed Arthus reactions inguinea pigs, although the 7yM did not give PCA(133). More yM than yG was required to elicit areaction.

Reactions Involving Rheumatoid Factors

One means of demonstrating factors in rheu-matoid arthritis serum which react with zy-globu-lin is to show that such a serum will bring aboutthe agglutination of erythrocytes sensitized withsubagglutinating amounts of specific antibody(156). Rheumatoid factor reacts with the 'y-globu-lin fixed to the cells as antibody. In studying thereaction of rheumatoid arthritis sera with sheepcells sensitized with various sheep cell agglutinins,two kinds of agglutinins were found which werenot affected by rheumatoid factor. One was pres-ent in infectious mononucleosis serum (94), theother, in the serum of rabbits during the earlystages of the immune response to the injection oferythrocytes (95). It then became apparent thatonly those antierythrocyte sera that containedappreciable amounts of -yG antibody wouldsensitize cells for agglutination in rheumatoidarthritis serum (108). Infectious mononucleosisserum and early rabbit sera did not react withrheumatoid factor because the agglutinins wereof yM type.There are other ways in which the hetero-

geneity of immunoglobulin is involved in reac-tivity with rheumatoid factors. When yG isseparated into three fragments by papain diges-tion, one fragment, the Fc, which contains onlyfragments of heavy chains, has no antibody ac-tivity, but does carry the antigenic determinantswhich enable it to combine with rheumatoidfactor (49). The heterogeneity of these combiningsites is reflected in the discovery that rheumatoidfactors are actually a family of yM globulins,some of which have specificity for geneticallydetermined antigenic groups present on the heavychains of yG globulin (43). The differences thatrheumatoid factors exhibit with respect to elut-ability from human -y-globulin suggested hetero-geneity with respect to affinity (88). The possiblerelation of these differences to specificity forparticular antigenic sites has not been determined.Although yG from a number of different speciesreacts with rheumatoid factor, reactivity is vari-able, and again indicates a multiplicity of rheu-matoid factors (96). Other aspects of the multiplenature of rheumatoid factors have been reviewed(73).

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A IMMUNoGLoBuLiN

The third principal immunoglobulin now desig-nated yA is more difficult to identify than yGand yM (57, 145). It is usually inactivated bymercaptoethanol (64). As late as 1962, there wasdoubt as to the possible antibody activity ofthese globulins (35), although several investi-gators had reported antibody activity in serumfractions which contained yA and probably otherglobulins. Activity has now been demonstratedin yA from man (57), horse (70), bovine (153,154),- rabbit (87, 153), and guinea pig (115).Table 3 lists instances in which serological reac-tions have been ascribed toyA, as well as failures,to demonstrate such reactivity. Vaerman et at.(145) described a method for obtaining yA in arelatively pure form and demonstrated Brucella

TABLE 3. Immunological activity of yAimmunoglobulin

Activity Reaction observed No reaction observed

Agglutination Brucella (57,153), Escher-ichia coli (2),diphtheriatoxin (57,104), humanerythrocytes(6, 64, 72,100, 101, 138),streptococcalM protein(104), azobenzene sul-fonate (24b)

Precipitation Vibrio fttus p-Azophenyl-endotoxin fi-lactoside(154), blood (70), azogroup A sub- benzene sul-stance (64) fonate (24b)

Complement Brucella (57),fixation human eryth-

rocytes (6,61a), p-azo-phenyl-f,-lactoside (70)

Lysis Human eryth-rocytes (64,138), E. coli(2)

Neutralization Poliovirus (12,59), influenzavirus (12),rhinovirus(114)

Passive cuta- Blood groupneous ana- A substancephylaxis (64)

agglutiniins in all hree human inmunoglobulins,including yA (57). Wilkinson (153) confirmedthis observation and also showed Brucella ag-glutirin in analogous fractions of bovine andrabbit sera.

Evidence that yA globulin of human serumafter typhoid vaccination combined with typhoidbacilli as antibody was presented by Rowe andTurner (116), but there was no direct evidencethat any of the agglutinating activity of the serumwas due to this globulin. The presence of yAantidiphtheria toxin in human serum was alsoshown by passive hemagglutination (57), butability of yA to neutralize toxin was not studied.It has been suggested, however, that the com-ponent of immune horse serum which containsmost of the antitoxic activity for diphtheria toxinis the equine equivalent of yA (57). Most of theprecipitating activity of bovine serum for Vibriofetus endotoxin was found in a fraction with thecharacteristics of yA (154). A portion of theinsulin-binding antibodies (155) and some of theantithyroglobulin activity (45) of human serumwas shown to be TA. Ishizaka et al. (64) ob-served precipitin reactions by the interfacialring test with all three human immunoglobulinsand blood group A substance. Reactions of TA,however, did not occur with all preparations,and they developed more slowly with yA thanwith yG and yM. Larger amounts of yA wererequired to bring about a reaction.There are several reports indicating that some

of the iso-hemagglutinins are yA (64, 72, 100,101). Ishizaka et al. (64) compared the activitiesof different classes of human group A iso-hemag-glutinins and found yA antibodies to be inter-mediate between -yG and TM in their agglutinat-ing activity, but this globulin failed to hemolyzeerythrocytes and to give PCA. Klinman et al.(70) reported that equine antihapten TA failedto fix complement and to cause precipitation. Itdid, however, possess a high affinity for antigenand inhibited the precipitation of yA. Heremanset al. (57) had previously shown that humananti-Brucella yA did not fix complement. Coldhemagglutinins of the TA class in one individualdid not fix complement or cause lysis (6). Roth-man has recently described methods for isolatingTyA globulin from guinea pig serum (115), andshowed that contact allergy to 2,4-dinitrochloro-benzene could be passively transferred by meansof it. The possible relation of this antibody to theguinea pig 7S T1 described earlier (14) has notbeen determined.The evidence which tends to associate yA anti-

body with reagin, the heat-labile antibody foundin the serum of patients with atopic allergy, has

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been summarized by Chase (26). More recently,Ishizaka et al. (62, 63) have presented evidencethat reagin can be distinguished from yA im-munologically, although the physicochemicalproperties of reagins and yA are similar. Theysuggest that reaginic activity resides in a newininunoglobulin which they call 'yE. The possi-bility that reaginic activity may not be confinedto this immunoglobulin has had recent support(79a, 91a).One of the most intriguing characteristics of

yA antibody is its tendency to be concentrated incertain secretions. Most of the neutralizing ac-tivity of human nasal secretions for poliovirus(12), influenza virus (12, 113), and rhinovirus(114) was due to yA antibody, even though thepredominant neutralizing antibody in the serumwas yG. The neutralizing antibody of humancolostrum for the poliovirus (59) and the iso-hemagglutinin found in human saliva and colos-trum (138) were likewise 'yA. There is evidencethat reaginic activity of saliva (61) and nasalsecretions (104) of pollen-sensitive individuals ispresent in 'yA globulin, but the identification ofserum reagin with 'yE (62, 63) suggests that thereagin of secretions may also belong to this class.Antibodies for Francisella tularensis in humannasal secretions were associated with yA. Theagglutinins for E. coil in human colostrum werefound to be yA, but these antibodies were notlytic in the absence of lysozyme (2). There is veryrecent evidence that susceptibility of man toherpesvirus infection is, at least in part, due to adeficiency in the production of specific 'yA (137).Herpes infection, with its predilection for muco-cutaneous tissue, is a situation where the neu-tralizing antibody of secretions might be expectedto play an important protective role. Althoughrabbit serum contains a relatively low concentra-tion of yA globulin, rabbit colostrum was foundto contain a much higher concentration (25).That such a globulin may have antibody activityin the rabbit as well as in man has also beendemonstrated (87, 153).The origin of -yA antibody in secretions is a

question of some interest. In the rabbit, the goat,and the cow, there is evidence (92) that immuneglobulin is transferred from the plasma to thecolostrum with considerable concentration beingeffected during the transfer. On the other hand,in man, Tomasi et al. (138) were unable to obtaintransfer of 131I-labeled 'yA from serum to saliva,and presented evidence for the local glandularsynthesis of 'yA. They also showed that the yAof human colostrum and saliva differed from themajor portion of serum yA in immunologicalspecificity and in the degree of polymerization.

These observations led them to conclude thatexternal secretions derive antibodies from animmunological system separate from that of cir-culating antibody. This conclusion was supportedby Adinolfi et al. (2), who found yA antibodiesfor E. coli in human colostrum in the absence ofserum antibodies of this type. Whether the glandselectively concentrates yA or synthesizes it tothe relative exclusion of yG and yM, an interest-ing problem is presented.

OUTLOOKThe recognition of different classes of immuno-

globulins and the determination of their capaci-ties to bring about the various immunologicalreactions have already led to a better understand-ing of several immunological phenomena. Thenecessity for considering the diverse propertiesof immunoglobulins in studying antibody produc-tion is being recognized more and more. Althoughthe patterns of antibody formation and functionin various species are generally similar, enoughdifferences have been discovered to indicate thatwhat is observed in one species is not necessarilytrue for others. It is inevitable that additionalsubtle differences in antibody properties will becorrelated with immunological reactivities.

Although a change in the predominant class ofantibody present in a serum can affect its sero-logical activity, for example, complement fixa-tion or agglutination, more information is neededregarding the possible changes in the activity ofeach class of antibody with time. It has been wellestablished that the ability of antibody to forma stable union with antigen increases with time,but the extent to which this increased stabilitymay be reflected in the observed result of theantigen-antibody union has not been fully deter-mined. There are also indications that differencesin the antibodies of individual animals may resultin differences in observed activity (89).The heterogeneity of the antibody globulins

that are intermediate between yG and yM hasnot been completely defined. Information islacking regarding the serological activity of yDand yE immunoglobulins, but the association ofthe latter with human reagin (63) suggests thatit may also participate in other demonstrablereactions. With the aid of refined techniques,additional immunoglobulins present in low con-centration may be identified. The relation of theintermediate antibodies of the guinea pig (8a, 14,115, 148) to similar antibodies of other animalsand man (20a) has yet to be clarified.

SUMMARYIn Table 4, an attempt has been made to indi-

cate the comparative effectiveness of the principal

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TABLE 4. Summary of the activity of theimmunoglobulinsa

Reaction -YG yM yA

Agglutination Weak Strong +Precipitation Strong Variable :1:Complement fixa- Strong Weak -

tionLysis Weak StrongNeutralizationViruses + + +Toxins and en- + ?zymes

Passive cutaneous _.b _anaphylaxis

Arthus reaction + +Avidity Variable Variable Strong

a Information not available regarding possibleserological activity of yD and -yE.

b Reaction in foreign species, not in homol-ogous species.

immunoglobulins in various reactions on thebasis of information currently available. There isno intent to imply that there are no exceptions tothe generalizations used in the table. As alreadyindicated, there is conflicting evidence in someareas, and in others the data are subject to differ-ent interpretations. One can conclude, however,that yG antibodies, which are usually more ap-parent in later stages of immunization, are highlyeffective precipitins and, in most instances, ac-count for the major portion of the complement-fixing activity of serum. They are effective inneutralizing viruses, exotoxins, and enzymes;they induce the Arthus reaction, and their abilityto form a stable union with antigen increaseswith time after immunization.

In contrast to -yG, -yM antibodies, which areoften the first to be detected, are most active inagglutination and lytic reactions. They are not asreadily detected by precipitation and complementfixation as are yG. They also have virus-neu-tralizing capacity, but they apparently fail toneutralize toxins and enzymes. They seem to beless effective than yG in the Arthus reaction, andthey fail to give PCA. In some instances, theyhave been dissociated from antigen less readilythan yG, but in other reports the reverse has beennoted.The demonstrable serological activity of yA is

more limited. This antibody has been demon-strated most often by direct or passive agglutina-tion. Its ability to neutralize viruses and its rela-tively high concentration in secretions point to itssignificance in the defense mechanism.

ACKNOWLEDGMENTSI am grateful to Paul Donaldson for suggestions

and criticism. The original work cited was supportedin part by National Science Foundation grants G-8767and GB-4590.

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1965. Studies on the nature of immunogenicityemploying soluble and particulate bacterialproteins, p. 31-42. In J. Sterzl [ed.], Molecu-lar and cellular basis of antibody formation.Czeckoslovak Academy of Sciences, Prague.

2. ADINOLFI, M., A. A. GLYNN, M. LINDSAY,AND C. M. MILNE. 1966. Serological proper-ties of TA antibodies to Escherichia colipresent in human colostrum. Immunology10:517-526.

3. ADLER, F. L. 1965. Studies on mouse antibodies.I. The response to sheep red cells. J. Immunol.95:26-38.

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6. ANGEVINE, C. D., B. R. ANDERSON, AND E. V.BARNETT. 1966. A cold agglutinin of the IgAclass. J. Immunol. 96:578-586.

7. ASHERSON, G. L., AND D. C. DRUMONDE. 1962.Characterization of autoantibodies producedin the rabbit by the injection of rat liver. Brit.J. Exptl. Pathol. 43:12-20.

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8a. ASKONAS, B. A., R. G. WHITE, AND P. C.WILKINSON. 1965. Production of -yl- and y2-antiovalbumin by various lymphoid tissues ofthe guinea pig. Immunochemistry 2:329-336.

9. BAUER, D. C. 1963. The molecular properties ofantibodies synthesized in response to simplehaptenes. J. Immunol. 91:323-330.

10. BAUER, D. C., M. J. MATHIES, AND A. B. STA-VITSKY. 1963. Sequences of synthesis of -y-1macroglobulin and ey-2 globulin antibodies andsecondary responses to proteins, salmonellaantigens and phage. J. Exptl. Med. 117:889-907.

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