Gamma globulins of bovine lacteal secretions

ARCHIVES

OF

BIOCHEMISTRY

AND

Gamma FREDERICK

From the Departments

BIOPHYSICS

Globulins

108,

230-239

of Bovine

(1964)

Lacteal

Secretions

A. MURPHY,l OLE AALUND, JOHN AND EDWARD J. CARROLL

of Veterinary

Microbiology University

and Clinical of California,

Received

Pathology,

W. OSEBOLD,

School of Veterinary

Medicine,

Davis

June 5, 1964

Gamma globulin components of bovine colostrum and dry secretion were compared with their counterparts in serum by means of immunoelectrophoresis, anion exchange chromatography, and double-diffusion antigenic analysis. Although comparable components appeared immunochemically and physicochemically similar no matter where they originated, the proportional amount of each component varied greatly according to the character of the secretion. Thus, electrophoretically fast and slow 75 gamma globulin components were found to selectively accumulate in dry secretion relative to other serum proteins. Colostrum formation involved the same discrimination relative to other serum proteins and also a virtually complete discrimination against slow 75 gamma globulin in favor of a fast component. Bovine gamma-1M and gamma-lh globulins were found in lacteal secretions.

confirmed with radioactive isotope labeled gamma globulins (3). The selectivity of transport of gamma globulins to colostrum relative to other serum proteins (e.g., albumin) was charac terized by Dixon et al. (4) as a function of the dam’s mammary acinar epithelium. Moreover, the selectivity of transport within the gamma globulin spectrum as studied by Carroll (5), who used DEAE cellulose chromatography, was shown to vary with the character of the secretion. Thus, immune globulins not adsorbed to the anion exchanger under the conditions used predominated in mammary secretions obtained during the period of nonlactation (dry secretion). As synthesis of colostrum progressed, the proportion of “nonadsorbed immune globulin was markedly decreased and the bulk of the immune globulins appeared as a peak eluting with 0.054.075 M NaCl” to the extent of 48 % of total colostral whey protein. In this study physicochemical and immunochemical characterization of the gamma globulin components of bovine colostrum and dry secretion further eluci-

INTRODUCTION

Physicochemical macroheterogeneity of the gamma globulins present in the serum of several species has been rather well characterized. That the distribution of electrophoretically distinct subgroups of the gamma globulin continuum in secretions of the bovine mammary gland is different from that in serum has been shown by Smith (1) and Larson (2) in studies of the transfer of antibody activity and proteins from maternal to newborn calf and lamb circulations. Electrophoretically slow gamma globulin of bovine serum does not appear in colostrum, whereas gamma globulins of higher mobility may represent as much as 50-60% of the total protein in colostrum and 90% of colostral whey protein. Smith recognized t’hat electrophoretically fast migrating gamma globulins become a major component of the calf’s serum following ingestion of colostrum. The transfer of gamma globulins from cow to calf via colostrum has been 1 Postdoctoral Fellow of the National Institute of Allergy and Infectious Diseases. Present address: Virology Section, Communicable Disease Center, Atlanta, Georgia. 230

BOVINE

GAMMA

date the change in selectivity for the 7s gamma globulins in the progression from lactation, to nonlactation (dry secretion), to colostrum synthesis. In addition to characterization and quantitation of the slow 7s gamma and fast 7s gamma globulins, the main components of the electrophoretic continuum, demonstration of the presence of the other immunoglobulins, gamma-1RI and gamma-IA, in these secretions is noted. MATERIALS

AND

METHODS

Lacteal secretions were drawn from healthy Holstein cattle. Samples of dry secretion were taken late in the nonlactating period but prior to the beginning of colostrum accumulation. Colostrum samples were drawn immediately prepartum. Normal bovine adult sera and post nursing calf sera served as the references for chromatographic and immunoelectrophoretic procedures. Anion exchange chromatography was performed according to the method of Fahey et al. (6) with the following modifications. I)EAE-Sephadex A-50 medium (Pharmacia Fine Chemicals, Inc., New York) replaced DEAE cellulose as the anion exchanger. Two distinct gradient elution schemes were employed. Gradient 1 was delivered by the cylinder-cone apparatus described by Fahey and Horbett (7). It provided a gently concave gradient of buffer with increasing phosphate concentration and concomitant decreasing pH. The initial buffer (110 ml of 0.02 M sodium phosphate, pH 8.0) was contained in a 109ml beaker equipped with a stirrer, and the limit buffer (55 ml of 0.37 M phosphate, pH 4.5) in a 50-ml Erlenmeyer flask at the same level. Siphons connected the flask to the beaker and the beaker to the column. This delivery system was used in conjunction with columns containing 1 gm (dry weight) of DEAE-Sephadex, to which loads of up to 200 mg of protein could be applied. Fraction volume was 4.0 ml. This gradient was especially effective in separating the nonadsorbed gamma globulins appearing in the breakthrough peak from those eluted at “the point in the effluent at which the gradient began to dominate” (8). Gradient 2, delivered by an inverted cone-cone reservoir system, was sigmoid shaped with an almost linear midportion. The mixing chamber containing initial buffer (110 ml of 0.02 M sodium phosphate, pH 8.0) was an inverted 125.ml Erlenmeyer flask. It was connected via siphons to the:column and to a second 125.ml Erlenmeyer flask in an upright position at the same level which contained limit buffer (135 ml of 0.3 M phosphate, pH 8.0). This delivery system was used in conjunction with columns containing

231

GLOBULINS

2.8 gm of DEAE-Sephadex to which a maximum of 600 mg of protein could be bound. Fraction size was 3.5 ml. The good resolution of this gradient in the middle portion of bovine serum chromatograms was of particular value in spreading the intermediary and fast 7s gamma globulin components. All chromatography was conducted at room temperature. Flow rate varied between 30 and 50 ml per hour with 1 meter of hydrostatic pressure applied by elevation of buffer vessels. Protein concentration of effluent fractions was determined by measuring optical density at 280 w. Immunoelectrophoresis was performed according to the micromethod of Scheidegger (9) with minor modification. Constant dimensions of wells and trenches served to control volumes of reagents applied. Rabbit antisera were prepared against bovine serum, bovine 7s gamma globulin (protein eluted as peak I in DEAE-Sephadex chromatography of whole serum), and bovine macroglobulins [protein eluted as peak I from gel filtration of whole serum on Sephadex G-200 according to the method of Flodin (10) and Gelotte et al. (ll)]. Antigens, whether sera, lacteal secretions, or chromatographic fractions, were applied directly to immunoelectrophoretic wells without concentration or dialysis. The double dijksion in gel technique of Ouchterlony (12) was carried out in a macroformat. RESULTS

IMMUNOELECTROPHORETIC CHARACTERIZATIONOFCOLOSTRALAND DRYSECRETION GAMMA GLOBULIN Five different ant’isera, each also used to develop the same whole serum reference, were employed to compare bovine lacteal secretions. Experiment’s relating physicochemically and immunochemically characterized immunoglobulins as they occur in bovine serum to particular immunoelectrophoretic arcs have been presented (13, 14). Such characterization, involving analytical ultracentJrifugation, gel filtration, anion exchange chromatography, determination of mercaptoethanol sensitivity, and antigenic analysis of whole proteins and their papain digestion products, is the basis for naming bovine “fast and slow 7s gamma,” “gammalN1,” and “gamma-lA” globulins and their respective imnmnoelectrophoretic arcs in terms of their counterparts in human serum. Rabbit antibovine sera 9 and 231 developed complex 7s gamma globulin pat-

232

MURPHY

FIG. 1. Immunoelectrophoresis of dry secretion whey 1229. BS, Bovine serum; RABS, rabbit antibovine serum; RABMACRO, rabbit antibovine macroglobulin; RArG, rabbit antibovine gamma globulin.

terns from dry secretions (Fig. 1). The latter also best developed the gamma-IA arc. Comparative arc brightness indicated that the gamma-lh content of dry secretion is lower than in serum. These two antisera, together with rabbit antibovine serum 229, served to demonstrate a major 75 gamma globulin component seen as an arc approaching the length and brightness of its counterpart in whole serum. Gamma-1M globulin was present in dry secretion in a concentration slightly less than t,hat in serum, as determined on the basis of the brightness of the distinctive gamma-1M arc and its proximity to the antiserum trench (antimacroglobulin 78). It may be noted that no alpha-2 macroglobulin was detected although antiserum 78 was especially sensitive in detecting this protein. The complexity of the major 7s gamma globulin arc was best developed by rabbit antiserum 97 (antibovine 75 gamma globulin). A characteristic spur on the 75 gamma globulin arc in whole serum immunoelectrophoresis analogous to the spurring and splitting of the human gamma globulin arc, as described by Edelman et al. (15), was an invariable property of this antiserum. Because of the development of thii spur when immunoelectrophoresing 7s gamma globulin free of gamma-1A and gamma-1M globu-

ET AL.

lins (via DEAE-Sephadex chromatography), it is contended that this spur related to antigenie differences or deficiencies within the slow and fast components of the 7s gamma globulins themselves. This same spur occurred in immunoelectrophoretic patterns of dry secretion whey, but in addition, an overriding spur, noted with consistency, originated in the cathodal part of t’he main gamma globulin arc and extended toward anode so that the two spurs crossed (Fig. 2). Similar immunoelectrophoretic analysis was perfomed on bovine colostrum (Fig. 3). The main gamma globulin arc of colost,rum in each case was notably shorter than that of the serum run in parallel and shorter than the gamma globulin arc of dry secretion. IGLOBULINS OF SERUM ( arcERENCE)

0

I I’% rrw ha

-8

fIMI YIA

0(t)

FIG. 2. Composite phoresis whey.

I

of the gamma

t-1

tracing of immunoelectroglobulins of dry secretion

FIG. 3. Immunoelectrophorgsis of colostral whey 2414. BS, Bovine serum; RABS, rabbit antibovine serum;- RABMACRO, rabbit antibovine macroglobulin; RA-yG, rabbit antibovine gamma globulin.

BOVINE

GAMMA

This finding is consonant with the fact that colostrum is deficient in those gamma globulin molecules of slowest ele&ophoretie mobility. As in dry secretions, two spurs originated in the main gamma globulin arc and crossed in the gamma-l area. Gamma-1A and gamma-M globulins were present in colostrum in concentrations lower than in serum. CHROMAT~GRAPHI~ CHARACTERIZATION OF COLOSTRAL AND DRY SECRETION GAMMA GLOBULINS Protein was eluted from DEAE-Sephadex columns in two major positions when dry secretion was fractionated using both gradients (Fig. 4). Immunolectrophoresis of DEAE-Sephadex fractions of whole serum had revealed that the same spectrum of mobilities as described by Fahey and Horbett (7) for human 75 gamma globulins was also

GRADENT

2

I ; 1.6ml. WHEY;

233

GLOBULINS

present in bovine 7s gamma globulins (13). Peak I protein of such serum chromatograms produced only one immunoelectrophoretic arc, that of the slowest migrating 75 gamma globulin. Serial fractions of peak II contained gamma globulins characterized by gradually increasing electrophoretic mobilities, the slowest corresponding with t’he spur on the gamma globulin arc of reference whole serum. Immunoelectrophoretic analyysis of DEAE-Sephadex fractions of dry secretion wheys revealed an ident’ical distribution of 75 gamma globulins with a spectrum of mobilities (Fig. 5). Gamma globulin molecules with particular net electrical properties migrate equally on immunoelectrophoretic slides and are eluted in the same position in DEAE-Sephadex chromatography whether they originate in serum or have passed to mammary secretions. Planimetry revealed that 40 % of the

SERUM REFERENCE

=Lbml.

-+64E

/ I

1

&I PERCENT

f’\ \

I

I(

OF EFFLUENT

FIG. 4. Anion exchange chromatograms of dry secretion whey 1229. Two gradient elution schemes were used, and each is compared to a whole serum reference chromatogram.

234

MURPHY

whey protein was eluted in the breakthrough peak and 60% in the second peak (gradient 1, Fig. 4). Comparable analysis of whole serum revealed approximately 15% of total protein in the breakthrough peak and 35 % in the second peak (consisting of approximately 50% fast 7s gamma globulin and 50 % transferrin). Therefore, the propor-

FIG. 5. Partial superimposition of immunoelectrophoretic patterns of fractions from DEAESephadex chromatography of dry secretion whey (gradient 2). BS, Bovine serum; RABS, rabbit antibovine serum.

GRADIENT

I ; 0.75 ml. WHEY;

ET AL.

tional distribution of gamma globulins between peaks I and II is the same in serum as it is in dry secretion. Those serum proteins eluted from DEAE-Sephadex as peak III were found to be either completely lacking (e.g., several alpha globulins) or present in very small amounts (e.g., albumin) in dry secretion (see Figs. 1 and 4). Recovery of from 85% (gradient 2) to 93% (gradient 1) of applied dry secretion protein from the DEAE-Sephadex columns, together with the characterization of the major eluted proteins as known serum components, precludes any possibility of the accumulation of a yet undefined whey protein fraction in the bovine mammary gland at the time of nonlactation as suggested by Larson (2). DEAE-Sephadex chromatography of colostral whey (Fig. 6) demonstrated the virtual absence of nonadsorbing gamma globulin, the slow 7s gamma, and the predominance of proteins eluted at that point where the

SERUM REFEFtENCE=l6ml:#

G4E ;

\

z-

SERUM REFERENCE = 6.0 ml. -#95

50

K

PERCENT OF EFFLUENT

FIG. 6. Anion exchange chromatograms of colostral whey 2414. Two gradient schemes were used, and each is compared to a whole serum reference chromatogram.

elution

BOVINE

GAMMA

gradient began to dominate (peak II, fast 7s gamma globulin). In immunoelectrophoresis, colostral slow 75 gamma globulin and fast 7s gamma globulins behaved as their serum and dry secretion counterparts. Planimetry revealed that fast 7s gamma globulin makes up more than 90% of the total colostral whey protein. COLOSTRAL TRANSFER OF IMMuN~GLoB~LINs A difficulty of building upon early descripCons (1) of gamma globulin transport based upon free electrophoresis lies in the ambiguity left by the limited resolution of multiple gamma globulin components of nearly identical electrophoretical mobility. Gamma globulin behavior in anion exchange chromatography brings another physicochemical parameter into the definition of particular components. In this case, DEAESephadex chromatography of the sera of dams and their calves upon the same gradient

GLOBULINS

(number 1) used for colostral whey chromatography served to further resolve the phenomenon of selective colostral transport of gamma globulins in terms of current immunochemically defined components. The distinctive shape of protein distribution curves of postnursing calf sera (Fig. 7) confirms the findings of Smith and Helm (16). Slow 75 gamma globulin (peak I), although present in dam’s serum, was not transported to calf serum by the ingestion of colostrum. On the other hand, faster components (peak II) were transferred in large amounts. A chromatogram of a colostral whey sample (2414, not from the dam in Fig. 7) is included in the figure as a point of reference. By comparison of the three protein curves in Fig. 7, the predominance of peak II protein, comparable to Smith’s “immune lactoglobulin” (1) and characterized here as fast 7s gamma globulin, in colostrum, easily accounts for the predominance of this protein in the serum of the calf following

PERCENT OF EFFLUENT

FIG. 7. Anion exchange chromatograms whey

(2414).

235

of maternal

(218) and calf (36) sera and colostral

236

MURPHY

ET AL.

FIG. 8. Ouchterlony double-diffusion analysis (left vs. right). Antigens: (1) (2) dry secretion vs. colostrum; (3) (4) colostrum vs. DEAE peak II of serum; (5) DEAE peak I of serum vs. DEAE peak I of dry secretion; (6) DEAE peak II of serum vs. DEAE peak II of dry secretion; (7) DEAE peak I of serum vs. DEAE peak I of colostrum; (8) DEAE peak II of serum vs. DEAE peak II of colostrum; (9) DEAE peak I of colostrum vs. DEAE peak I of dry secretion; (10) DEAE peak II of dry secretion vs. DEAE peak II of colostrum; (11) DEAE peak I of serum vs. DEAE peak II of serum; (12) DEAE peak I of dry secretion vs. DEAE peak II of dry secretion. Antisera: (1, 3, 6, 8, 10, 11) developed with rabbit antibovine gamma globulin 97; (2,4,5,7,9, 12) developed with rabbit antibovine serum 229.

nursing. Immunoelectrophoresis of DAE-E Sephadex fractions from calf serum chromatography confirmed the identity of the peak I and II proteins. ANTIGENIC COMPARISONS OF GAMMA GLOBULINS FROM COLOSTRUM, DRY SECRETION, AND SERUM

Previous work had not indicated any antigenie difference between slow and fast 7s gamma globulins as they occur in bovine serum (13), with the exception of the manifestation of immunoelectrophoretic spurs occurring as described above. Nevertheless, antigenic comparisons of these two components as they occur in dry secretion and colostrum were performed by the double diffusion in-gel technique (Fig. 8). This technique is sensitive in detecting minor antigenic differences, but in all cases complete arc confluence, the reaction of antigenie identity, was seen. Thus, fast and slow 7S gamma globulin components of serum, dry secretion, and colostrum were shown to be identical to the extent of the resolving power of the antisera employed.

DISCUSSION

Gamma globulins are actively and heterogeneously transported from serum to other fluids of the body. In the bovine species it was found that although transport of serum proteins into mammary gland dry secretion discriminated in favor of 75 gamma globulins and against several other proteins (e.g., albumin and alpha-2M globulin), t)here was no apparent selectivity between slow 7S gamma and fast 75 gamma globulin. On the other hand, the accumulative process active in colostrum formation was ext.raordinarily selective in favor of the fast components. As Dixon et al. (4) had shown, the preferential removal and transfer of gamma globulin during colostrum formation is of such efficiency that “the concentration of gamma globulin in colostrum over serum reached > 100 times the comparable ratio for albumin.” From the chromatographic dat,a presented here it is possible to extend this statement to conclude that the concentration of fast 75 gamma globulin in colostrum over serum reached >lOO times the comparable ratio for slow 75 gamma globulin. This selectivity

BOVINE

GAMMA

of transport of fast 7s gamma and retention of slow 75 gamma globulin is all the more remarkable in light of the similarities of the two proteins in the bovine species, both physicochemically and immunochemically. As described here, analysis by the double diffusion-in-gel technique failed to reveal any antigenic differences between these two prot’eins as they occur in mammary secreCons. Those differences that have been described (13) include: (a) minor elution position differences of the F fragments of the t’wo proteins upon chromatographic analysis of papain digestion products (alt,hough no antigenic differences between comparable fragments were detected), (b) a minor ultracentrifugal sedimentation coefficient difference between the two proteins as they occur in serum (slow 7s gamma = 6.5 szo,w; fast 7s gamma =6.2 S20,w, (c) the net electrical charge difference manifest in electrophoretic mobility (as seen in immunoelectrophoresis) and elution position difference in anion exchange chromatography, and (d) localization of the complement-fixing antibody activity in the fast 7s gamma globulins exclusively in the one system tested (anti-Anaplasma marginale antibodies). All of these parameters describe differences amounting to only a small band of the spectrum of gamma globulin heterogeneity and emphasize the close similarity of t,he two proteins rather than the differences. The antigenic complexity of colostral and dry secretion gamma globulins implicit in the double spur phenomenon but unconfirmed by Ouchterlony analysis may be compared wit’h the findings of Edelman et al. (15). They found that in human 75 gamma globulin the anodal spur represents precipitation of gamma globulin molecules by antibodies directed against determinants located on the S fragments of papain digestion and t,hat the main gamma arc is developed by antibodies against F fragment determinants. Whether splitting indicates “heterogeneity of gamma globulin in relation to the F and S antigenic components, or whether different antigenic groups on one molecule can give rise to separate lines in certain instances” remains unknown. Explanation of the spur originating in the cathodal end of the gamma

GLOBULINS

237

globulin arc from bovine dry secretion and colostrum likewise depends upon the eventual answer to this question. An electrophoretically heterogeneous 75 gamma globulin spectrum has been reported in several species (7, 17-22) but most notably in the guinea pig (23,24). Extensive description of the heterogeneous distribution between guinea pig fast and slow 7s gamma globulins of antigenic determinants (25) and of the capacity to mediate particular biological functions has become the basis for defining the degree of independence of these two major components of the 7s gamma globulin charge continuum. For example, it has been shown that slow 75 gamma antibodies fix complement but are unable to provoke anaphylactic reactions, whereas fast 7s gamma antibodies do mediate anaphylaxis but do not fix complement or sensitize antigen-coated erthrocytes for lysis in the presence of complement (26, 27). The different ontogenesis of each protein component (28) and the different proportional distribution of particular antibody activities following various antigenic challenges further imply a degree of independence of synthesis (29). But in testing the possibility that “biologic properties associated with only one or the other of the [7S gamma] antibodies would favor transmission or rejection by fetal membranes” (30), no degree of independence of transfer was found. In the guinea pig 7s antihaptene antibodies of both mobilities were transmitted from actively and passively immunized dams to t’heir young in significant amounts. However, the transport selectivity noted in the bovine species here is consonant with similar selective immunoglobulin transport phenomena in other body secretions. Tomasi and Zigelbaum (31) reported a highly selective accumulation of gamma-1A globulin in human parotid saliva, tears, bile, and small intestinal secretions. Likewise, the predominating immunoglobulin in human colostrum and milk is gamma-la (32-34). De Muralt et al. (35) estimated that 90% of the immunoglobulin in human colostrum is gamma-1A and gamma-lM, and 10% is fast gamma globulin. Studies with 1131labeled intact gamma-1NI globulin in man

238

MURPHY

indicate that it does not penetrate into extravascular tissues but remains confined to the circulation (36). Homologous gamma globulin is transported across rabbit and guinea pig fetal membranes more efficiently than heterologous proteins (30, 37). The work of Brambell et al. (38) on the transfer of homologous gamma globulin and antibody activity from the uterine cavity to fetal circulation in the rabbit has demonstrated the important role of the F fragment (Porter’s papain digestion product III) in the transfer mechanism. It was “suggested that fraction III, which contains most of the antigenic groups of the original molecules, also has the configuration recognized by the cells as homologous gamma globulin.” In carrying this concept a step further, it may be concluded that even within homologous gamma globulins, properties of the slightly different F fragments are involved in transfer discrimination. Thus, at one level of discrimination, in the human, 7s gamma globulins are allowed to reach the fetal circulation, but gamma-1A and gamma-114 globulins are retained by the mother (39). And at perhaps an even finer level of discrimination the minor differences between the F fragments of slow and fast bovine gamma globulin as described above appear to be recognized by the active transport mechanism of bovine mammary gland acinar epithelial cells. REFERENCES 1. SMITH, E. L., J. Dairy Sci. 31, 127 (1948). 2. LARSON, B. L., J. Dairy Sci. 41,1033 (1958). Biol. 3. LARSON, B.L., BND GILLESPIE, D.C.,J. Chem. 437, 565 (1957).

4. DIXON, F. J., WEIGLE, W. O., AND VAZQUEZ, J. J., Lab. Invest. 10, 216 (1961). 5. CARROLL, E. J., J. Dairy Sci. 44, 2194 (1961). 6. FAHEY, J. L., MCCOY, P. F., AND GOULIAN, M., J. Clin. Invest. 37, 272 (1958). 7. FAHEY, J. L., END HORBETT, A. P., J. Biol. Chem. 234, 2645 (1959). 8. PETERSON, E. A., AND CHIAZZE, E. A., Arch. Biochem. Biophys. 99, 136 (1962). 9. SCHEIDEGGER, J. J., Intern. Arch. Allergy Appl. Immunol. 7, 102 (1955). 10. FLODIN, P., “Dextran Gels and Their Applications in Gel Filtration,” p. 75. Meijels Bokindustri, Halmstad, Sweden, 1962.

ET AL. I11. GELOTTE, B., FLODIN, P., AND KILLBNDER, J., Arch. Biochem. Biophys. Suppl. 1,319 (1962). 12. OUCHTERLONY, O., in “Progress in Allergy” (P. Kallos and B. H. Waksman, eds.), Vol. VI, p. 48. S. Karger, Basel, 1962. of 13. MURPHY, F. A., Ph.D. Thesis, University California, Davis, 1964. University Microfilms, Inc., Ann Arbor, Michigan. 14. MURPHY, F. A., AAL~ND, O., .IND OSEBOLD, J. W., Proc. Sot. Exptl. Biol. Med. In press (1965). 15. EDELM.IN, G. M., HEREMANS, J. F., HEREMANS, M.-TH., AND KUNKEL, H. G., J. Exptl. Med. 112, 203 (1960). 16. SMITH, E. L., AND HOLM, A., J. Biol. Chem.

176, 349 (1948). 17. FAHEY, J. L., AND LAWRENCE, M., Federation Proc. 21, 19 (1962). 18. F.~HEY, J. L., AND ASKONAS, B. A., J. Exptl. Med. 116, 623 (1962). 19. BENEDICT, A.A., BROWN, R. J., ANDAYENG.IR, R., J. Exptl. Med. 116, 195 (1962). 20. AMIRAIAN, K., AND LEIKHIM, E. J., J. Immunol. 87, 301 (1961). 21. ONOUE, K., Y.4~1, Y., AND PRESSMAN, D., J. Immunol. 92, 173 (1964). of Hyper22. R.iYNauD, M., in “Mechanisms sensitivity” (J. Shaffer, G. LoGrippo, and M. Chase, eds.), p. 27. Little, Brown, Boston, Massachusetts, 1959. 23. YAGI, Y., MAIER, P., AND PRESSMAN, D., J. Immunol. 89, 442 (1962). 24. BENACERRAF, B., OVARY, Z., BLOCH, K. J., AND FRANKLIN, E. C., J. Exptl. Med. 117,

937 (1963). 25. THORBECKE, G. J., BENACERR.~F, B., .IND OVARY, Z., J. Zmmunol. 91, 670 (1963). 26. OVARY, Z., BENACERRAF, B., AND BLOCH, K. J., J. Exptl. Med. 117, 951 (1963). 27. BLOCH, K. J., KOURILSKY, F. M., OVARY, Z., AND BENACERRAF, B., J. Exptl. Med. 117,

965 (1963). Proc. 23, 346 28. THORBECKE, G. J., Federation (1964). 29. BLOCH, K. J., KOURILSKY, F. M., OVARY, Z., AND BENACERRAF, B., Proc. Sot. Exptl. Biol. Med. 114, 52 (1963). 30. BLOCH, K. J., OVARY, Z., KOURILSRY, F. M., AND BENACERRAF, B., Proc. Sot. Exptl. BioZ. Med. 114, 79 (1963). 31. TOMASI, T. B., AND ZIGELBAUM, S., J. Clin. Invest. 42, 1552 (1963). 32. HANSON, L. A., Intern. Arch. Allergy Appl. Zmmunol. 17, 45 (1960). 33. HANSON, L. A., Intern. Arch. Allergy Applied Immunol. 18, 241 (1961). 34. HANSON, L. A., AND BERGGARD, I., C&n. Chim. Acta 7, 828 (1962).

BOVINE

GAMMA

35. DE MUR.~LT, G., GUGLER, E., AND ROULET, D. L. A., in “Protides of the Biological Fluids, 7th Colloquium” (H. Peeters, ed.), p. 166. Elsevier, Amsterdam, 1960. 36. COHEN, S., AND FREEMAN, T., in “Protides of the Biological Fluids, 7th Colloquium” (H. Peeters, ed.), p. 264. Elsevier, Amsterdam, 1960.

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37. BATTY, I., BR.LMBELL, F. W. R., HEMMINGS, W. A., .IND O~~KLEY,C. L., Proc. Roy. Sot. (London), Ser. B 143, 452 (1954). 38. BRAMBELL, F. W. R., HEMMINGS, W. A., OAKLEY, C. L., AND PORTER, R. R., Proc. Roy. Sot. (London), Ser. B 161, 478 (1960). 39. FREDI, T'. J., Am. J. Obstet. Gynecol. 84, 1756 (1962).