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Plasma proteins and immunoglobulins in penguins (Pygoscelis adeliae)
Biochimica et Biophysica Acta, 400 (1975) 137-142 © Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands
BBA 37094 PLASMA PROTEINS A N D I M M U N O G L O B U L I N S IN P E N G U I N S (PYGOSCELIS A D E L I A E ) OSVALDO D. CASTRELOS, MARGARITA ROMERO MERCADO, CARMEN ABAT~NGELO and RICARDO A. MARGNI Department of Microbiology, Chair of Immunology, Faculty of Pharmacy and Biochemistry, Buenos Aires University, Junin 956, Buenos Aires (Argentina) (Received December 12th, 1974)
SUMMARY Total and fractionated proteins were evaluated in the serum of normal penguins. Four proteins with antibody activity, determined by passive haemagglutination, were isolated by Sephadex G-200 filtration and DEAE-cellulose chromatography, from the serum of penguins (Pygoscelis adeliae) inoculated with dinitrophenylated human y-globulin. Immunoelectrophoresis and immunodiffusion showed that all these proteins exhibited both their own and shared antigenic determinants. On account of their electrophoretic mobility, elution conditions and characteristics of the precipitation arcs, they have been tentatively denominated IgG2 (Y2), IgG1 (yx), IgM (YM) and IgX (probably IgA).
INTRODUCTION In previous papers we have analyzed different immunobiologic aspects of the Antarctic continent [1-5]. Recently we have carried out qualitative and quantitative studies of serum proteins, glycoproteins and lipoproteins of the antarctic seal [6], as well as of their immunoglobulin system [7]. Following along this line of investigation and with the idea of enlarging upon studies already undertaken by Allison and Feeney [8], we decided to analyze the plasma proteins in an endeavour to isolate and study the immunoglobulins of Pygoscelis adeliae, for which purpose we used the serum available from penguins inoculated with dinitrophenylated human v-globulin. The antigen was the same that we employed when studying precipitating and non-precipitating antibodies of different animal species [9-12]. MATERIAL AND METHODS Antisera. These were obtained from Pygoscelis adeliae, captured at the Argentine Cabo Primavera Antarctic Base (64 ° 10' S, 60 ° 57' W) and inoculated in both feet with 1 ml dinitrophenylated human v-globulin in 0.5~o concentration,
138 mixed with an equal volume of Freund's complete adjuvant. The inoculation was repeated 10 days later and the birds were bled three weeks after that. Little and Eisen's method [13] was used lbr the preparation ofdinitrophenylated human v-globulin. Quantification of the total and fractionated protehvs. The total serum proteins were measured in keeping with the method reported by Lowry et al. [14]. Quantification of the protein fractions was carried out by cellulose acetate electrophoresis [15]. The time of run was 60 min, using veronal buffer 0.05 M, pH 8.6. Protehz fractionation. The immune serum was precipitated with ammonium sulfate at 5 0 ~ saturation. The precipitate was dialyzed against HCI-Tris buffer 0.1 M, pH 8 and passed through a Sephadex G-200 column, elution being undertaken with the same buffer. The protein peaks with haemagglutinating activity, as determinated by spectrophotometric readings at 280 nm, were chromatographed on DEAE-cellulose column, elution being undertaken with phosphate buffer, pH 7.8 at different molarities. Immunodiffusion. Ouchterlony's method [16] on glass slides was used. Immunoeleetrophoresis. This was performed in accordance with Scheidegger's technique [17]. Veronal buffer 0.05 M, pH 8.2 and 1.25~o agar were used.
Precipitathzg antisera Rabbit anti-penguin serum. This was obtained by inoculating rabbits weighing 3 kg with 1 ml of a 1:6 dilution of penguin serum in saline, emulsified with a equal volume of complete Freund's adjuvant. Two injections at 15 days interval were made in the four footpads of each animal, bleeding being carried out 2 weeks later. Rat anti-penguhz 9'2 serum. This was obtained by inoculation of albino rats with 0.5 ml of a 0.1 ~ protein solution, mixed with an equal amount of Freund's complete adjuvant on days 1, 7 and 28. They were bled 10 days later. The antigen used was the protein fraction with IgG2 (72) characteristics which eluted at 0.005,/0.01 M buffer phosphate, pH 7.8. Part of the serum was used whole and the rest adsorbed with the other isolated proteins with antibody activity, to make it monospecific. Passive haemagglutination Dinitrophenylated sheep red cells, obtained in keeping with the method described by Bullock and Kantor [18] were used as antigen. The reaction was developed by adding 0.05 ml of a 2 ~o suspension of red cells to 0.5 ml of serum dilutions or immunoglobulin solutions (1-500/~g/ml). Readings were taken after incubation at room temperature during 18 h. Haemagglutination with the protein fractions previously treated with mercaptoethanol was done by the method described in [19]. RESULTS Fig. 1 shows fractionation in cellulose acetate of a pool of Pygoscelis adeliae normal serum. The protein values for each fraction, as determined by elution, are given in Table I. Sephadex G-200 filtration of immune penguin serum (I :1600 haemagglutinating activity) produced four protein peaks which were denominated $1, $2, $3 and $4 (Fig.
139
/I
j
Fig. 1. Penguin serum proteins fractionated by cellulose acetate electrophoresis. TABLEI
Penguin serum proteins g/100ml Total proteins Albumin at globulins az globulins fl-globulins ~-globulins
5.60 -k 0.50 3.56 + 0.38 0.34:1_ 0.05 0.64 ± 0.11 0.40 ± 0.06 0.66 ± 0.13
2A). The first two peaks and the ascending portion of the third evidenced antibody activity as determined by passive haemagglutination. The Sl peak, submitted to immunoelectrophoresis analysis with anti-penguin serum as precipitating reagent, contained a protein with the characteristics of IgM, contaminated with other proteins. This peak was submitted to chromatography on DEAE-cellulose column, elution being undertaken with phosphate buffer 0.05/0.2 M, pH 7.8. The protein peak, which eluted with the buffer of highest molarity, exhibits the electro and immunoelectrophoretic characteristics of IgM (~M) and shows agglutination of dinitrophenylated sheep red cells. This activity is sensitive to mercaptoethanol treatment. After concentration, the S2 peak and the ascending portion of S3 were submitted to chromatography on DEAE-cellulose with a phosphate gradient 0.005/0.1 M, pH 7.8. Three fractions with haemagglutinating activity were obtained and they eluted between 0.005 and 0.01 M (peak A); 0.03 and 0.05 M (peak B), and 0.06-0.08 M (Fig. 2B, peak C). Fig. 3 shows the immunoelectrophoretic characteristics of the different isolated protein fraction with haemagglutinating capacity, when rabbit anti-penguin serum was used as precipitating reagent. When dinitrophenylated bovine serum albumin was employed, light precipitin lines with the same immunoelectophoretic characteristics were obtained. Upon immunodiffusion analysis, all the proteins with immunoglobulin characteristics showed partial antigenic identity. A fused precipitation line with spurs was obtained when rabbit anti-penguin serum was used as precipitating reagent. When diffused against non-adsorbed rat anti-penguin 72 serum, each protein yielded a precipitation band fusing with the adjoining bands corresponding to the other proteins. Only ~2 showed a hardly perceptible spur corresponding to its own specific antigenic determinants (Fig. 4B). The ~2 fraction was the only one to give a precipitation arc with monospecific anti-y2 serum.
0
.u
B
i
,” 2.0;
‘1 0 /’
A
,’
,’
,’
,’
,’
,’
,,’
3.20
x5
7 :
h 3.10 -: 0 4 105
1005 20
40
60
a0 Effluent
100 120 (tubes)
140
Fig. 2. (A) Sephadex G-200 filtration of immune penguin serum, previously precipitated with ammonium sulfate at 50% saturation. Position of standard marker proteins; (- - -); (B) Chromatography on DEAE-cellulose of S, peak and ascending portion of S,, obtained by filtration through Sephadex G-200.
Fig. 3. lmmunoelectrophoretic characteristics of the different isolated protein fractions with antibody activity. PS, immune penguin serum; Precipitating reagent, rabbit anti-penguin serum.
141 All the fractions agglutinated the dinitrophenylated sheep red cells at concentrations ranging between 1 and 5/~g/ml. DISCUSSION The results obtained indicated that the concentration and distribution of
Pygoscelis adeliae serum proteins are similar to those of other birds and vertebrates. From the serum of penguins, inoculated with dinitrophenylated human yglobulin it was possible to isolate four protein fractions with antibody activity which, on account of their immunodiffusion behaviour and immunoelectrophoretic characteristics, can be likened to immunoglobulins. The protein fraction with antibody activity corresponding to peak $1 and purified by chromatography on DEAE-cellulose, possesses the immunoelectrophoretic characteristics of lgM (TM). It also has its own antigenic determinants as well as others shared with the other protein fractions with antibody activity as deduced from Fig. 4. Its haemagglutinating activity is sensitive to mercaptoethanol treatment. Similarly, the A, B and C fractions obtained from peaks $2 and $3 by DEAEcellulose chromatography also exhibit immunoglobulin characteristics. Of the three
IgG~
IgG I
0
0
IgG2
IgX
IgG2
IgX
Q
Q
TgM
IgM
Fig. 4. Immunodiffusion analysis. All the protcins with immunoglobulin characteristics show partial antigenic identity. A fused precipitation line with spurs is obtained when rabbit anti-penguin serum is used as precipitating reagent, (A). When diffusion is developed with non-adsorbed rat anti-penguin ~'2serum, each protein yields a precipitation line fusing with the adjoining bands corresponding to the other proteins. Only ~'2 showed a hardly perceptible spur corresponding to its own specific antigenic determinant, (B).
142 peaks, two appear to be lgG with different electrophoretic mobility. The faster (B) with the mobility of ~'l, was d e n o m i n a t e d lgG1, while that with the mobility of72 (A) was identified as lgG2. The third peak (C) was tentatively named lgX. Due to its elution conditions, its electrophoretic mobility and the characteristics of the precipitation band similar to that of h u m a n and mouse igA, this peak is probably lgA. This investigation indicated that the penguins possess an i m m u n o g l o b u l i n system, constituted by four protein fractions with the immunoelectrophoretic characteristics of IgM (TM), lgGl (7-fast), lgG2 (y-slow) and lgX (probably IgA). Similarly as occurs with i m m u n o g l o b u l i n s of other animal species, they possess their own antigenic d e t e r m i n a n t s and others which are shared a m o n g them. All of them possess a n t i b o d y activity as determined by passive haemagglutination. ACKNOWLEDGMENTS This work was developed with the aid of the lnstituto AntS.rtico Argentino, Direcci6n Nacional del Ant~irtico. The authors express their gratitude to Luis Del Prete who inoculated and bled the penguins. REFERENCES I Margni, R. A. and Castrelos, O. D. (1964) Instituto Ant~irtico Argentino, Buenos Aires, Contribuci6n No. 75, pp. 1-13 2 Margni, R. A. and Castrelos, O. D. (1964) lnstituto Ant~irtico Argentino, Buenos Aires, Contribuci6n No. 76, pp. 1-14 3 Margni, R. A. and Castrelos, O. D. (1964) in Biologic Antarctique (Carrick, R., Holgate, M. and Prevost, J., eds), pp. 121-139, Herman ,Paris 4 Margni, R. A., Castrclos, O. D. and Herrera, M. E. (1966) Instituto Ant~irtico Argentino, Buenos Aires, Contribt, ci6n No. 102, pp. 1-19 5 Margni, R. A., Castelos, O. D. (1971), Instituto Antfirtico Argentino, Buenos Aires, Contribuci6n No. 141, pp. 1-19 6 Margni, R. A., Hajos, S. E. and Romero Mercado, M. (1972) Experientia 28, 862 7 Margni, R. A., Hajos, S. E. and Romero Mercado, M. (1971) Instituto Antfirtico Argentino, Buenos Aires, Contribuci6n No. 139, pp. 1-13 8 Allison, R. G. and Feeney, R. E. (1968) Arch. Biochem. Biophys. 124, 548-555 9 Margni, R. A. and Binaghi, R. A. (1972) Immunology 22, 557-563 10 Margni, R. A. and Hajos, S. E. (1973) Immunology 24, 435-443 11 Margni, R. A., Castrelos, O. D. and Paz, C. B. (1973) Immunology 24, 781-789 12 Cordal, M. E. and Margni, R. A. (1974)Immunochemistry 11, 765-770 13 Little, J. and Eisen, H. N. (1967) in Methods in Immunology and Imrnunochemistry (Williams, C. A. and Chase, W. M., eds), Vol. I, pp. 128-133, Academic Press, New York 14 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 15 Hcer, E. E. and Margni, R. A. (1971) Electro e inmunoelectroforesis (Fcrn~indez, G., ed.) pp. 48-57, Buenos Aires 16 Ouchterlony, O. (1958) Progress in Allergy 5, I 78 17 Scheidegger, J. J. (1955) Int. Arch. Allergy Appl. lmmunol. 7, 103-110 18 Bullock, W. F. and Kantor, F. S. (1965) J. lmmunol. 94, 317-322 19 Onoue. K., Kishimoto, T. and Yamamura, Y. (1967), J. Immunol. 94, 303-313
BBA 37094 PLASMA PROTEINS A N D I M M U N O G L O B U L I N S IN P E N G U I N S (PYGOSCELIS A D E L I A E ) OSVALDO D. CASTRELOS, MARGARITA ROMERO MERCADO, CARMEN ABAT~NGELO and RICARDO A. MARGNI Department of Microbiology, Chair of Immunology, Faculty of Pharmacy and Biochemistry, Buenos Aires University, Junin 956, Buenos Aires (Argentina) (Received December 12th, 1974)
SUMMARY Total and fractionated proteins were evaluated in the serum of normal penguins. Four proteins with antibody activity, determined by passive haemagglutination, were isolated by Sephadex G-200 filtration and DEAE-cellulose chromatography, from the serum of penguins (Pygoscelis adeliae) inoculated with dinitrophenylated human y-globulin. Immunoelectrophoresis and immunodiffusion showed that all these proteins exhibited both their own and shared antigenic determinants. On account of their electrophoretic mobility, elution conditions and characteristics of the precipitation arcs, they have been tentatively denominated IgG2 (Y2), IgG1 (yx), IgM (YM) and IgX (probably IgA).
INTRODUCTION In previous papers we have analyzed different immunobiologic aspects of the Antarctic continent [1-5]. Recently we have carried out qualitative and quantitative studies of serum proteins, glycoproteins and lipoproteins of the antarctic seal [6], as well as of their immunoglobulin system [7]. Following along this line of investigation and with the idea of enlarging upon studies already undertaken by Allison and Feeney [8], we decided to analyze the plasma proteins in an endeavour to isolate and study the immunoglobulins of Pygoscelis adeliae, for which purpose we used the serum available from penguins inoculated with dinitrophenylated human v-globulin. The antigen was the same that we employed when studying precipitating and non-precipitating antibodies of different animal species [9-12]. MATERIAL AND METHODS Antisera. These were obtained from Pygoscelis adeliae, captured at the Argentine Cabo Primavera Antarctic Base (64 ° 10' S, 60 ° 57' W) and inoculated in both feet with 1 ml dinitrophenylated human v-globulin in 0.5~o concentration,
138 mixed with an equal volume of Freund's complete adjuvant. The inoculation was repeated 10 days later and the birds were bled three weeks after that. Little and Eisen's method [13] was used lbr the preparation ofdinitrophenylated human v-globulin. Quantification of the total and fractionated protehvs. The total serum proteins were measured in keeping with the method reported by Lowry et al. [14]. Quantification of the protein fractions was carried out by cellulose acetate electrophoresis [15]. The time of run was 60 min, using veronal buffer 0.05 M, pH 8.6. Protehz fractionation. The immune serum was precipitated with ammonium sulfate at 5 0 ~ saturation. The precipitate was dialyzed against HCI-Tris buffer 0.1 M, pH 8 and passed through a Sephadex G-200 column, elution being undertaken with the same buffer. The protein peaks with haemagglutinating activity, as determinated by spectrophotometric readings at 280 nm, were chromatographed on DEAE-cellulose column, elution being undertaken with phosphate buffer, pH 7.8 at different molarities. Immunodiffusion. Ouchterlony's method [16] on glass slides was used. Immunoeleetrophoresis. This was performed in accordance with Scheidegger's technique [17]. Veronal buffer 0.05 M, pH 8.2 and 1.25~o agar were used.
Precipitathzg antisera Rabbit anti-penguin serum. This was obtained by inoculating rabbits weighing 3 kg with 1 ml of a 1:6 dilution of penguin serum in saline, emulsified with a equal volume of complete Freund's adjuvant. Two injections at 15 days interval were made in the four footpads of each animal, bleeding being carried out 2 weeks later. Rat anti-penguhz 9'2 serum. This was obtained by inoculation of albino rats with 0.5 ml of a 0.1 ~ protein solution, mixed with an equal amount of Freund's complete adjuvant on days 1, 7 and 28. They were bled 10 days later. The antigen used was the protein fraction with IgG2 (72) characteristics which eluted at 0.005,/0.01 M buffer phosphate, pH 7.8. Part of the serum was used whole and the rest adsorbed with the other isolated proteins with antibody activity, to make it monospecific. Passive haemagglutination Dinitrophenylated sheep red cells, obtained in keeping with the method described by Bullock and Kantor [18] were used as antigen. The reaction was developed by adding 0.05 ml of a 2 ~o suspension of red cells to 0.5 ml of serum dilutions or immunoglobulin solutions (1-500/~g/ml). Readings were taken after incubation at room temperature during 18 h. Haemagglutination with the protein fractions previously treated with mercaptoethanol was done by the method described in [19]. RESULTS Fig. 1 shows fractionation in cellulose acetate of a pool of Pygoscelis adeliae normal serum. The protein values for each fraction, as determined by elution, are given in Table I. Sephadex G-200 filtration of immune penguin serum (I :1600 haemagglutinating activity) produced four protein peaks which were denominated $1, $2, $3 and $4 (Fig.
139
/I
j
Fig. 1. Penguin serum proteins fractionated by cellulose acetate electrophoresis. TABLEI
Penguin serum proteins g/100ml Total proteins Albumin at globulins az globulins fl-globulins ~-globulins
5.60 -k 0.50 3.56 + 0.38 0.34:1_ 0.05 0.64 ± 0.11 0.40 ± 0.06 0.66 ± 0.13
2A). The first two peaks and the ascending portion of the third evidenced antibody activity as determined by passive haemagglutination. The Sl peak, submitted to immunoelectrophoresis analysis with anti-penguin serum as precipitating reagent, contained a protein with the characteristics of IgM, contaminated with other proteins. This peak was submitted to chromatography on DEAE-cellulose column, elution being undertaken with phosphate buffer 0.05/0.2 M, pH 7.8. The protein peak, which eluted with the buffer of highest molarity, exhibits the electro and immunoelectrophoretic characteristics of IgM (~M) and shows agglutination of dinitrophenylated sheep red cells. This activity is sensitive to mercaptoethanol treatment. After concentration, the S2 peak and the ascending portion of S3 were submitted to chromatography on DEAE-cellulose with a phosphate gradient 0.005/0.1 M, pH 7.8. Three fractions with haemagglutinating activity were obtained and they eluted between 0.005 and 0.01 M (peak A); 0.03 and 0.05 M (peak B), and 0.06-0.08 M (Fig. 2B, peak C). Fig. 3 shows the immunoelectrophoretic characteristics of the different isolated protein fraction with haemagglutinating capacity, when rabbit anti-penguin serum was used as precipitating reagent. When dinitrophenylated bovine serum albumin was employed, light precipitin lines with the same immunoelectophoretic characteristics were obtained. Upon immunodiffusion analysis, all the proteins with immunoglobulin characteristics showed partial antigenic identity. A fused precipitation line with spurs was obtained when rabbit anti-penguin serum was used as precipitating reagent. When diffused against non-adsorbed rat anti-penguin 72 serum, each protein yielded a precipitation band fusing with the adjoining bands corresponding to the other proteins. Only ~2 showed a hardly perceptible spur corresponding to its own specific antigenic determinants (Fig. 4B). The ~2 fraction was the only one to give a precipitation arc with monospecific anti-y2 serum.
0
.u
B
i
,” 2.0;
‘1 0 /’
A
,’
,’
,’
,’
,’
,’
,,’
3.20
x5
7 :
h 3.10 -: 0 4 105
1005 20
40
60
a0 Effluent
100 120 (tubes)
140
Fig. 2. (A) Sephadex G-200 filtration of immune penguin serum, previously precipitated with ammonium sulfate at 50% saturation. Position of standard marker proteins; (- - -); (B) Chromatography on DEAE-cellulose of S, peak and ascending portion of S,, obtained by filtration through Sephadex G-200.
Fig. 3. lmmunoelectrophoretic characteristics of the different isolated protein fractions with antibody activity. PS, immune penguin serum; Precipitating reagent, rabbit anti-penguin serum.
141 All the fractions agglutinated the dinitrophenylated sheep red cells at concentrations ranging between 1 and 5/~g/ml. DISCUSSION The results obtained indicated that the concentration and distribution of
Pygoscelis adeliae serum proteins are similar to those of other birds and vertebrates. From the serum of penguins, inoculated with dinitrophenylated human yglobulin it was possible to isolate four protein fractions with antibody activity which, on account of their immunodiffusion behaviour and immunoelectrophoretic characteristics, can be likened to immunoglobulins. The protein fraction with antibody activity corresponding to peak $1 and purified by chromatography on DEAE-cellulose, possesses the immunoelectrophoretic characteristics of lgM (TM). It also has its own antigenic determinants as well as others shared with the other protein fractions with antibody activity as deduced from Fig. 4. Its haemagglutinating activity is sensitive to mercaptoethanol treatment. Similarly, the A, B and C fractions obtained from peaks $2 and $3 by DEAEcellulose chromatography also exhibit immunoglobulin characteristics. Of the three
IgG~
IgG I
0
0
IgG2
IgX
IgG2
IgX
Q
Q
TgM
IgM
Fig. 4. Immunodiffusion analysis. All the protcins with immunoglobulin characteristics show partial antigenic identity. A fused precipitation line with spurs is obtained when rabbit anti-penguin serum is used as precipitating reagent, (A). When diffusion is developed with non-adsorbed rat anti-penguin ~'2serum, each protein yields a precipitation line fusing with the adjoining bands corresponding to the other proteins. Only ~'2 showed a hardly perceptible spur corresponding to its own specific antigenic determinant, (B).
142 peaks, two appear to be lgG with different electrophoretic mobility. The faster (B) with the mobility of ~'l, was d e n o m i n a t e d lgG1, while that with the mobility of72 (A) was identified as lgG2. The third peak (C) was tentatively named lgX. Due to its elution conditions, its electrophoretic mobility and the characteristics of the precipitation band similar to that of h u m a n and mouse igA, this peak is probably lgA. This investigation indicated that the penguins possess an i m m u n o g l o b u l i n system, constituted by four protein fractions with the immunoelectrophoretic characteristics of IgM (TM), lgGl (7-fast), lgG2 (y-slow) and lgX (probably IgA). Similarly as occurs with i m m u n o g l o b u l i n s of other animal species, they possess their own antigenic d e t e r m i n a n t s and others which are shared a m o n g them. All of them possess a n t i b o d y activity as determined by passive haemagglutination. ACKNOWLEDGMENTS This work was developed with the aid of the lnstituto AntS.rtico Argentino, Direcci6n Nacional del Ant~irtico. The authors express their gratitude to Luis Del Prete who inoculated and bled the penguins. REFERENCES I Margni, R. A. and Castrelos, O. D. (1964) Instituto Ant~irtico Argentino, Buenos Aires, Contribuci6n No. 75, pp. 1-13 2 Margni, R. A. and Castrelos, O. D. (1964) lnstituto Ant~irtico Argentino, Buenos Aires, Contribuci6n No. 76, pp. 1-14 3 Margni, R. A. and Castrelos, O. D. (1964) in Biologic Antarctique (Carrick, R., Holgate, M. and Prevost, J., eds), pp. 121-139, Herman ,Paris 4 Margni, R. A., Castrclos, O. D. and Herrera, M. E. (1966) Instituto Ant~irtico Argentino, Buenos Aires, Contribt, ci6n No. 102, pp. 1-19 5 Margni, R. A., Castelos, O. D. (1971), Instituto Antfirtico Argentino, Buenos Aires, Contribuci6n No. 141, pp. 1-19 6 Margni, R. A., Hajos, S. E. and Romero Mercado, M. (1972) Experientia 28, 862 7 Margni, R. A., Hajos, S. E. and Romero Mercado, M. (1971) Instituto Antfirtico Argentino, Buenos Aires, Contribuci6n No. 139, pp. 1-13 8 Allison, R. G. and Feeney, R. E. (1968) Arch. Biochem. Biophys. 124, 548-555 9 Margni, R. A. and Binaghi, R. A. (1972) Immunology 22, 557-563 10 Margni, R. A. and Hajos, S. E. (1973) Immunology 24, 435-443 11 Margni, R. A., Castrelos, O. D. and Paz, C. B. (1973) Immunology 24, 781-789 12 Cordal, M. E. and Margni, R. A. (1974)Immunochemistry 11, 765-770 13 Little, J. and Eisen, H. N. (1967) in Methods in Immunology and Imrnunochemistry (Williams, C. A. and Chase, W. M., eds), Vol. I, pp. 128-133, Academic Press, New York 14 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 15 Hcer, E. E. and Margni, R. A. (1971) Electro e inmunoelectroforesis (Fcrn~indez, G., ed.) pp. 48-57, Buenos Aires 16 Ouchterlony, O. (1958) Progress in Allergy 5, I 78 17 Scheidegger, J. J. (1955) Int. Arch. Allergy Appl. lmmunol. 7, 103-110 18 Bullock, W. F. and Kantor, F. S. (1965) J. lmmunol. 94, 317-322 19 Onoue. K., Kishimoto, T. and Yamamura, Y. (1967), J. Immunol. 94, 303-313