- Email: [email protected]
A reliable method for generating antibodies against pancreozymin, secretin and gastrin
439
Clinica Chimica Acta, 13 (1976) 439-444 0 Elsevier/North-Holland Biomedical Press
CCA 8155
A RELIABLE METHOD FOR GENERATING ANTIBODIES AGAINST PANCREOZYMIN, SECRETIN AND GASTRIN
WERNER SCHLEGEL
* and SOTIRIOS RAPTIS
Department of Internal Medicine, Endocrinology Steinhiivelstrasse 9, 7900 Ulm/Donau (G.F.R.) (Received
and Metabolism, University of Ulm,
June 24th, 1976)
Summary
High specific antibodies against the gastrointestinal hormones pancreozymin, secretin, and gastrin were generated by coupling these peptides with N,N’-carbonyldiimidazole to bovine serum albumin. None of the antisera showed any cross reaction with gut and pancreatic hormones tested for cross reactivity. The dilution of antisera which were useful for the detection of 5-250 pg of hormone in human serum were 1 : 150 000 for secretin, 1 : 2000 for gastrin, and 1 : 2000 for pancreozymin. The NJV’carbonyldiimidazole reagent is therefore highly effective for binding labile peptides to protein carriers without destroying the immunogenic features.
Introduction
Several investigators have reported the development of specific radioimmunoassays sensitive enough to measure concentrations of pancreozymin, secretin and gastrin in serum [l-14]. Among these the radioimmunoassay for gastrin is doubtlessly the most validated one. Gastrin concentrations are measured routinely as they are known to produce the Zollinger-Ellison syndrome. Concerning the other two gastrointestinal hormones only a few radioimmunoassays were published displaying diverging results with respect to fasting serum levels [ 1,3,4,6]. In the development of radioimmunoassays for secretin and pancreozymin the limiting factor was the labelling of these peptides. In addition pancreozymin has poor immunogenic features. We are reporting now on a simple method for generating high specific antibodies against pancreozymin, secretin and gastrin. The principle of this method was already successfully introduced for raising antibodies against prostaglandins [ 151. * To whom correspondence should be addressed.
Materials and methods Gastrin was obtained from Imperial Chemical Ind. Ltd., Ph~maceutic~ Division, Macclesfield, Cheshire, Great Britain; highly purified secretin, 50% pure secretin, 99% + 50% pure pancreozymin, and the CCK-analogue, which is lengthened at its N-terminus by a hexapeptide, from Professor V. Mutt, the Gastrointestinal Hormone Laboratory of the Karolinska Institute, Stockholm, Sweden; and highly pure synthetic secretin from Professor E. Wiinsch, Miinthen, G.F.R.; and 6-tyrosyl-secretin from Schw~z/Mann, Orangeburg, U.S.A. Generation of antibodies The method used for generating antisera was basically the same as published by Orczyk and Behrman [ 161. The conjugates were obtained according to Axen [15], with several variations necessitating detailed description of this method. 3 mg peptide hormone are dissolved in 0.2 ml dimethylformamide (Merck, Darmstadt). While this solution is being shaken in a waterbath at 25°C in an atmosphere of dry nitrogen, 250 (ug N,N’-carbonyldiimidazole (Merck, Darmstadt) are added in a single portion. Shaking under nitrogen is continued for at least 20 min. A solution of 1 mg bovine serum albumin (Behringwerke AG, Marburg) in 0.3 ml bidistilled water is then added to the reaction mixture. The
Standardcurve Pancreozymin antiserua dilutton
MO
lopit
I : 2o(M(3009)
90.
80. 70. HI50. 40. XJ20.
10.
5 1
2
3
4
5 678910
2
3
4
5 6789wD
2
3
4 5 67e91~ &tube
Fig. 1. Dose-response curve with highly purified pancreozymin, using serum from rabbit 3009. The horizontal axis represents the log doses of added hormone and the vertical axis the logit of bound tracer B (corrected for non-specific binding) versus B, = value of Z3 with no unlabelled ligand in the system. The final antiserum dilution was 1 : 2000. 4000 dpm of the pancreozymin anaIogue were added as labelled compound.
441
reaction is allowed to continue for 5 h at 25°C while a weak stream of dry nitrogen is blown through the solution. One ml of a water/dimethylformamide mixture (3 : 2, v/v) is then added. The resulting solution or suspension according to the peptide is applied to collodion bags (Dialyzing system from Sartorius-Membranfilter GmbH, Gottingen) and dialyzed against bidistilled water for 30 min to 2 h in an ice bath. Dialysis and concentration yielded a conjugate which was free of unbound peptide. The effectiveness of conjugation was monitored by labelled hormones. The 100-500 c(g peptide-BSA-conjugates emulsified by equal volumes of complete Freund’s adjuvant (Difco Laboratories, Detroit) are injected intradermally and subcutaneously for ummunization [16] in 15 rabbits for each hormone. In the case of secretin and pancreozymin the semipurified peptide was used for the first 4 immunizations. 5 additional animals were treated with highly purified synthetic (Wiinsch) secretin-conjugates. When a titer of 1 : 100 was achieved all further immunizations were done with 99% purified or synthetic hormone. The animals were test-bled by ear vein puncture and l-5 ml blood was collected. The presence of specific antibodies to the hormones was checked by radioimmunoassay, using the procedure as described by Raptis et al. [12] for gastrin, by Raptis et al. [ 141 for secretin and by Harvey et al. [l] for pancreozymin. After 4 immunizations when a weak binding was obtained, such animals were given a booster injection of 500 pg conjugate and bled 9 days later. About 40 ml of blood was collected at each bleeding. The serum samples were divided into small aliquots (0.5-l ml) and stored in the deep freezer. The dose-response curves were calculated by using the curve-fitting program which was introduced by Rodbard and Lewald [ 171. Results The pancreozymin analogue, the 6-tyrosyl-secretin, and synthetic human gastrin I were radioiodinated and each of them was used as labelled antigen. Graded amounts of unlabelled 99% pure pancreozymin or highly purified secretin or synthetic human gastrin I were tested in the different assay systems to determine their ability in displacing the labelled peptides. Fig. 1 illustrates a typical standard curve for pancreozymin. There was no crossreaction with insulin, pancreatic glucagon, natural 99% pure secretin, synthetic secretin, gastrin, motilin and vasoactive intestinal polypeptide (VIP) up to 1 ng. The final dilution of the antibody was 1 : 2000, the sensitivity 5-8 pg per tube. Seven of the 15 animals developed antisera. Fig. 2 shows a standard curve for secretin. Motilin, gastrin, 99% pure pancreozymin, insulin, pancreatic glucagon and VIP up to 10 ng did not cross react with the antibody which was diluted to a final concentration of 1 : 150 000. The sensitivity was 5 pg per tube. All animals developed antisera. The gastrin antibody was also highly specific. There was no cross reaction with 99% pure pancreozymin, natural and synthetic secretin, insulin, pancreatic glucagon, motilin and VIP up to a concentration of 10 ng (Fig. 3). The final dilution for the antibody was 1 : 2000 and the sensitivity 5-8 pg/ml. Ten of the 15 animals developed antisera. The assay data yield straight lines with steep slopes when transformed into logit-log dose relationship. The dose-response
442
Secretin - Standardcurve
6AO
1ogit
antiserol
95_
dilufton
1 : 15o.m
Eo13)
90.
80. M&O50. 40. 30.
a. 10* 5
1
2
3
4
5678910
2
3
4
56789100
2
3
4
56
7891OlXl
P@Jb Fig. 2. Dose-response curve with highly purified secretin, using serum from rabbit 5013. The final antiserum dsution was 1 : 150 000. 4000 dpm of &tyrosyl-secretin were added as iabelled compound.
curves covered a range between 5 and 250 pg of added standard. titration curve of serum from rabbit 406 obtained 2 months injection of highly pure secretin-~~~nsch)-BSA~onjugate.
Fig. 4 shows a after the first
Anti - Gartrin Serum
Fig. 3. Dose-response curve with gastrfn, using serum from rabbit B 1002. The final antiserum dilution was 1 : 2000.4000 dpm were added as Iabelled compound.
443
[X bwnd
] ANTI- SECRETIN SERUM(WUnsch-Secretin) RABBIT406
l/lmO l/zMm 1/m
l/Em
1/160001/3x00 l/E4cOo [Finalantiserum dilution]
Fig. 4. Serial dilution curve of anti-secretin serum. Secretin antibodies were obtained after immunization with synthetic secretin (Wiinsch).
Discussion In order to generate specific antibodies for substances with low molecular weights, these substances must be conjugated covalently as haptens to water soluble carriers. Because of the high degree of solubility and purity, bovine serum albumin have been employed as the carrier protein. In addition albumin does not seem to interfere with the immunological response of the hapten. A detailed discussion of the various conjugation procedures has been published by Beiser et al. [ 181. Most of these methods (especially the binding with carbodiimide [ 191) were not useful for conjugating the labile hormones secretin and pancreozymin because a considerable amount denatured during conjugation. Fortunately it turned out that NJ’-carbonyldiimidazole was a highly effective reagent for binding peptides to protein carrier without destroying the immunogenic features.
444
Secretin and gastrin are described as very immunogenic by themselves 120,211. This does not apply to pancreozymin, though [ 211. Therefore, only few laboratories could describe a radioimmuno~say for this hormone so far [1,4,21]. The present method should help to generate highly specific antibodies against labile peptides like pancreozymin and others, which contain a reactive carboxylic or amino group. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft, SFB 87 Endokrinologie Ulm. The authors thank Mr. M. Leitze, Mrs. U. Mann and Miss U. Baumert for excellent technical assistance. The synthetic secretin was kindly provided by Professor E. W~nsch, Miinthen, We also greatefully acknowledge the courtesy of Dr. R.F. Harvey, Bristol and Dr. K.D. Buchanan, Belfast, for additional testing of our antibodies. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Harvey, R.F., Dowsett, L., Hartog, M. and Read, A.E. (1974) Gut X5,690--699 Henry, R.W., Flanagan, R.W.J. and Buchanan, K.D. (1975) Lancet ii, 202-203 Strauss, E., Urbach, H.-J. and Yalow, R.S. (1975) Biochem. Biophys. Res. Commun. 64,1036-1040 Thompson, C.J., Fender, H.R., Ramus, N.J., Villar. H.V. and Rayford, P.L. (1975) Ann. Surg. 182, 496-504 Reeder, D.D., Becker, H.D., Rayford, P.L. and Thompson, J.C. (1974) in Endocrinology of the Gut (Chey, W.Y. and Brooks, F.P. eds.), pp. 327-336, Cb. B. Slack Inc., Thorofare, N.J. Boden, G. and Chey, W.Y. (1973) Endocrinology 92,1617-1624 Boehm, M., Lee. Y. and Chey, W.Y. (1974) in Endoc~o~ogy of the Gut &hey, W.Y. and Brooks, F.P., eds.), PP. 310-319, Ch. B. Slack Inc., Thorofare, N.J. Boden, G. (1974) in Method of Hormone Radioimmunoassay (Jaffe, B.M. and Behrman, H.R., eds.), pp. 275-288, Academic Press, New York and London McGuigan, J.E. and Herbs& Ch. E. (1975) in Gastrointestinal Hormones (Thompson, J.C., ed.), PP. 85-98. University of Texas Press, Austin and London Stadil, F. and Rehfeld, J.F. (1972) Stand. J. Clin. Lab. Invest. 39, 361-368 Yafow, R.S. and Berson, .%A. (1970) Gastroenterology 58, l-14 Raptis, S., DoBinger, H.C., Von Berger, L., Schlegel, W., Schrijder, K.E. and Pfeiffer, E.F. (1975) Digestion 13,15-26 Bloom, S.R. (1975) in Gastrointestinal Hormones (Thompson, J.C., ed.). PP. 257-268. University of Texas Press, Austin and London Raptis, S.. Leitze, M., Schlegel. W. and Pfeiffer, E.F. (1975) Horm. Metab. Res. 7, 447 Axen, U. (1974) Prostaglandins 5,45-47 Orczyk, G.P. and Behrman, HR. (1972) Prostaglandins 1,3-20 Rotbard, D. and Lewald, J.E. (1970) Acta Endocrinol. (Copenhagen) Suppl. 147. 79-98 Beiser, S.M., Butler, Jr., V.P. and E&urger, B.F. (1968) in Textbook of Immunopathology (Miescher, P.A. and Miiller-Eberhard, H.J., eds.), pp. 15-24, Grune und Stratton, New York Godfriend, T.L.. Levine, L. and Fasman. G.D. (1964) Science 144,1344-1346 Buchanan, K.D., Teale, J.D. and Horn, G. (1972) Human Metab. Res. 4, 507-508 Bloom, S.R. (1974) Br. Med. Bull. 30.62-67
Clinica Chimica Acta, 13 (1976) 439-444 0 Elsevier/North-Holland Biomedical Press
CCA 8155
A RELIABLE METHOD FOR GENERATING ANTIBODIES AGAINST PANCREOZYMIN, SECRETIN AND GASTRIN
WERNER SCHLEGEL
* and SOTIRIOS RAPTIS
Department of Internal Medicine, Endocrinology Steinhiivelstrasse 9, 7900 Ulm/Donau (G.F.R.) (Received
and Metabolism, University of Ulm,
June 24th, 1976)
Summary
High specific antibodies against the gastrointestinal hormones pancreozymin, secretin, and gastrin were generated by coupling these peptides with N,N’-carbonyldiimidazole to bovine serum albumin. None of the antisera showed any cross reaction with gut and pancreatic hormones tested for cross reactivity. The dilution of antisera which were useful for the detection of 5-250 pg of hormone in human serum were 1 : 150 000 for secretin, 1 : 2000 for gastrin, and 1 : 2000 for pancreozymin. The NJV’carbonyldiimidazole reagent is therefore highly effective for binding labile peptides to protein carriers without destroying the immunogenic features.
Introduction
Several investigators have reported the development of specific radioimmunoassays sensitive enough to measure concentrations of pancreozymin, secretin and gastrin in serum [l-14]. Among these the radioimmunoassay for gastrin is doubtlessly the most validated one. Gastrin concentrations are measured routinely as they are known to produce the Zollinger-Ellison syndrome. Concerning the other two gastrointestinal hormones only a few radioimmunoassays were published displaying diverging results with respect to fasting serum levels [ 1,3,4,6]. In the development of radioimmunoassays for secretin and pancreozymin the limiting factor was the labelling of these peptides. In addition pancreozymin has poor immunogenic features. We are reporting now on a simple method for generating high specific antibodies against pancreozymin, secretin and gastrin. The principle of this method was already successfully introduced for raising antibodies against prostaglandins [ 151. * To whom correspondence should be addressed.
Materials and methods Gastrin was obtained from Imperial Chemical Ind. Ltd., Ph~maceutic~ Division, Macclesfield, Cheshire, Great Britain; highly purified secretin, 50% pure secretin, 99% + 50% pure pancreozymin, and the CCK-analogue, which is lengthened at its N-terminus by a hexapeptide, from Professor V. Mutt, the Gastrointestinal Hormone Laboratory of the Karolinska Institute, Stockholm, Sweden; and highly pure synthetic secretin from Professor E. Wiinsch, Miinthen, G.F.R.; and 6-tyrosyl-secretin from Schw~z/Mann, Orangeburg, U.S.A. Generation of antibodies The method used for generating antisera was basically the same as published by Orczyk and Behrman [ 161. The conjugates were obtained according to Axen [15], with several variations necessitating detailed description of this method. 3 mg peptide hormone are dissolved in 0.2 ml dimethylformamide (Merck, Darmstadt). While this solution is being shaken in a waterbath at 25°C in an atmosphere of dry nitrogen, 250 (ug N,N’-carbonyldiimidazole (Merck, Darmstadt) are added in a single portion. Shaking under nitrogen is continued for at least 20 min. A solution of 1 mg bovine serum albumin (Behringwerke AG, Marburg) in 0.3 ml bidistilled water is then added to the reaction mixture. The
Standardcurve Pancreozymin antiserua dilutton
MO
lopit
I : 2o(M(3009)
90.
80. 70. HI50. 40. XJ20.
10.
5 1
2
3
4
5 678910
2
3
4
5 6789wD
2
3
4 5 67e91~ &tube
Fig. 1. Dose-response curve with highly purified pancreozymin, using serum from rabbit 3009. The horizontal axis represents the log doses of added hormone and the vertical axis the logit of bound tracer B (corrected for non-specific binding) versus B, = value of Z3 with no unlabelled ligand in the system. The final antiserum dilution was 1 : 2000. 4000 dpm of the pancreozymin anaIogue were added as labelled compound.
441
reaction is allowed to continue for 5 h at 25°C while a weak stream of dry nitrogen is blown through the solution. One ml of a water/dimethylformamide mixture (3 : 2, v/v) is then added. The resulting solution or suspension according to the peptide is applied to collodion bags (Dialyzing system from Sartorius-Membranfilter GmbH, Gottingen) and dialyzed against bidistilled water for 30 min to 2 h in an ice bath. Dialysis and concentration yielded a conjugate which was free of unbound peptide. The effectiveness of conjugation was monitored by labelled hormones. The 100-500 c(g peptide-BSA-conjugates emulsified by equal volumes of complete Freund’s adjuvant (Difco Laboratories, Detroit) are injected intradermally and subcutaneously for ummunization [16] in 15 rabbits for each hormone. In the case of secretin and pancreozymin the semipurified peptide was used for the first 4 immunizations. 5 additional animals were treated with highly purified synthetic (Wiinsch) secretin-conjugates. When a titer of 1 : 100 was achieved all further immunizations were done with 99% purified or synthetic hormone. The animals were test-bled by ear vein puncture and l-5 ml blood was collected. The presence of specific antibodies to the hormones was checked by radioimmunoassay, using the procedure as described by Raptis et al. [12] for gastrin, by Raptis et al. [ 141 for secretin and by Harvey et al. [l] for pancreozymin. After 4 immunizations when a weak binding was obtained, such animals were given a booster injection of 500 pg conjugate and bled 9 days later. About 40 ml of blood was collected at each bleeding. The serum samples were divided into small aliquots (0.5-l ml) and stored in the deep freezer. The dose-response curves were calculated by using the curve-fitting program which was introduced by Rodbard and Lewald [ 171. Results The pancreozymin analogue, the 6-tyrosyl-secretin, and synthetic human gastrin I were radioiodinated and each of them was used as labelled antigen. Graded amounts of unlabelled 99% pure pancreozymin or highly purified secretin or synthetic human gastrin I were tested in the different assay systems to determine their ability in displacing the labelled peptides. Fig. 1 illustrates a typical standard curve for pancreozymin. There was no crossreaction with insulin, pancreatic glucagon, natural 99% pure secretin, synthetic secretin, gastrin, motilin and vasoactive intestinal polypeptide (VIP) up to 1 ng. The final dilution of the antibody was 1 : 2000, the sensitivity 5-8 pg per tube. Seven of the 15 animals developed antisera. Fig. 2 shows a standard curve for secretin. Motilin, gastrin, 99% pure pancreozymin, insulin, pancreatic glucagon and VIP up to 10 ng did not cross react with the antibody which was diluted to a final concentration of 1 : 150 000. The sensitivity was 5 pg per tube. All animals developed antisera. The gastrin antibody was also highly specific. There was no cross reaction with 99% pure pancreozymin, natural and synthetic secretin, insulin, pancreatic glucagon, motilin and VIP up to a concentration of 10 ng (Fig. 3). The final dilution for the antibody was 1 : 2000 and the sensitivity 5-8 pg/ml. Ten of the 15 animals developed antisera. The assay data yield straight lines with steep slopes when transformed into logit-log dose relationship. The dose-response
442
Secretin - Standardcurve
6AO
1ogit
antiserol
95_
dilufton
1 : 15o.m
Eo13)
90.
80. M&O50. 40. 30.
a. 10* 5
1
2
3
4
5678910
2
3
4
56789100
2
3
4
56
7891OlXl
P@Jb Fig. 2. Dose-response curve with highly purified secretin, using serum from rabbit 5013. The final antiserum dsution was 1 : 150 000. 4000 dpm of &tyrosyl-secretin were added as iabelled compound.
curves covered a range between 5 and 250 pg of added standard. titration curve of serum from rabbit 406 obtained 2 months injection of highly pure secretin-~~~nsch)-BSA~onjugate.
Fig. 4 shows a after the first
Anti - Gartrin Serum
Fig. 3. Dose-response curve with gastrfn, using serum from rabbit B 1002. The final antiserum dilution was 1 : 2000.4000 dpm were added as Iabelled compound.
443
[X bwnd
] ANTI- SECRETIN SERUM(WUnsch-Secretin) RABBIT406
l/lmO l/zMm 1/m
l/Em
1/160001/3x00 l/E4cOo [Finalantiserum dilution]
Fig. 4. Serial dilution curve of anti-secretin serum. Secretin antibodies were obtained after immunization with synthetic secretin (Wiinsch).
Discussion In order to generate specific antibodies for substances with low molecular weights, these substances must be conjugated covalently as haptens to water soluble carriers. Because of the high degree of solubility and purity, bovine serum albumin have been employed as the carrier protein. In addition albumin does not seem to interfere with the immunological response of the hapten. A detailed discussion of the various conjugation procedures has been published by Beiser et al. [ 181. Most of these methods (especially the binding with carbodiimide [ 191) were not useful for conjugating the labile hormones secretin and pancreozymin because a considerable amount denatured during conjugation. Fortunately it turned out that NJ’-carbonyldiimidazole was a highly effective reagent for binding peptides to protein carrier without destroying the immunogenic features.
444
Secretin and gastrin are described as very immunogenic by themselves 120,211. This does not apply to pancreozymin, though [ 211. Therefore, only few laboratories could describe a radioimmuno~say for this hormone so far [1,4,21]. The present method should help to generate highly specific antibodies against labile peptides like pancreozymin and others, which contain a reactive carboxylic or amino group. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft, SFB 87 Endokrinologie Ulm. The authors thank Mr. M. Leitze, Mrs. U. Mann and Miss U. Baumert for excellent technical assistance. The synthetic secretin was kindly provided by Professor E. W~nsch, Miinthen, We also greatefully acknowledge the courtesy of Dr. R.F. Harvey, Bristol and Dr. K.D. Buchanan, Belfast, for additional testing of our antibodies. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Harvey, R.F., Dowsett, L., Hartog, M. and Read, A.E. (1974) Gut X5,690--699 Henry, R.W., Flanagan, R.W.J. and Buchanan, K.D. (1975) Lancet ii, 202-203 Strauss, E., Urbach, H.-J. and Yalow, R.S. (1975) Biochem. Biophys. Res. Commun. 64,1036-1040 Thompson, C.J., Fender, H.R., Ramus, N.J., Villar. H.V. and Rayford, P.L. (1975) Ann. Surg. 182, 496-504 Reeder, D.D., Becker, H.D., Rayford, P.L. and Thompson, J.C. (1974) in Endocrinology of the Gut (Chey, W.Y. and Brooks, F.P. eds.), pp. 327-336, Cb. B. Slack Inc., Thorofare, N.J. Boden, G. and Chey, W.Y. (1973) Endocrinology 92,1617-1624 Boehm, M., Lee. Y. and Chey, W.Y. (1974) in Endoc~o~ogy of the Gut &hey, W.Y. and Brooks, F.P., eds.), PP. 310-319, Ch. B. Slack Inc., Thorofare, N.J. Boden, G. (1974) in Method of Hormone Radioimmunoassay (Jaffe, B.M. and Behrman, H.R., eds.), pp. 275-288, Academic Press, New York and London McGuigan, J.E. and Herbs& Ch. E. (1975) in Gastrointestinal Hormones (Thompson, J.C., ed.), PP. 85-98. University of Texas Press, Austin and London Stadil, F. and Rehfeld, J.F. (1972) Stand. J. Clin. Lab. Invest. 39, 361-368 Yafow, R.S. and Berson, .%A. (1970) Gastroenterology 58, l-14 Raptis, S., DoBinger, H.C., Von Berger, L., Schlegel, W., Schrijder, K.E. and Pfeiffer, E.F. (1975) Digestion 13,15-26 Bloom, S.R. (1975) in Gastrointestinal Hormones (Thompson, J.C., ed.). PP. 257-268. University of Texas Press, Austin and London Raptis, S.. Leitze, M., Schlegel. W. and Pfeiffer, E.F. (1975) Horm. Metab. Res. 7, 447 Axen, U. (1974) Prostaglandins 5,45-47 Orczyk, G.P. and Behrman, HR. (1972) Prostaglandins 1,3-20 Rotbard, D. and Lewald, J.E. (1970) Acta Endocrinol. (Copenhagen) Suppl. 147. 79-98 Beiser, S.M., Butler, Jr., V.P. and E&urger, B.F. (1968) in Textbook of Immunopathology (Miescher, P.A. and Miiller-Eberhard, H.J., eds.), pp. 15-24, Grune und Stratton, New York Godfriend, T.L.. Levine, L. and Fasman. G.D. (1964) Science 144,1344-1346 Buchanan, K.D., Teale, J.D. and Horn, G. (1972) Human Metab. Res. 4, 507-508 Bloom, S.R. (1974) Br. Med. Bull. 30.62-67