Summary
The reactions between four insulin antisera and eighteen insulin derivatives with modifications at the A1, B1 and B29 positions have been studied using a standard double-antibody radioimmunoassay procedure. The derivatives studied had: a) single modifications at A1, B1 or B29; b) modifications at two sites with or without a crosslink between them; c) modifications at all three sites with or without a crosslink. Analysis of the results showed a clear difference in the reactivity of the antisera. One antiserum (GP 5) was highly sensitive to modifications of the B1 residue and another (Ab 1) was sensitive to A1 and B29 modifications. Thus, immunological potencies of insulin analogues derived on the basis of these reactions with the antisera give widely varying results. These antisera were used in discriminatory radioimmunoassays of chemically modified insulins in biological fluids for estimation of in vivo hypoglycaemic potencies by an infusion technique, where the knowledge of the specificity of the antisera was useful in assessing the immunological identity of immunoreactive material in plasma with the analogue infused.
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Blundell, T. L., Cutfield, J. F., Cutfield, S. N., Dodson, E. T., Dodson, G. G., Hodgkin, D. C., Mercola, D. A., Vajayan, M.: Atomic positions in rhombohedral 2-zinc insulin crystals. Nature231, 506–511 (1971)
Freychet, P., Brandenburg, D., Wollmer, A.: Receptor-binding assay of chemically modified insulins. Comparison ofin vitro andin vivo bioassays. Diabetologia10, 1–5 (1974)
Gliemann, J., Gammeltoft, S.: The biological activity and binding affinity of modified insulins determined on fat cells. Diabetologia10, 105–113 (1974)
Jones, R. H., Dron, D. I., Ellis, M. J., Sönksen, P. H., Brandenburg, D.: Biological properties of chemically modified insulins. 1. Biological activity of proinsulin and insulin modified at A1-glycine and B29-lysine. Diabetologia12, 601–608 (1976)
Lindsay, D. G., Shall, S.: Monosubstituted 2,2-dimethyl-3-for-myl-L-thiazolidine-4-carbonyl insulins. Eur. J. Biochem.15, 547–554 (1970)
Schwartz, G., Katsoyannis, P. G.: Synthesis of human [9-leucine-B] insulin. Biochemistry15, 4071–4076 (1976)
Busse, W. D., Carpenter, F. H.: Synthesis and properties of carbonylbis (methionyl) insulin, a proinsulin analogue which is convertible to insulin by cyanogen bromide cleavage. Biochemistry15, 1649–1657 (1976)
Paselk, R. A., Levy, D.: Preparation of several trifluoroacetyl insulin derivatives. Biochim. Biophys. Acta359, 215–221 (1974)
Brandenburg, D., Gattner, H. G., Wollmer, A.: Darstellung und Eigenschaften von Acetylderivaten des Rinderinsulins. 1. Hoppe Seylers Z. Physiol. Chem.353, 599–617 (1972)
Brandenburg, D., Busse, W. D., Gattner, H. G., Zahn, H., Wollmer, A., Gliemann, J., Puls, W.: Structure-function studies with chemically modified insulins. Peptides 72: Proceedings of the Twelfth European Symposium, Reinhardsbrunn Castle, German Democratic Republic, September 1972, pp. 270–283. Amsterdam: North Holland Publishing Company 1973
Brandenburg, D., Gattner, H. G., Schermutzki, W., Schüttler, A., Uschkoreit, J., Weimann, J., Wollmer, A.: Crosslinked insulins: preparation, properties and applications. Adv. Exp. Med. Biol. 86 a, 261–282 (1977)
Lindsay. D. G., Shall, S.: Acetoacetylation of insulin. Biochem. J.115, 587–595 (1969)
Lindsay, D. G.: Intramolecular crosslinked insulin. FEBS Lett.21, 105–108 (1971)
Lindsay, D. G., Loge, O., Losert, W., Shall, S.: Carbamyl and methylthiocarbamylinsulins. Biochim. Biophys. Acta263, 658–665 (1972)
Greenwood, F. C., Hunter, W. M., Glover, J. S.: The preparation of131I-labelled human growth hormone of high specific radioactivity. Biochem. J.89, 114–123 (1963)
Morgan, C. R., Lazarow, A.: Immunoassay of insulin: two antibody system. Plasma insulin levels in normal, subdiabetic and diabetic rats. Diabetes12, 115–126 (1963)
Sönksen, P. H.: Separation of antibody-bound from free hormone by the double-antibody technique. In: Hormones in human blood. Detection and assay. Antoniades, H. N. (Ed.), pp. 121–138. Cambridge, Mass.: Harvard Univ. Press 1976
Finney, D. J.: Statistical methods in biological assay. Second Edition, pp. 99–138. London: Griffin 1964
Papachristodoulou, D., Brandenburg, D., Dron, D. I., Jones, R. H., Sönksen, P. H., Thomas, J. H.: Studies on the relationship between the molecular structure and the catabolism of insulin. Acta Biol. Med. Ger.36, 1705–1712 (1977)
Yalow, R. S.: Application of radioimmunologic methods to problems in insulin antigenicity and hormonal assay. Acta Med. Scand. [Suppl]601, 149–196 (1977)
Ellis, M. J., Darby, S. C., Jones, R. H., Sönksen, P. H.:In vitro bioactivity of insulin analogues: Lipogenic and anti-lipolytic potency and their interaction with the effect of native insulin. Diabetologia15, 403–410 (1978)
Pullen, R. A., Lindsay, D. G., Wood, S. P., Tickle, I. J., Blundell, T. L., Wollmer, A., Krail, G., Brandenburg, D., Zahn, H., Gliemann. J., Gammeltoft, S.: Receptor-binding region of insulin. Nature259, 369–373 (1976)
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Dron, D.I., Jones, R.H., Sönksen, P.H. et al. Radioimmunoassay of chemically modified insulins. Diabetologia 18, 59–63 (1980). https://doi.org/10.1007/BF01228304
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DOI: https://doi.org/10.1007/BF01228304