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Human C-peptide

Part I: Radioimmunoassay

Humanes C-Peptid

I. Radioimmunologische Bestimmung

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Zusammenfassung

Synthetisches humanes C-Peptid mit einer Tyrosin-Gruppe am Aminoende wird unter Einsatz von Chloramin T oder von Wasserstoffperoxid und Lactoperoxidase mit125Jod markiert. Die mit beiden Methoden erzielten Ergebnisse werden verglichen. Antiserum gegen synthetisches humanes C-Peptid (ohne Tyrosin), das zum Teil an Kaninchenalbumin gekoppelt wurde, wird in Meerschweinchen und Ziegen erzeugt. Ziegen eignen sich zur Antikörperbildung besser als Meerschweinchen. Bei der Herstellung von Standardkurven für den Radioimmunoassay tritt das sogenannte „hook effect“ Phänomen auf. Durch Verdünnen von Antiseren mit hohem Antikörpertiter, die nach wiederholtem Immunisieren bei Ziegen gewonnen wurden, gelingt es Standardkurven mit monotonem Abfall zu erhalten.

Freies und an Antikörper gebundenes C-Peptid werden mit Hilfe des Anionaustauscher Harzes Amberlite getrennt. Durch Anwendung dieses Trennverfahrens wird eine unspezifische Bindung von markiertem C-Peptid an Eiweißfraktionen in Diabetikerseren ausgeschlossen.

Die Empfindlichkeit unseres Radioimmunoassays beträgt ungefähr 0,3 ng C-Peptid/ml Serum. Intraund Interassayvariabilität liegen unter 10%. Eine Kreuzreaktion mit unserem Antiserum wird nur für humanes Proinsulin gefunden.

Summary

Synthetic human C-peptide bearing a Tyrosine group at its amino end is labelled with125iodine using chloramin T or hydrogen peroxide and lactoperoxidase. The results are compared applying both methods. Antiserum to synthetic human C-peptide (without Tyrosine) which was partially coupled to rabbit albumin, is raised in guinea pigs and goats. Goats show to be superior to guinea pigs concerning antibody production. The so-called “hook effect” phenomenon is observed in setting up the standard curves for the radioimmunoassay. Monotonically decreasing standard curves are obtained on dilution of antiserum with a high antibody titer which was produced by repeated immunization in goats.

Free C-peptide and C-peptide bound to antiserum are separated with the anion exchange resin Amberlite. Using this separation technique we excluded unspecific binding of labelled C-peptide to protein fractions in serum of diabetics.

The sensitivity of our radioimmunoassay is approx. 0.3 ng C-peptide/ml serum. Intra- and interassay variability are below 10%. Human proinsulin is the only substance found to crossreact with the antiserum.

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References

  1. Bartels, H.J., Hesch, R.D., Hüfner, M.: Das Phänomen ansteigender B/F Kurven bei radioimmunologischen Bestimmungsmethoden. Z. klin. Chem.10, 351 (1972)

    Google Scholar 

  2. Beischer, W., Heinze, E., Keller, L., Raptis, S., Pfeiffer, E.F.: Human C-peptide, part II: clinical studies. Klin. Wschr.54, 717 (1976)

    PubMed  Google Scholar 

  3. Block, M.B., Mako, M.E., Steiner, D.F., Rubenstein, A.H.: Circulating C-peptide immunoreactivity. Studies in normals and in diabetic patients. Diabetes21, 1013 (1972)

    PubMed  Google Scholar 

  4. Cresto, C., Yalow, R.S.: Anomalously ascending standard curves in the radioimmunoassay of insulin, human growth hormone and ACTH. Horm. Metab. Res., Suppl.5, 1 (1974)

    Google Scholar 

  5. Geiger, R., Jäger, G., König, W.: Darstellung der Gesamtsequenz des human-Proinsulin-C-Peptids und seines Glu9, Gln11 Analogen. Chem. Ber.106, 2347 (1973)

    Google Scholar 

  6. Goodfriend, T.L., Levine, L., Fasman, G.D.: Antibodies to bradykinin and angiotensin: a use of carbodiimides in immunology. Science144, 1344 (1964)

    PubMed  Google Scholar 

  7. Greenwood, F.C., Hunter, W.M.: The preparation of131Ilabelled human growth hormone of high specific radioactivity. Biochem. J.89, 114 (1963)

    PubMed  Google Scholar 

  8. Heding, L.G.: Radioimmunological determination of human C-peptide in serum. Diabetologia11, 541 (1975)

    PubMed  Google Scholar 

  9. Heding, L.G., Larsen, U.D., Markussen, J., Jørgensen, K.H., Hallund, O.: Radioimmunoassays for human, pork and ox C-peptides and related substances. Horm. Metab. Res., Suppl.5, 40 (1974)

    Google Scholar 

  10. Heding, L.G., Rasmussen, S.M.: Human C-peptide in normal and diabetic subjects. Diabetologia11, 201 (1975)

    PubMed  Google Scholar 

  11. Holohan, K.N., Murphy, R.F., Buchanan, K.D., Elmore, D.T.: Enzymic iodination of polypeptide hormones for radioimmunoassay. Clin. chim. Acta,45, 153 (1973)

    PubMed  Google Scholar 

  12. Horwitz, D.L., Starr, J.I., Mako, M.E., Blackard, W.G., Rubenstein, A.H.: Proinsulin, insulin, and C-peptide concentrations in human portal and peripheral blood. J. clin. Invest.55, 1278 (1975)

    PubMed  Google Scholar 

  13. Imura, H., Nakai, Y., Matsukura, S., Hirata, Y.: The paradoxical binding phenomenon in radioimmunoassay of ACTH. Horm. Metab. Res., Suppl.5, 7 (1974)

    Google Scholar 

  14. Kancko, T., Oka, H., Munemura, M., Oda, T., Yamashita, K., Suzuki, S., Yanaihara, N., Hashimoto, T., Yanaihara, Ch.: Radioimmunoassay of human proinsulin C-peptide using synthetic human connecting peptide. Endocr. jap.21, 141 (1974)

    Google Scholar 

  15. Markussen, J., Heding, L.G., Jørgensen, K.H., Sundby, F.: Proinsulin, insulin and C-peptide. Horm. Metab. Res., Suppl.3, 33 (1971)

    Google Scholar 

  16. Matsukura, S., West, C.D., Ichikawa, Y., Jubiz, W., Harada, G., Tyler, F.H.: A new phenomenon of usefulness in the radioimmunoassay of plasma adrenocorticotropic hormone. J. Lab. clin. Med.77, 490 (1971)

    PubMed  Google Scholar 

  17. Melani, F., Ditschuneit, H., Bartelt, K.M., Friedrich, H., Pfeiffer, E.F.: Über die radioimmunologische Bestimmung von Insulin im Blut. Klin. Wschr.43, 1000 (1965)

    PubMed  Google Scholar 

  18. Melani, F., Rubenstein, A.H., Oyer, P.E., Steiner, D.F.: Identification of proinsulin and C-peptide in human serum by a specific immunoassay. Proc. nat. Acad. Sci.67, 148 (1970)

    PubMed  Google Scholar 

  19. Rodbard, D., Lewald, J.E.: Computer analysis of radioligand assay and radioimmunoassay data. Acta endocr. (Kbh.), Suppl.147, 79 (1970)

    Google Scholar 

  20. Rubenstein, A.H., Clark, J.L., Melani, F., Steiner, D.F.: Secretion of proinsulin C-peptide by pancreatic β-cell and its circulation in blood. Nature224, 697 (1969)

    Google Scholar 

  21. Schmidt, F.H.: Enzymatische Methode zur Bestimmung von Blut- und Harnzucker unter Berücksichtigung von Vergleichsuntersuchungen mit klassischen Methoden. Internist (Berl.)4, 554 (1963)

    Google Scholar 

  22. Thorell, J.I., Johansson, B.G.: Enzymatic iodination of polypeptides with125I to high specific activity. Biochim. Biophys. Acta251, 363 (1971)

    PubMed  Google Scholar 

  23. Weintraub, B.D., Rosen, S.W., McCammon, J.A., Perlmann, R.L.: Apparent cooperativity in radioimmunoassay of human chorionic gonadotropin. Endocrinology92, 1250 (1973)

    PubMed  Google Scholar 

  24. Werner, W., Rey, H.G., Wielinger, H.: Über die Eigenschaften eines neuen Chromogens für die Blutzuckerbestimmung nach der GOD/POD-Methode. Z. Anal. Chem.252, 224 (1970)

    Google Scholar 

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Authors and Affiliations

  1. Department of Internal Medicine, Endocrinology and Metabolism, University of Ulm, Ulm, Germany

    W. Beischer, L. Keller, M. Maas, E. Schiefer & E. F. Pfeiffer

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  1. W. Beischer
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  2. L. Keller
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  3. M. Maas
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  4. E. Schiefer
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  5. E. F. Pfeiffer
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Supported by the Deutsche Forschungsgemeinschaft, (SFB 87)

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Beischer, W., Keller, L., Maas, M. et al. Human C-peptide. Klin Wochenschr 54, 709–715 (1976). https://doi.org/10.1007/BF01470462

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