Gross, WL

Kekow, J.

Müller-Ruchholtz, W.

Ulrichs, Karin

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Pancreas Transplantation: A Study of Insulin Secretion in Isolated Islets of Langerhans and in Sera Using a New Enzyme-Linked Immunosorbent Assay J. Kekow, K. Ulrichs, W. Muller-Ruchholtz, and W.L. Gross RADIOIMMUNOASSA YS (RIAs) are routinely used for the detection of immunoreactive insulin (IRI).l Their dis-advantages are limited sensitivity and ac-curacy and their dependence on instable radionucleotides, requiring a special labora-tory. However, for monitoring graft function in pancreas transplantation when immuno-modulatory treatment is required a fast, sensi-tive, constantly available assay is needed. A new enzyme-linked immunosorbent assay (ELISA) method was therefore developed es-pecially for detecting IRI in small volume specimens. MATERIALS AND METHODS EL/SA Procedure The ELISA is based on the principle of competitive saturation (Fig I). Microtiter plates with 96 round-bottomed wells (Nunc, Denmark) were coated using a sandwich principle. First, the plates were coated with a rabbit anti-guinea pig antibody (antiglobulin antibody) at a pH of 9.6 in a two-hour incubation period at 37°C (150 ILL/well). The plates were washed and then incubated with an anti-insulin antibody (Novo, Denmark) developed in guinea pigs at a final dilution of 1 :72,000 and a pH of 7.4, using phosphate-buffered saline (PBS) plus 1 % bovine serum albumin (BSA) (lOO ILL/well). Appropriate standards were prepared from the stock solution, for example, starting with 100.0 ng/mL. One hundred microliters of the standard were added in tripli-cate to the plates after free antibodies had been removed by repeated washings. The samples, rat sera or superna-tants from cultivated rat islets, were also diluted in PBS containing 6% BSA and used in triplicate (100 ILL each/well). The plates were allowed to incubate over-night. Next day, 100 ILL of enzyme-la be led insulin (Sig-ma, St Louis) was added to each well without washing the plates. The incubation lasted for 4 hours at 4°C. Then the plates were washed and 100 ILL orthophenylenediamine dihydrochloride (OPD) solution was added per well. The enzyme reaction was measured at 492 nm. RESULTS For a high quality ELISA using an anti-insulin antibody fixed to a solid phase, opti-mum insulin binding to the anti-insulin anti-body is a critical point. It was possible to apply the ELISA principle2 to the measurement of insulin by fixing the anti-insulin antibody with an antiglobulin antibody. Curve C in Fig 2 illustrates a typical dose-response curve start-ing at 100 ng/mL rat insulin. The standard could be diluted to a concentration of 0.049 ng/mL rat insulin, and the curves were clearly dose-dependent. When only buffer was added instead of insulin standard, the optical density (OD) was approximately 0.804. The blanks were no higher than 0.007. This was also true of a standard curve for rat insulin omitting the anti-insulin antibody in the coating procedure. To demonstrate the efficiency of the indirect coating method, curves A and B in Fig 2 show the standard curves that resulted when the anti-insulin antibody was coated directly onto the microtiter plates without using an anti-globulin antibody. These standard curves express only a poor increase in the OD in relation to the amount of insulin. For routine measuring of supernatants from cultivated rat islets, we used a standard curve ranging from 100 ng/mL to 0.78 ng/ mL rat insulin. Using a 1 :50 dilution for the culture supernatants, it was possible to detect from 39.0 to 5,000 ng/mL of insulin in one run. For detecting insulin in rat sera, we preferred a standard curve ranging from 12.5 ng/mL to 0.049 ng/mL rat insulin. This means that for sera diluted 1 :5, between 0.24 and 60.0 ng/mL of rat insulin could be detected. Further statistical analysis showed that the degree of precision was acceptable, From the Departments of Internal Medicine and Immunology, Medical School. University of Kiel. FRG. Address reprint requests to Dr Jorn Kekow. I. Medi-zinische Universitats-klinik Kiel. Schittenhelmstr. 12. D 2300 Kiel. FRG. © /987 by Grune & Stratton. Inc. 0041-1345/87/1905-0/6/ $03.00/0 Transplantation Proceedings. Vol XIX. No 5 (October). 1987: pp 3921-3922 3921 3922 8 rabbit anti-guinea pig ab o b) • W gUinea pig anti-insultn ab [::::1 insultn (unlabeled I ~ insulin (peraxidase-Iabeled I o OPO (substratel Fig 1. Determination of rat insulin by ElISA: (a) coating of the microtiter plate with the antiglobulin antibody; (b) anti-insulin antibody is bound by the antiglobulin antibody; (c) insulin (from sera. culture supernatants or standard) is bound by the anti-insulin antibody; (d) competitive saturation of the anti-insulin antibody with either unlabeled or enzyme-Iabeled insulin; and (e) measurement of substrate degradation after removal of all unbound insulin (Iabeled!unla-beled). the variation within a test having a standard deviation of .:s 15. I % and that between dif-ferent tests of .:s 15.6%. A study on the recov-ery of rat insulin in serum or culture superna-tants was also performed. When the expected values were compared with the ones we mea-sured, the recovery ranged from 80% to 166%. CONCLUSIONS Total IRI is conventionally determined by RIA. The known advantages of ELlS As can also be applied to IRI measurement, as dem-onstrated by a new modified ELlSA. It is characterized by indirect binding of the anti-insulin antibody by a second antibody and uses the principle of competitive saturation. KEKOW ET Al OD 492 nm 07 0.6 0.5 0.4 03 0.2 0.1 L __ --. ___ -.-___ -.-___ ,- rat insulin 01 10 100 ng/ml Fig 2. Standard curves: influence of the coating procedure. Curves A and B: the anti-insulin antibody is coated directly onto the microtiter plate without using the antiglobulin antibody (dilution of the anti-insulin antibody for curve B is 1 :18.000 and for curve A 1 :72.000); Curve C: optimization of the standard curve by fixing the anti-insulin antibody (dilution 1 :72.000) with an antiglobulin antibody. 00 expressed as mean ± SO of triplicate samples. This ELlSA is sufficiently accurate, repro-ducible, and very sensitive. This is the case in detecting both high levels of insulin in culture supernatants and very low levels in rat sera. With the specimens being handled in the microtiter plate system and the lack of depen-dence on fresh radionucleotides, larger num-bers of specimens can be processed at any given time. The new assay thus allows opti-mized insulin measurement in both in vivo and in vitro experiments, which is essential for planning strategies for pancreas transplanta-tion, for example, immunomodulatory treat-ment. REFERENCES I. Heding LG: Diabetologia 8:260, 1972 2. Voller A, Bartlett A, BidweII DE: J Clin Pathol 31:507,1978 

Pancreas transplantation: a study of insulin secretion in isolated islets of Langerhans and in sera using a new enzyme-linked immunosorbent assay

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Pancreas transplantation: a study of insulin secretion in isolated islets of Langerhans and in sera using a new enzyme-linked immunosorbent assay

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