Development of an Immunoassay to Detect Insulin in Serum and Buffer Matrices Using ORIGENO® Electrochemiluminescent Technology

Abstract

Immunoassays were developed to detect insulin in serum and buffer matrices using the ORIGEN® electrochemiluminescent (ECL) technology. A sandwich immunoassay format utilizing two monoclonal antibodies was used. The assay was able to detect insulin from several species including rat, human, cow and pig. Sample volume remained small for the serum and buffer matrix assays at 25 μl and 50 μl respectively. Assay "turn around" time was approximately four hours for both the insulin in buffer matrix ECL assay and the insulin in serum matrix ECL assay. The insulin in buffer ECL assay had an assay range of 0.1 to 12.8 ng/ml with a lowest detectable limit (LDL) of 0.047 ng/ml. The mouse/rat and human insulin in serum matrix ECL assays provided assay ranges of 0.78 to 50 ng/ml or 0.78 to 200 ng/ml respectively with LDL's of ≤ 0.54 ng/ml. Inter-assay and intra-assay precision for both the serum and buffer ECL assays averaged percent coefficient of variation (% C.V.'s) below 10 %. In feasibility studies (Harrington et al. 1997) the ECL assay compared favorably, out performing their commonly used radioimmunoassay (RIA, Linco, St. Louis MO) and scintillation proximity assay (SPA®, Amersham International, Arlington Heights IL; Bosworth and Towers 1989). The ECL assay achieved linear ranges at least 10 fold greater than SPA or RIA. The ECL assay had a much faster "turn around" time of less than four hours as compared to RIA and SPA with assay turn around times of 24 hours and 18 hours respectively. ECL inter-assay precision fell within the range of 2.1 - 5.2 % C.V., which was comparable to both the RIA and the SPA insulin assays. Functional sensitivity was slightly better on the ECL assay compared with ELISA and remained slightly better on the RIA and SPA as compared to the ECL assay.