Development of a novel ELISA for human insulin using monoclonal antibodies produced in serum-free cell culture medium.

Abstract Objectives: Development of an ELISA for human insulin that utilizes monoclonal antibodies (mAbs) produced in serum-free medium. Design and methods: Insulin mAbs were produced in vitro by hybridomas in serum-free medium. A two-step ELISA was developed to replace bovine insulin (standard) and bovine serum albumin (assay buffer) with non-animal reagents. Results: The sensitivity of the insulin ELISA was 0.73 uU/mL with a dynamic range of 2–200 uU/mL. No cross-reactivity with either human C-peptide or human proinsulin was observed. The intra- and inter-assay CVs were n  = 12) compared to lean subjects ( n  = 10, p p p y  = 1.06 x  − 0.44, r  = 1.00, n  = 44). Conclusions: This novel insulin ELISA provides precision and reliability equal to methods currently used in clinical research and serves as a guide for the development of other serum-free immunoassays.