A monoclonal-based, two-site enzyme immunoassay of human insulin

A procedure is described for the efficient production of insulin-specific monoclonal antibodies, which involves primary and secondary immunization of BALB/c mice in the hind footpads with bovine or porcine insulin and fusion of lymphocytes from popliteal lymph nodes with a P3×63 murine myeloma line. With this protocol, over 200 positive hybrids were obtained from four separate fusions. Dissociation constants of 31 purified monoclonals, cross-reacting with human insulin, were determined by two different methods and ranged between 4 × 10 −10 and 2 × 10 −6 mol/1.24 monoclonals were biotinylated, paired in all possible combinations and tested by ELISA for their capacity to simultaneously bind to human insulin in a two-site assay. More than 40 monoclonal pairs were found which formed a sandwich with the hormone. The development of a simple and rapid one-step enzyme immunoassay is described, which involves a first monoclonal bound to the wells of a microtiter plate and a second monoclonal conjugated to alkaline phosphatase. With this assay, insulin can be determined in a range between 0.08 and 7.5 ng/ml in 3–4 h.