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 P3x63 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 X 10(-10) and 2 X 10(-6) mol/l. 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.
D000469:Alkaline Phosphatase; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000915:Antibody Affinity; D001667:Binding, Competitive; D003429:Cross Reactions; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007124:Immunoenzyme Techniques; D007328:Insulin / Q000032:analysis*; D008722:Methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008832:Microchemistry
D000911:Antibodies, Monoclonal; D007328:Insulin; D000469:Alkaline Phosphatase