THE OPTIMIZATION OF ELISA FOR METHAMPHETAMINE DETERMINATION - THE EFFECT OF IMMUNOGEN, TRACER AND ANTIBODY PURIFICATION METHOD ON THE SENSITIVITY

To obtain more sensitive immunoassay for methamphetamine (MA) determin ation, the optimum condition of enzyme-linked immunosorbent assay (ELI SA) was investigated in regard to immunogens, antibody purification me thods and coating tracers. Activated MA, N-(4-aminobutyl)methamphetami ne (4-ABMA), was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) and used as immunogen. The antibodies were pu rified by protein G chromatography or various immunoaffinity chromatog raphy-linked MA-protein ligands, such as MA-BSA, MA-KLH or MA-ovalbumi n (OVA). Each purified antibody was characterized by means of sensitiv ity and cross-reactivity using the three MA-protein coating tracers, M A-BSA, MA-KLH and MA-OVA. The best sensitivity of each antibody was ac quired with the MA-OVA tracer although the tracer concentration and th e antibody titer level at optimum condition were varied. The antibody with high titer level did not always yield good sensitivity. At optimu m condition, immunoaffinity chromatography-purified antibodies were be tter for sensitivity and for specificity than protein C-purified antib odies. The cross-reactivity of the purified antibodies seemed to be af fected by immunogen structure and showed somewhat different patterns a ccording to the immunoaffinity ligand utilized. These data show that t he antibody purification method as well as choice of coating tracer an d immunogen is essential for the sensitivity and specificity of EIA; t he optimum condition for assay should be discovered using various meth ods and combinations.