Stabilization of enzyme immunoassays for atrazine

Reagent stability is an important issue in immunoassay technology. Enzyme immunoassays (EIA) for atrazine are used as an example for investigating the influence of different stabilizing agents on the activity of polyclonal and monoclonal antibodies (Ab), enzyme tracer (ET) as well as enzyme substrate after storage at different temperatures. When lyophilized Ab were stored for two weeks at 30°C, ca. 70% of the original activity in the EIA (lyophilized Ab stored at -20°C) was retained. In contrast, the activity of Ab-coated plates decreased very fast during storage at 30°C. This could be prevented by a post-coating step, especially with bovine serum albumin (BSA), polyvinyl alcohol (PVA) or a commercial solution (PAN/Coat(TM)) as additives. The best stabilization of the ET was achieved with PVA (5%), BSA (1%), fetal calf serum (FCS, 0.5%) and two commercial stabilizing solutions. After storage at 4°C for 10 weeks, 100-175% of the activity of the control (ET stored at 4°C without additives) was observed. Activities similar to the control were also found after 10 weeks of storage at 30°C when two commercial stabilizing solutions were used. BSA (1%) was equally effective up to 6 weeks, subsequently the activity decreased to ca. 50% of the control. The enzyme substrate solutions (hydrogen peroxide and tetramethylbenzidine) should be stored separately. Under these conditions, they did not show any loss of activity after storage for two weeks at 30°C. The results obtained for the EIA were transferred to an immunofiltration assay, a membrane-based field test. Addition of BSA and PVA to the ET stored at 30°C led to the same color development as with the ET stored at 4°C without additives. The improved stability of the components of the EIA allows postal transfer without refrigeration and reliable performance of the tests in, and outside of the laboratory.