An enzyme linked immunosorbent assay for nitrate reductase using monoclonal antibodies

An indirect sandwich enzyme linked immunosorbent assay (ELISA), which used both rabbit polyclonal and rat monoclonal antibodies to nitrate reductase (NR) (E.C.1.6.6.1), was adapted to measure NR protein in crude extracts of spinach (Spinacea oleracea) plants. Conditions were optimised for maximum dose response with respect to coating times and dilutions of antibodies and antigen. These were polyclonal, 1 hr and 1/3000; antigen, 1 hr; monoclonal, 1 hr and 50μg protein/ml for MAC 74; and antispecies, 40 min at 27° and 1/2000 sheep anti-rat. The ELISA signal was a linear function of the amount of NR up to ca 1.5 ng and of the log of the amount of NR over the range 20–1200 ng. All our monoclonals (AFRC MAC 74–79) gave positive dose responses and the method is illustrated with MAC 74. Changes in antigenic NR in leaves of spinach plants on nutrient-nitrate removal followed by resupply and the distribution of NR in roots, petioles and leaves of spinach plants, were measured by ELISA and shown to be related to changes in NADH-NR activity.