A VERY RAPID ENZYME-IMMUNOASSAY FOR CONFIRMATION OF RHIZOBIAL IDENTITY AND ESTIMATION OF CELL NUMBERS IN FRESH BROTH CULTURE

An enzyme immunoassay has been developed that confirms identity and es timates cell numbers of Bradyrhizobium japonicum and Rhizobium legumin osarum by. vicine in broth culture. Total testing time required is les s than 10 min and no specialized equipment is required. The assay invo lves a simultaneous and brief reaction of cells, primary antibody, and secondary antibody - enzyme conjugate in aqueous suspension, after wh ich the cells are captured on a 0.45-mu m-pore-size syringe filter mem brane. Upon addition of enzyme substrate, cell-bound antibody-enzyme c onjugate converts the substrate to a blue-green reaction product withi n the transparent body of the syringe filter. The assay can be quantif ied by stopping the reaction with acid, extracting the coloured substr ate reaction product from the syringe filter, and measuring absorbance using a spectrophotometer. The assay was developed using cultures of rhizobia and bradyrhizobia but should be applicable to liquid cultures of most microorganisms.