Immunoassays are routinely used for the rapid detection of foodborne pathogens and spoilage/indicator bacteria. Antibodies used for detection need to have high specificity and affinity towards live bacterial cells. Assays combining the use of immunomagnetic separation (IMS) and 'lux' and 'gfp' gene labels were developed for the determination of antibody affinity. Antibody affinities were determined and compared to enzyme-linked immunosorbent assay (ELISA) sensitivities in varying pH, sodium chloride, and temperature conditions. The bioluminescence assay was sensitive for determining affinity. For assays using bioluminescent 'S.' Enteritidis and 'E. coli' O157:H7, increases in sodium chloride levels resulted in higher affinities, however changes in temperatures had no significant effect. A higher affinity and ELISA sensitivity was obtained for anti-'E. coli' O157:H7 antibodies at pH 3 compared to pH 5 and 7. No relationship was found between affinity and ELISA sensitivities using various sodium chloride and temperature conditions.