Enzyme immunoassay for detection of antibody-coated bacteria

To quantitatively evaluate factors potentially affecting antibody coating of bacteria in urine, we developed an assay with enzyme-linked rather than fluorescein-conjugated immunoglobulin. Using the enzyme immunoassay (EIA) in an in vitro system in which concentrations of serotype O44 Escherichia coli and antibody titer to E. coli Orr O44 O antigen were known, we compared specimens run in parallel with a fluorescent antibody (FA) assay. At greater than or equal to 10(5) bacteria per ml, antibody titer to homologous O antigen correlated directly with absorbance in the EIA. Both tests had sensitivities exceeding 95% in specimens containing greater than or equal to 10(5) bacteria per ml, but the FA test detected 23 of 27 positive specimens with less than 10(5) bacteria per ml compared with 21 of 43 detected by EIA (P = 0.002). However, nonspecific fluorescence caused false positives in 8% of negative tests run by FA compared with 1% of simultaneous EIA tests (P = 0.05). pH alterations and pretreatment of bacteria with antibiotics did not affect either test. Heterologous E. coli strains showed no cross-reactivity with O44 antiserum, but all Staphylococcus aureus isolates tested caused false positives in both assays, and one Klebsiella strain repeatedly caused a false-positive FA assay. The EIA appears to be a simple, quantitative, and specific technique for detection of antibody-coated bacteria in this experimental system.