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A one-step modification of the membrane-filtration enzyme immunoassay (MF EIA) (Barnett et al., J. Clin. Microbiol., 23:385-399, 1987), for estimation of virus-specific antibody is described. The modified MF EIA allowed serum, antigen and enzyme-conjugated anti-globulin to be incubated together in membrane-based 96-well plates to enable the formation of immune complexes in solution at 37 degrees C. The assay required only 45 min for completion and polyethylene glycol was shown to be an essential component in reaction mixtures for IgG assays to enhance immune complex formation. The modified MF EIA was as sensitive as the previous two-step method for monitoring responses to influenza vaccine, and control antigen backgrounds were significantly reduced. The one-step procedure was also shown to be suitable for the rapid serodiagnosis of naturally acquired influenza A and B infections. However, MF EIA detected cross-reactive H1N1 responses in 57.7% of naturally-acquired H3N2 infections, suggesting that responses to common internal antigens were being measured. Cross-reactive responses to influenza A viruses could not be detected in volunteers receiving subunit vaccines.