Amplified enzyme-linked-immunofilter assays enable detection of 50-10(5) bacterial cells within 1 hour

Two enhanced enzyme-linked-immunofilter assay (ELIFA) methods for the rapid and quantitative detection of whole bacterial cells are described. In the first method, specific antibody bound to bacterial cells was amplified using a secondary antibody and detected by the conjugated enzyme activity (peroxidase) of a third antibody in a chemiluminescent assay. In the second method, a chromogenic substrate was used in conjunction with a biotinylated secondary antibody and avidin. Both assays were conducted within 55 min using a 96-well continuous flow immunofilter apparatus. The assay values were determined either as the reflectance of developed X-ray film placed over chemiluminescent membranes or of precipitated chromogen on the membrane surface. The biotin/avidin method enabled quantitative detection of approximately 60 to 10(5) cells. The detection limit (blank + 2 SD) of the chemiluminescent assay with a 30-s film exposure time was 50 cells. The ELIFA methods described represent a considerable advance in sensitivity over previous immunological methods of detecting whole bacterial cells and suggest that immunological methods may approach PCR in sensitivity.