Detection of bacteria by hybridization of rRNA with DNA-latex and immunodetection of hybrids.

A novel nucleic acid hybridization assay with a DNA probe immobilized on 1.25-micron-diameter latex particles was developed. Hybridization of the immobilized probe DNA with sample rRNA was complete in 10 to 15 min. Alkaline phosphatase-labeled anti-DNA-RNA was allowed to bind to the DNA-RNA hybrids on the latex particles. Then the latex was collected on a small glass fiber filter pad, and bound alkaline phosphatase was quantitated by reflectance rate measurement. The method detected a broad range of bacterial species and had a detection limit of 500 cells per assay. The assay was used to screen urine samples for bacteriuria and had a sensitivity of 96.2% compared with conventional culture at a decision level of greater than or equal to 10(4) CFU/ml. The hybridization method could have broad application to the detection of bacteria and viruses.

Immunochemiluminometric assay for hepatitis B surface antigen.

A novel "sandwich" immunoassay for hepatitis B surface antigen monitored by chemiluminescence is described. The method involves use of an antibody-coated microtitration plate and requires 100 micro L of test specimen. The antigen binds to the antibody during the first 2-h incubation and, after an intermediate wash step, the sandwich is completed by 2-h incubation with antibody to antigen that has been labeled with an isoluminol derivative. A final wash step follows. A luminometer, built in-house, adds "microperoxidase" and peroxide, to initiate chemiluminescence, and provides automated readout at 10-s intervals. Results compare well in specificity and sensitivity with those of a comparison radioimmunoassay procedure. Within- and between-assay variability (CV) is 7 to 13% (n = 6). All reagents are stable at 4 degrees C for at least several months. Use of a non-radioisotopic label in this assay avoids the stability problems and inconvenience associated with radioactivity.