INDIRECT SANDWICH ENZYME-LINKED IMMUNOSORBENT ASSAY FOR RAPID DETECTION OF HEMOPHILUS-INFLUENZAE TYPE-B INFECTION

The development and testing of an enzyme-linked immunosorbent assay with excellent sensitivity for the detection of H. influenzae type b (HIb) antigen in clinical specimens from patients with HIb meningitis is reported. The assay, an indirect sandwich technique, uses polystyrene balls as a solid phase and an alkaline phosphatase-labeled goat anti-rabbit globulin conjugate. Specimens are incubated with polystyrene balls armed with burro anti-HIb antiserum and recognition antibody is visualized by addition of alkaline phosphatase-labeled anti-globulin together with the enzyme substrate p-nitrophenyl phosphate. Concentrations of antigen are determined from standard curves prepared by using purified HIb capsular antigen polyribophosphate. The reproducibly detects polyribophosphate at concentrations between 1-5 ng/ml. Cross-reactions have not as yet been encountered in simulated and authentic clinical specimens containing other species including Escherichia coli, Klebsiella pneumoniae, group B Streptococcus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis and Listeria monocytogenes. In preliminary tests with 11 spinal fluid specimens, 2 serum specimens and 5 urine specimens from patients with culture-proved HIb meningitis, antigen was detected in all specimens in concentrations ranging from 1-7000 ng/ml. Antigen was not detected in any of 62 clinical specimens which were culture negative for HIb, including 11 spinal fluid specimens from patients with bacterial meningitis caused by microorganisms other than HIb. The enzyme-linked immunosorbent assay technique described here is considerably simpler than radioimmunoassay and, based on concurrent tests with 14 positive clinical specimens, may be more sensitive than counterimmunoelectrophoresis. It holds considerable promise for clinical use in rapid detection of systemic HIb infections.