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Levels of antibodies against Haemophilus influenzae type b capsular polysaccharide were determined in acute-phase and convalescent-phase serum samples obtained from 21 children with invasive H. influenzae type b infections and from 44 children vaccinated with two H. influenzae type b vaccines. Amounts of immunoglobulin G (IgG), IgM, and IgA antibodies were measured by direct and indirect enzyme-linked immunosorbent assay (ELISA), and the total amount of antibodies was measured by radioimmunoassay (RIA). Results obtained by ELISA were calculated by multiple-point parallel-line comparison and by endpoint analysis. A very good correlation was obtained between direct and indirect ELISA values. In the lower range of antibody concentrations, the correlation between ELISA values obtained by endpoint analysis and those obtained by multiple-point parallel-line comparison was poor, since the latter method of calculation yielded values of up to 1 microgram/ml in sera that were negative according to endpoint analysis. These sera with negative endpoint titers also had undetectable or very low antibody concentrations as measured by RIA. Consistent with this finding, in acute-phase and prevaccination sera with undetectable or low antibody concentrations as measured by RIA, ELISA values calculated by multiple-point parallel-line comparison were much higher. In sera with higher antibody concentrations, however, parallel-line comparisons showed good correlation between RIA and ELISA values. Although no reference method for measuring true antibody concentrations is available, ELISA values as calculated by multiple-point parallel-line comparison appear to overestimate antibody concentrations in sera containing low antibody concentrations, whereas ELISA values obtained by endpoint analysis are less well correlated with RIA values at higher concentrations.

journal of clinical microbiology

Comparison between radioimmunoassay and direct and indirect enzyme-linked immunosorbent assays for determination of antibodies against Haemophilus influenzae type b capsular polysaccharide