• A comparison of ELISA methods for the determination of human serum antibodies to Haemophilus influenzae type b

      van Gageldonk PGM; Mariani M; Berbers GAM; LVO-BI; Centro Ricerche; CHIRON S.p.A.; Siena; Italy/Laboratorio di Immunochimica e Sierologia Sperimentale/Departimento Immunologia (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 1998-10-16)
      A comparison between the non-competitive ELISA from Phipps et al., the established radio-antigen binding assay (RABA) and the newly developed Chiron competitive ELISA for measuring anti-Hib polysaccharide (HibPs) antibody concentrations in human sera is described. For this study 4 different serum panels, all from the same clinical trial, were used and the results were compared with the original trial data, determined with the Phipps' non-competitive ELISA. Reliability, reproducibility plus a good correlation with the RABA demonstrate that both the Chiron competitive ELISA and the Phipps non-competitive ELISA can replace the classical RABA for anti-Hib total Ig determination. However, the clear advantage of the somewhat more laborious competitive ELISA, as proposed by Mariani et al., is that overestimation of the percentage of subjects with antibody titers > 0.15 mug/ml is absent, as compared to the non-competitive ELISA. Competition with free HibPs, appeared necessary to avoid false positive binding for anti Hib antibody concentrations 1.0 mug/ml. Although the problem might be restricted for a large part to pre-immune sera it can still influence the amount of subjects falsely considered sufficiently protected against Hib. The Phipps' non-competitive ELISA could be improved by changing the incubation time, incubation temperature , incubation buffers, conjugate and ELISA plate towards the conditions of the Chiron competitive ELISA. Based on the results we propose an improved ELISA for determining Hib antibodies. Basic elements are that samples are tested in a twofold serial dilution and control wells with free soluble Haemophilus influenzae type b polysaccharide are used for samples with Hib antibody concentrations ó 1.0 mug/ml.<br>