In a recent article, Mariani and collaborators (2) described a competitive enzyme-linked immunosorbent assay (ELISA) for the quantitation of serum antibodies to Haemophilus influenzae type b (Hib) which correlated very well with the traditional radioantigen binding assay (RABA) and a previously described direct ELISA (3).
While their analyses were very comprehensive, the basis for their comparison of methods was data generated in the same laboratory. Mariani et al. did not reference an interlaboratory study evaluating the direct ELISA which indicated that the reproducibility of the HbO-HA assay is laboratory dependent (1). In the HbO-HA direct ELISA interlaboratory study, 7 of 11 laboratories reported higher antibody concentrations for low-titered sera than expected based on the RABA. Based on the data generated by Mariani et al., it appears that their laboratory encountered this previously described difficulty. The fact that four laboratories in the previous interlaboratory study generated data by the HbO-HA direct ELISA that were consistent with the RABA indicates that the variation is a laboratory-specific phenomenon.
The reasons why some laboratories encounter difficulty with low-titered sera in the HbO-HA ELISA are unclear. We speculate that direct ELISAs are sensitive to low levels of endotoxin contamination, which are introduced via buffers and glassware used during the antigen-coating steps; human sera contain antibodies which can bind to these contaminants.
Another possibility is that the Mariani et al. study used sera which were more concentrated (1:20 dilution) than the 1:50 dilution recommended by Phipps et al., which may enhance the nonspecific binding of human immunoglobulins (2). In our experience, a starting dilution of 1:50 provides sufficient sensitivity in the HbO-HA ELISA to quantitate to 0.1 μg/ml. Thirdly, we have noted that plates vary by lot and by manufacturer in their performance characteristics, requiring prescreening for optimal and specific antigen-binding capacity.
For those laboratories that are unable to correct this background binding which affects low-titered specimens, the Mariani et al. competitive ELISA appears to provide a sensitive alternative method for quantitation of human antibodies to Hib polysaccharide.
REFERENCES
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