Variable quantitation of Haemophilus influenzae type b anticapsular antibody by radioantigen binding assay

Abstract

The measurement of antibody to Haemophilus influenzae type b capsular polysaccharide is important in the study of natural immunity and in the immunogenicity evaluation of H. influenzae type b vaccines. Several radioantigen binding assays (RABA) have been developed to measure H. influenzae type b anticapsular antibody, but recent immunogenicity data obtained with structurally similar vaccines suggest major differences in antibody quantitation in different laboratories. To evaluate interlaboratory variability in the measurement of anticapsular antibody levels, we blindly evaluated a sample of 40 pre- and postimmunization sera by eight RABAs in different laboratories. Evaluation of RABA methods revealed differences in polysaccharide antigens, radiolabeling methods, concentration and volume of antigen and antibody, and other assay methods. The reported results of assays varied significantly between laboratories (up to sixfold differences in geometric means), in part because of differences in assay sensitivity and different proportions of samples having undetectable levels of antibody (0 to 65% of specimens with undetectable levels). After standardizing the limit of sensitivity for all assays (0.125 microgram/ml), the results of all combinations of paired analyses of RABA assays correlated well (r = 0.88 to 0.99) but the geometric mean levels still varied as much as twofold. For individual sera, the differences between paired assays often were substantial (P less than or equal to 0.0001, paired t test), with some results varying as much as 64-fold. Differences were greatest for lower levels of antibody. There was good comparability and interlaboratory reproducibility of some assays but not of others. Intrinsic or extrinsic labeling of the antigen was not a major determinant of comparability. In most instances, the current variation in the quantitation of antibody levels by these assays precludes interassay comparisons. A standardized measurement of antibody needs to be developed to adequately compare results between different H. influenzae type b immunogenicity studies.

Variability in the Functional Activity of Vaccine-Induced Antibody to Haemophilus influenzae Type b

“…The reason for this difference is not known but may relate to the greater antibody concentration present in the RABA-2 reaction vial because of the smaller volume of diluent used. These data emphasize the importance of previous suggestions that the conditions of the RABA can markedly influence the anti-Hib PS antibody concentrations assigned to a serum (9,36,39), particularly if the antibody is of low avidity (9). Finally, our results also suggest that measurements of antibody avidity may help predict the functional activity of antibody to Hib PS.…”

Interlaboratory study evaluating quantitation of antibodies to Haemophilus influenzae type b polysaccharide by enzyme-linked immunosorbent assay

“…All 12 participating laboratories were provided with 20 coded serum samples and were asked to analyze the samples by HbO-HA ELISA and/or RABA. As RABA has been the traditional assay method and has been previously standardized (5,18), the data obtained by this method served as the baseline for comparison with the data generated by the new ELISA method (12). The RABA was performed by five participating laboratories.…”

“…Quantitation of antibodies specific for the capsule of Haemophilus influenzae type b (Hib) has been an active area of clinical research since the 1970s (6,10,11,15,17), continuing even after the introduction of Hib vaccines for infants in the United States in the 1990s (3). The radioactive antigen binding assay (RABA) for Hib polysaccharide (PS) antibodies was standardized following reports that data derived by RABA in different laboratories were quite variable (4,5,18). In fact, the need for reliable standardized assay methods is now paramount, as the availability of these vaccines creates new opportunities for immunization.…”

“…The Hib capsular PS, polyribosylribitol phosphate, is the primary immunogen in all Hib vaccines. Traditionally, the immune status and response to vaccination for antibodies specific to the Hib PS in human sera have been quantitated by RABA (5,18). The antibody levels have provided a surrogate marker for predicting the minimum antibody concentrations associated with protection from invasive disease (9).…”

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

“…Since the first description of an RIA for this purpose (12), two distinctly different assays (1, 9) and several modifications (15) have been described. Two recent studies have demonstrated that RIA results vary too much among laboratories to permit comparisons among results of vaccine studies that use serum samples assayed in different laboratories (5,15).…”