Boies. 1986. Antibody responses to Haemophilus influenza type b polysaccharide vaccine in relation to Km(1) and G2m(23) immunoglobulin allotypes. J. Infect.
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1988, p. 72-78 0095-1137/88/010072-07$02.00/0 Copyright © 1988, American Society for Microbiology
Vol. 26, No. 1
Variable Quantitation of Haemophilus influenza Type b Anticapsular Antibody by Radioantigen Binding Assay JOEL I. WARD,1* DAVID P. GREENBERG,' PORTER W. ANDERSON,2 KELLY S. BURKART,' PETER D. CHRISTENSON,' LANCE K. GORDON,3 HELENA KAYHTY,4 JOSEPH S. C. KUO,5 AND PHILIP VELLA6 Department of Pediatrics, Harbor-UCLA Medical Center, University of California, Los Angeles School of Medicine, Torrance, California 90509'; Department of Pediatrics, University of Rochester, Rochester, New York 146272; Connaught Laboratories, Swiftwater, Pennsylvania 183703; National Public Health Institute, Helsinki, Finland4; Lederle Laboratories, Pearl River, New York 109655; and Merck Sharp and Dohme Research Laboratories, West Point,
Pennsylvania 194866 Received 29 May 1987/Accepted 21 September 1987
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 ,ig/ml), the results of ail 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 c 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. influehzae type b immunogenicity studies.
Antibody to the capsular antigen of Haemophilus influtype b (polyribosylribitol phosphate [PRP]) has been measured by a variety of methods to study immunity to disease (6, 17, 26) and to evaluate the immunogenicity of several polysaccharide vaccines (7, 18, 24, 28). Most antiPRP antibody assays use radioactive antigen-binding assay (RABA) methods and principles described by Farr (11). With either intrinsically ([3H]PRP) or extrinsically ([1251]PRP) labeled antigens, antibody binding is measured by adding dilutions of sera to radiolabeled antigen and then separating and counting the bound antigen. The percent binding is calculated, and this is compared to that of a serum standard that has been quantitated by precipitation assay (25). Three different radioimmunoassays have been developed (3, 21, 25), and several modifications of these assays also have been described (12, 15, 18, 23). It has been assumed that antibody levels measured in different laboratories by different methods were similar, because all assays used a U.S. Food and Drug Administration (FDA) serum standard for quantitation of antibody binding. However, marked differences in the immunogenicity of nearly identical polysaccharide vaccines produced by different manufacturers have recently been noted (9, 16). This suggests that the quantitation of antibody
in various laboratories using different assay methods differs significantly (9). A standardized and reproducible antibody assay is essential for comparing the immunogenicity of different H. influenzae type b vaccines in different laboratories and to correlate antibody levels with protection. The purpose of this study was to evaluate potential differences in antibody quantitation among existing RABAs, A major objective of this blinded multicentered collaborative study was to assess the comparability of antibody quantitation between laboratories and to assess the reproducibility of two assays in different laboratories. The iniplidations of these findings for evaluation of H. influenza type b vaccines are discussed. Variables in the assay that may account for some of the observed differences have been evaluated in another study (5).
enzae
*
MATERIALS AND METHODS Study design. Sera for this evaluation were obtained from a study of the immunogenicity of two PRP vaccines that were administered blindly and randomly to 18- and 24month-old children (16). After informed consent was obtained, blood specimens were collected from each subject just before vaccine administration and 4 to 6 weeks postimmunization; all sera were stored at -70°C in coded vials. Samples of pre- and postimmunization sera were selected
Corresponding author. 72
VOL. 26, 1988
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