Development and technical and clinical validation of a quantitative enzyme-linked immunosorbent assay for the detection of human antibodies to hepatitis B surface antigen in recipients of recombinant hepatitis B virus vaccine

Abstract

Pending removal from the market of a commercial assay (the AUSAB [Abbott Laboratories] enzyme immunoassay [EIA]) for the determination of antibodies to hepatitis B surface antigen (HBsAg), a new in-house quantitative enzyme-linked immunosorbent assay (ELISA) to measure antibodies against HBsAg (anti-HBs) was developed (anti-HBs in-house). Specific anti-HBs antibodies were sandwiched between the precoated HBsAg ad and ay subtypes purified from plasma from hepatitis B virus (HBV) human carriers and the recombinant HBsAg adw2 subtype tagged with horseradish peroxidase. The assay was calibrated against the 1st International Reference Preparation for anti-hepatitis B immunoglobulin (lot 1977-W1042). Analytical sensitivity and the limit of quantitation were estimated at 0.43 mIU/ml and 2.0 mIU/ml, respectively. Overall reproducibility was 11.86%, and accuracy was estimated to be 94.89%. More than 4,000 samples from seven clinical trials were tested with the anti-HBs in-house assay and compared to results generated with AUSAB EIA and AUSAB radioimmunoassay (RIA). During the technical validation, the anti-HBs in-house assay was compared to the AUSAB RIA as a reference (n = 919). Overall assessment of concordance and Deming's regression analysis were performed. The coefficient of correlation between the AUSAB RIA and anti-HBs in-house assay was 0.9815 with a slope of 0.9187. The overall agreement between anti-HBs in-house and AUSAB RIA was 97.61%, considering the clinical cutoffs at 3.3 mIU/ml and 1.0 mIU/ml for the respective assays. From a clinical perspective, seroprotection rates and anti-HBs geometric mean antibody concentrations for individual studies calculated with either the in-house assay or the reference assays were similar. Conclusions of individual studies were confirmed. The performance characteristics of the in-house assay are acceptable. There is no evidence that use of the new assay would lead to different clinical conclusions from the reference method.

FIG. 1.

Linear regression between FBS and human-plasma anti-HBs free as diluent. Hum-plasma, human plasma.

FIG. 2.

Observed values and the model predictions for linearity assessment. For groups 1 and 4, the prediction line for each sample is superimposed with the prediction line for the mean sample within each group.

FIG. 3.

OD response of serial dilutions of three highly concentrated anti-HBs samples.

FIG. 4.

Deming's regression of the anti-HBs in-house assay and AUSAB RIA with CVs of 11.86% for both the RIA and anti-HBs in-house assay. UL, upper limit; LL, lower limit.

FIG. 5.

GMCs (mIU/ml) at each blood sampling time point in study A (elderly patients), study E (adolescents), and study F (infants) for the anti-HBs in-house assay versus reference assays. PI to PIV repesent postdose months 1 to 4, respectively, and m1, m2, etc., represent study months 1, 2, etc.