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

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.Hepatitis B infection is a global health problem but most acutely affects developing countries (16). Currently there is no effective therapy against hepatitis B, whose disease spectrum ranges from asymptomatic disease to chronic liver diseases, including cirrhosis and hepatocellular carcinoma. Prevention of the illness through vaccination remains the method of choice for its control and eradication. Active immunization against hepatitis B infection can be achieved using vaccines containing either inactivated hepatitis B virus (HBV) surface protein (HBsAg) physicochemically purified from plasma from HBV human carriers or recombinant surface antigen produced by transfer of the S gene of HBV coding for HBsAg to an appropriate plasmid that is then inserted into the desired expression vector. The recombinant vaccine manufactured by GlaxoSmithKline Biologicals (GSK) is produced in yeast and is antigenically similar to plasma-derived HBsAg (4). Clinical studies of this recombinant vaccine either formulated as a single-component vaccine (Engerix-B; trademark of GSK) or formulated in combination with other antigens such as hepatitis A vaccine (Twinrix; trademark of GSK) or pediatric diphtheria-tetanus-pertussis-based vaccines (such as Infanrix hexa and Tritanrix HepB; trademarks of GSK) have proven its efficacy and immunogenicity (5).To date, commercial assays from Abbott Laboratories have been used at GSK to quantify the immune response to HBV vaccines in terms of antibodies against HBsAg (anti-HBs). However, since these assays are no longer commercially available in Europe, GSK has developed an in-house assay with adequate technical and clinical performance to ensure long-term supply of an assay with consistent quality. This paper describes the development, technical validation, and the clinical assessment of the new anti-HBs in-house assay.