Hydrogen peroxide (Merck, India)-activated chromogen (3,3,5,5-tetramethylbenzidine; Sigma) was added to all wells

Hydrogen peroxide (Merck, India)-activated chromogen (3,3,5,5-tetramethylbenzidine; Sigma) was added to all wells. were comparable to thein vivovalues when compared with the estimates of AxSYM. The IC-ELISA can therefore be considered to be a reliable test for derivingin vitrorelative potency and antigen concentration in vaccine batches for batch control and release. Hepatitis B is usually a major global health problem caused by hepatitis B computer virus (HBV), and the disease is usually characterized by the most serious type of viral hepatitis resulting in cirrhosis and hepatocellular carcinoma. Worldwide, an estimated 2 billion people have been infected with HBV, and more than 350 million have chronic liver infections (26). HBV has a double-stranded DNA (dsDNA) 3.2 kb in size with four reading frames encoding several overlapping viral proteins, including pre-S1, S2, S, core, HBe, X, and polymerase proteins. Hepatitis B envelope protein or surface antigen (HBsAg) is composed of three related envelope proteins covalently linked together. HBV contamination can be prevented and controlled by prophylactic vaccination. The earlier generation of vaccine for immunization programs was prepared from human plasma-derived antigen. With the introduction of recombinant DNA technology and state of the art expression platforms, HBsAg subunit vaccines were made available for both adult and pediatric use worldwide (20). Thein vivopotency of hepatitis B vaccine as a part of quality control is usually evaluated in laboratory animals, which is correlated with the results of the vaccine clinical trials (25). In addition to this, relative potency is also assessed for each vaccine batchin vitroby immunoassays which have been validated using parallel-line assays. The Gatifloxacin disadvantages of thein vivopotency screening are the inherent variation in results and cost of the animal experiments (11). In addition, the antigenic complexity of HBsAg complicates the evaluation, as estimations of at least 50% seroconversion against HBsAg vary depending upon the subtype of the antigen used in the test (24). HBsAg contains the common immunodominant a determinant shared by all serotypes and genotypes of HBV and two sets of mutually unique subtype determinants designated d/y and w/r, resulting in four major subtypes of HBsAg: adw, adr, ayw, and ayr (5,18). Alteration of residues in the a determinant can result in reduced antigenicity and reduced levels of protein expression (13). Measurement of anti-a antibodies rather than the antibodies to total HBsAg is usually believed to be a true indicator of the immunity against HBsAg in the vaccinated subjects (14). Enzyme-linked immunosorbent assay (ELISA)-based methods have been developed for the quantification of group-specific a antigen in monovalent hepatitis B vaccine (27) and combination vaccines (10). Commercial ELISA kits, especially the Auszyme kit developed by Abbott Laboratories, Gatifloxacin have long been used widely for quantifying HBsAg content. The manufacturer has discontinued the kit and replaced it with an expensive method for automated analysisin vitro. Subsequent to this, an alternativein vitropotency method based on an inhibition ELISA for evaluation of Gatifloxacin vaccines containing HBsAg has been reported (4). Development of similar in-house ELISA-based procedures for assessing the vaccine’s potencyin vitrowould render the quality assessment of vaccines including the batch release economical and avoid the need for relying on expensive kits and gear. Monoclonal antibodies (MAbs) play a pivotal role in developing rapid and sensitive Gatifloxacin ELISA-based methods for antigen and antibody detection, quantification, and characterization of antigen in vaccine research and development (3).In vitroproduction of MAbs has CAPN2 become simpler and inexpensive without having to use laboratory animals for large-scale production. The polyclonal antibodies of immune human and animal origins can be replaced with the HBsAg-specific MAbs for development of highly sensitive and specific ELISAs. In the present study, we report the development of HBsAg determinant a-specific MAbs and the use of one such MAb for the development of an immunocapture ELISA (IC-ELISA) for assessment of thein vitropotency of hepatitis B vaccine formulations. == MATERIALS AND METHODS == == Hepatitis B purified antigen (HBsAg) and reference standard. == Recombinant HBsAg (subtype adw2) expressed inPichia pastorisobtained from the production department of the Human Biologicals Institute (HBI), Hyderabad, India, was used as a capture antigen for screening the hybridomas. Elovac-B vaccine containing 20 g of HBsAg obtained from HBI, Hyderabad, was used for hyperimmunization of BALB/c mice to develop hybridomas secreting MAbs and also as the.