Establishment and validation of an ELISA for the quantitation of HBsAg in recombinant hepatitis B vaccines.

Commercial enzyme-linked immunosorbent assay (ELISA) kits for the determination of the in vitro potency of recombinant hepatitis B vaccines, which detect hepatitis B surface antigen (HBsAg), have been used frequently as an alternative for traditional in vivo potency tests. With the constant need for validation procedures, an ELISA that could be employed to determine the in vitro potency of five recombinant hepatitis B vaccines simultaneously was established using two monoclonal antibodies. The use of two monoclonal antibodies produced "in house" specific for the small envelope protein S of the hepatitis B virus (HBV) resulted in the production of a highly specific, sensitive and stable ELISA. The standard ELISA parameters used in this study, considering the HBsAg content of each recombinant hepatitis B vaccine evaluated, resulted in a standard curve that could be applied for potency evaluations of different, commercial hepatitis B vaccine lots.

Generation of monoclonal antibodies against human recombinant interferon beta using genetic immunization with simultaneous expression of IgM and IgG isotypes.

Monoclonal antibodies (MAbs) against human recombinant interferon beta (hrIFNbeta) were generated by genetic immunization (GI). In order to test two viral promoters frequently used in mammalian expression plasmid vectors, mice were inoculated four times by intramuscular injection, without adjuvant, with 100 microg of either pcDNA 3.1hrIFNbeta or pZeoSV2IFNbeta containing the entire human interferon beta gene and under the control of, respectively, human cytomegalovirus (HCMV) immediate-early promoter or early SV-40 enhancer/promoter. Only serum samples from mice immunized with pZeoSV2IFNbeta were positive to anti-hrIFNbeta. The spleens of the immunized mice were fused with myeloma Sp2/0 cells and the hybridoma clones generated screened by an in house enzyme-linked immunosorbent assay (ELISA). Fourteen MAbs were selected as reactive with hrIFNbeta. Western blot analysis was performed and only one recognized the 18 kDa isoform (non-glycosylated) of hrIFNbeta. All MAbs were subjected to antibody isotype characterization with a commercial ELISA and showed unusual profile with simultaneous expression of both IgM and IgG2a isotypes. This observation is further supported by RT-PCR amplification of the IgM CH4 domain using total RNA from hybridomas.

Screening of CHO cell clones expressing histidine-tagged major S hepatitis B surface protein using a semi-quantitative PCR protocol.

Different mammalian cells have been used successfully for the expression of the hepatitis B surface antigen (HBsAg). The patterns of expression of the HBsAg seem to depend on the cell type and the number of gene copies integrated into cellular genome. The expression of an HBsAg fused to histidine tag (His-HBsAg), had not been reported in mammalian cells. This paper describes a semi-quantitative polymerase chain reaction (PCR) employed to investigate the patterns of expression of His-HBsAg in stably transfected Chinese hamster ovary (CHO) cell clones. pcDNA4CR20, a mammalian expressing vector, encoding His-HBsAg, was constructed. The correlation between His-HBsAg expression and the number of integrated copies of the HBsAg gene into cell clones genome was evaluated by the semi-quantitative PCR, with limit dilutions of the genomic DNA as template. The results show a positive correlation between the expression levels of His-HBsAg with the number of the HBsAg gene copies integrated into CHO cell clones. The approach of a semi-quantitative PCR proved to be a good and non-expensive alternative strategy to analyze the patterns of expression of integrated genes into CHO cells genome.