Balb/c mice were immunized with aluminium hydroxide [alum, Al (OH)3]-adjuvanted hepatitis B (HB) vaccines of subtypes adr, ayw or adw. Spleen cells from the immune animals were fused with SP2/O cells. Eight hybridoma clones producing antibodies specific for HB surface antigen (HBsAg) were selected. Monoclonal antibodies (mAbs) of four clones were specific for group-specific antigen/a, and the other of four clones were specific for subtype antigen/d, y, r, or w. The anti-HBs/a mAbs were classified into three non-competitive groups. Quantitation of group-specific determinant a of HBsAg (HBsAg/a) was performed by sandwich enzyme-linked immunosorbent assay (ELISA), in which a solid phase of anti-HBs guinea-pig polyclonal antibodies (pAb), the HBsAg for testing, anti-HBs/a mouse mAb and horseradish peroxidase (HRP)-conjugated anti-mouse IgG were used. The unadsorbed HBsAg was used to establish the standard curve of HBsAg/a. The lower detection limits were 0.5 to 1 ng/ml of HBsAg. Methods of solubilization of alum were investigated to quantify HBsAg/a in adsorbed HB vaccines. The recovery rate was more than 60% if vaccines were prediluted. The recovery of HBsAg/a in HB vaccines produced by the same manufacturer showed the similar recovery rate, and the contents of HBsAg/a in adsorbed HB vaccines could be estimated by the recovery rate determined for adsorbed HB vaccines.
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