A new reversed passive hemagglutination test for HBsAg, termed Raphadex
B, has been developed using immunochemically purified chimpanzee anti-HBs bound
to stabilized human erythrocytes. The test has been found to have equivalent sensitivity
to the Ausria 125 I radioimmunoassay, and detected a similar number of HBsAg-containing
specimens in screening of volunteer blood donors. This method offers an economical
approach to third generation methodology for hepatitis B screening of blood donors.
HBsAg/adw was purified from 2.6 liters of pooled plasma from a single chimpanzee carrier by polyethylene glycol (PEG) precipitation followed by isopycnic and rate zonal centrifugation. The different morphological populatilons of HBsAg separated in the final rate zonal centrifugation step were combined into seven pools: two fractions rich in filaments and Dane particles, two pools composed of filaments and 20--28-nm spheres, and three fractions containing mostly 20--28-nm spheres. The purified preparations of HBsAg analyzed for normal serum protein contaminants revealed albumin and traces of IgG. The same samples analyzed after Tween-80 treatment, revealed enhanced quantitites of the previous two contaminants, and in addition, transferrin, traces of alpha2-macroglobulin, IgM, and complement (C3/C3c). The residual contaminants were mostly removed by further purification and fractionation after detergent treatment using zone convection electrofocusing and rate zonal centrifugation. Our findings indicate that conventional purification techniques will not provide preparations of HBsAg free of traces of serum protein contaminants. Many of these are released only by detergent treatments and subsequent purification. It is not yet clear whether detergents release these contaminants from the interior of the particles or from firm association or incorporation within the membranes.
When highly purified HBsAg particles, separated by rate zonal centrifugation into populations differing in predominant size, were tested for HBeAg, the e1 specificity was detected preferentially in association with particle fractions containing large filaments and Dane particles. These results were obtained both by agar gel diffusion and by radioimmunoassay for e antigen. The e antigen activity present in these fractions was potentiated by prior treatment of particles with Tween 80, suggesting cryptic localization of e1 specificity within or under the outer membrane. The HBeAg released by detergent treatment from a purified preparation composed predominantly of small-particle forms of HBsAg was separated by electrofocusing into a peak of nonparticulate e antigen in the pH range of 5.7--6.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major polypeptides in this preparation with approximate molecular weights of 25,000, 55,000, and 70,000. Furthermore, two additional peaks of e antigen activity were detected which migrated in association with HBsAg particles at isoelectric points of 4.4 and 5.5--5.6. The major portion of e antigen remained in association with particles after further purification by rate zonal centrifugation.
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