Production of Potent Anti-Australia Antigen Sera of High Specificity and Sensitivity in Goats

Abstract

Potent antisera of high specificity and sensitivity were produced, in goats, to purified Australia antigen (Au). The antigen was prepared by one of three methods: (i) pelleting, low pH treatment, isopycnic centrifugation two times in CsCl, and rate zonal centrifugation in sucrose; (ii) same as procedure i, with the exception of the low pH treatment; or (iii) twice banding in CsCl by using a BXIV batch-type zonal centrifuge rotor with subsequent preparative Pevikon electrophoresis. The goat anti-Au sera contained high levels of precipitating antibody as tested by immunodiffusion in agar gel and discontinuous counterimmunoelectrophoresis (DCIE) as well as specific complement-fixing antibody and could be used for routine screening of sera for Au without prior adsorption with Au-negative normal human serum (NHS). Identification of 66 of 70 positive specimens (94.3%) in a panel of 98 coded sera (49 duplicates) with 100% reproducibility was made by using one of the goat anti-Au sera at a dilution of 1:16 in the DCIE method. No false positives were recorded. Low levels of antibody against NHS components were effectively removed by a single adsorption with glutaraldehyde cross-linked NHS.

Biophysical and Biochemical Heterogeneity of Purified Hepatitis B Antigen

“…Electron microscopy examination of negatively stained preparations consistently showed only the presence of particles measuring 17 to 27 nm in diameter. Similar preparations were described in our earlier report (9). Each preparation contained from 1013 to 1014 particles per ml and 70 to 200 MAg of protein per ml.…”

“…Plasma obtained from three anicteric hepatitis patients, with positive HB Ag complement-fixing (CF) titers of 1: 2560 or greater, were processed to yield purified HB Ag particles according to procedure I of Dreesman et al. (9). Briefly, 90 to 120 ml of plasma was pelleted by high-speed centrifugation, treated at pH 2.4 at room temperature for 1 hr, repelleted, and banded twice on isopycnic CsCl gradients, followed by a rate zonal centrifugation in a preformed linear sucrose gradient.…”

“…Blood was obtained by cardiac puncture on day 35. Each serum was examined for the presence of antibody to both HB Ag and to NHS by gel diffusion and by twodimensional CF titrations similar to that described previously (9). None of the preparations elicited an antibody response to NHS proteins.…”

Interaction Between Hepatitis B Surface Proteins and Monomeric Human Serum Albumin

“…The fact that pHSA binding to HBsAg requires chemical cross-linking by glutaraldehyde (16,17) but not by other reagents could mean that the phenomenon is an in uitro artifact. Moreover, there is disagreement in the literature (3,18) as to which HBs protein the pHSA is bound. The hypothetical binding of human serum albumin or pHSA to HBsAg has also been implied in models of how immune tolerance to HBsAg or immunopathogenesis of HBV could be induced (19).…”

Further Characterization of Internal Image‐Bearing Anti‐Idiotypic Antibodies: Specific Binding to Immunoglobulin Receptors on Murine Hybridoma Cells Secreting Antibodies to Hepatitis B Surface Antigen.

“…Isolation and purification of HBsAg. Purification of 22-nm HBsAg particles was performed as previously described [9], Chimpanzee plasma positive for HBsAg was ktndly provided by Dr Jorg Eiehberg (this department). The concentration of HBsAg was determined using an extinction coefficient of 37,26 for a 1% preparation at 281) nm.…”