Subtyping of hepatitis B antigen by use of monospecific antibody-coated cells.
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Biomedical subjects
Publications and source records attributed to F B Hollinger.
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Dilution of hyperimmune hepatitis type B antibody in undiluted homologous species normal serum has resulted in enhanced sensitivity for detecting hepatitis B antigen by a discontinuous counter-immunoelectrophoresis method compared to hepatitis type B antibody diluted in heterologous species normal serum or buffer solutions.
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.
Hepatitis B antigen of the D (a+, d+, y-) subtype was purified from plasma of apparently healthy persons and from hepatitis patients. The original samples contained 20- and 42-nm particles and tubular forms (20-nm diameter). Ultracentrifugation during the purification procedure yielded pellets which were then treated at pH 2.4. Both the large, 42-nm Dane particles and the tubular forms were lost during the acid treatment of the pelleted particles, yielding a preparation containing a mixture of particles approximately 20 and 25 nm in diameter. This difference in size was substantiated in that two distinct molecular weights were calculated from high-speed equilibrium data, 3.6 x 10(6) and 4.5 x 10(6). Further heterogeneity was observed in that hepatitis B antigenic activity was present in purified particles with an isoelectric pH of 4.0 and also in those with a pH of 4.4. No significant differences were observed in the gross amino acid composition of purified antigen obtained from plasma of three different persons. (125)I-labeled, purified antigen was found to contain six distinct polypeptides with molecular weights ranging from 10,000 to 39,000.
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A solid-phase, noncompetitive radioimmunoassay has been developed for aldolase B in human serum and tissues. Aldolase B was purified from human liver, and specific antisera to purified aldolase B were obtained from chickens. Specific antihuman aldolase B IgG was purified by affinity chromatography. Disposable polypropylene plates were coated with affinity purified specific IgG antibody and used for radioimmunoassay with 125I-specific IgG antibody to aldolase B. The nonspecific binding was minimized by saturating the binding sites of the plates with 2% ovalbumin in 0.1% Tween 20. This radioimmunoassay is specific for the aldolase B subunit, with no cross-reactivity with human aldolase A or aldolase C subunits. Aldolase B is predominantly found in normal liver. Relatively high aldolase B levels are also observed in kidney. Serum levels of aldolase B in 21 normal subjects ranged from 21 to 39 ng per ml, with a mean of 28.7 +/- 8.6 (2 S.D.) ng per ml. Forty of 42 (95%) patients with acute and chronic hepatitis without cirrhosis had serum aldolase B levels greater than 40 ng per ml. Serum aldolase B levels correlated well with total serum aldolase enzyme activities (r = 0.967) and SGPT (r = 0.951) in patients with liver diseases. In cancer patients, serum aldolase B was slightly elevated in 15 of 26 (58%) patients with cancer metastatic to the liver or primary liver cell carcinoma, whereas no elevation of serum aldolase B was observed in 16 cancer patients without liver metastasis. Measurements of aldolase B serum levels by radioimmunoassay appear to be a useful measure of liver cell necrosis from benign or malignant liver diseases.
An IgM-specific anti-[anti-HBs] antibody was detected by radioimmunoassay using anti-IgM-coated beads and 125I-labeled anti-HBs. This antiidiotype was found only in the sera of hepatitis B virus-infected patients, both acute and chronic. However, not all HBsAg-positive patients exhibited this reaction, and activity was correlated with the presence of HBeAg. Approximately 93% of sera that contained antiidiotype activity also contained HBeAg. Conversely, 70% of the sera positive for HBeAg reacted in the IgM assay. No correlation was observed between the presence of antiidiotype and rheumatoid factor or elevated SGPT levels. Two approaches were used to determine whether the reactive moiety was an IgM anti-[anti-HBs] as postulated or an IgM anti-HBs/HBsAg complex. It was shown that chicken anti-HBs sera, which does not share the common idiotype of human and other mammalian anti-HBs, did not block a positive reaction in this radioimmunoassay even though it specifically bound HBsAg. It was also demonstrated that treatment with polyethylene glycol, which will precipitate IgM anti-HBs/HBsAg activity, did not precipitate the reactive moiety in 6 of 7 sera tested, lending further evidence to the existence of an IgM antiidiotype in these patients. It is suggested that this antiidiotype directed against anti-HBs may be involved in a defective feedback mechanism resulting in the suppression of production of anti-HBs and maintenance of the carrier state.
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