A three-step solid-phase radioimmunoassay, HAVAB-M, was developed for use in clinical labs as an aid to diagnosing hepatitis A. Polystyrene beads were coated with anti-human IgM (mu-chain specific) and were incubated successively with serum specimen, HAV, and anti-HAV 125I. HAVAB-M was used to assay sera from patients with hepatitis A and was found to have high sensitivity for the IgM antibody to HAV. The antibody was detectable within a few days of onset of symptoms of hepatitis, and it reached maximum concentrations within one to three weeks. The test was designed so that most patients' sera would become negative approximately three months after onset. HAVAB-M was shown to be specific for IgM antibody, with virtually no detection of IgG anti-HAV. The test showed no interference fro rheumatoid factor and no cross-reactivity with sera from patients with hepatitis B or other infectious diseases.

The total carbohydrate content of purified hepatitis B antigen (HB Ag) ranged between 3.6 and 6.5% as determined by the phenol-sulfuric acid method. Disc-sodium dodecyl sulfate PAGE of purified HB Ag followed by periodate-Schiff staining revealed one major and two minor glycoproteins with molecular weights of 22,000,27,000 and 32,000 daltons, respectively. Both ad and ay subtypes of HB Ag contained these glycoproteins.

The morphological forms of hepatitis B antigen (HBsAg) present in an HBsAg-positive plasma (15–25 nm spheres, filaments 20–22 nm in diameter and 80–300 nm in length, and 42 nm Dane particles) were isolated and characterized biophysically. The HBsAg in this plasma was fractionated into five distinct populations, containing predominately 15–19 nm spheres, 20–22 nm spheres, 20 × 300 nm filaments, a mixture containing 23–25 nm spheres and rare filaments, and 42 nm Dane particles, respectively. These populations were labeled with 125I and electrofocused in a pH ampholyte gradient. Each population had a distinct isoelectric pH with values ranging from 3.65 to 4.70. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of each of the above labeled populations revealed the presence of 7, 10, 9, 5, and 9 polypeptides, respectively. The population containing predominately 23–25 nm spheres failed to react with antibody specific for HBsAg and probably was composed of inner cores of the Dane particles.

Monolayers of pancreatic p-cells from strains of mice susceptible @IL/J) and resistant (C57BL/&l) to the development of virus-induced diabetes mellitus were inoculated with the M variant of encephalomyocarditis (EMC) virus. Immunofluorescence showed that viral antigens appeared in up to 10 times more /3 cells from susceptible &IL/J mice than from resistant C57BLKJ mice. Infectious center assays revealed that lo-30 times more &IL/J /I cells contained infectious virus than C57BLFJ p cells. Viral attachment experiments showed no difference in the binding of EMC virus when embryonic fibroblasts, pancreatic fibroblasts, and kidney cells from SJLlJ and C57BL/&J mice were compared. However, at least twice as much virus attached to the pancreatic /3 cells from susceptible than from resistant strains of mice. Our data suggest that genetically determined differences in viral receptors on the surface of fi cells may be one of the factors controlling susceptibility to EMC-induced diabetes mellitus. 606

Comparative biophysical and biochemical analyses were performed on purified preparations of hepatitis B antigen (HBS Ag) subtypes adw and ayw, including isoelectric pH evaluations, analysis of the different morphological forms, molecular weight determinations, and analysis of the polypeptides by polyacrylamide gel electrophoresis. Both HB8 Ag-positive plasma and purified HB8 Ag were analyzed by electrofocusing in a sucrose ampholyte gradient. Four distinct populations of HB. Ag with a pH range of 4.5 0.1 to 5.4 + 0.1 for unfractionated plasma samples and 3.9 + 0.05 to 4.9 + 0.05 for purified samples were detected in both adw and ayw preparations. Electron microscopic studies of each population of purified HB. Ag revealed 19to 27-nm spheres in each fraction. Purified material labeled with "'I by the chloramine-T method behaved as one major vpopulation with an isoelectric pH value of 3.9 0.1. Purified adw preparations revealed a major population with a molecular weight of 3.7 x 106 and a second one of 4.6 x 106. Purified preparations of ayw contained one population with a molecular weight of 4.6 x 106. Polyacrylamide gel electrophoretic analysis of purified HB6 Ag revealed nine polypeptides for ayw and seven for adw particles. These studies indicate that purified preparations of HB6 Ag are heterogeneous and that distinct differences can be detected between the two subtypes.

Purified preparations of hepatitis B surface antigen (HB. Ag) were solubilized with sodium dodecyl sulfate and urea under reducing conditions and subsequently fractionated by preparative sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (PAGE).

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