Table 1 Serum samples with HBsAg-positive carriers and liver disease patients Fig. 1 Preparation of purified HBsAg particles Fig. 2 Methods for analys

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2 Table 1 Serum samples with HBsAg-positive carriers and liver disease patients Fig. 1 Preparation of purified HBsAg particles Fig. 2 Methods for analysis of HBV-associated particles

Table 2 Analysis of HBV-peptides in sera from HBsAg-positive carriers and liver dis ease patients a) % Concentration of protein of each peptide to 100% of total HBV-protein Fig.

4 Table 2 Analysis of HBV-peptides in sera from HBsAg-positive carriers and liver dis ease patients a) % Concentration of protein of each peptide to 100% of total HBV-protein Fig. 3 Patterns of SDS-PAGE of case 1 and case 11. Left showed marker proteins of 94Kd to 14.4Kd. Right showed sam ple. Silver staining was per formed. Fig. 3-a SDS-PAGE of HBsAg particles from case 1 Fig. 3-b SDS-PAGE of HBsAg particles from case 11

Fig. 4 Densitometry patterns of HBV-peptides from case 1 and case 11 Table 3 Western blotting pattern of HBV - peptides by HRPO-pHSA Table 4 Western blotting pattern of

5 Fig. 4 Densitometry patterns of HBV-peptides from case 1 and case 11 Table 3 Western blotting pattern of HBV - peptides by HRPO-pHSA Table 4 Western blotting pattern of HBV - peptides by HRPO-anti-HBs Fig. 5 Western-blotting pattern of peptides from case 5. Left: marker protein, Mid dle: stained by HRPO-anti-HBs, Right: stained by HRPO-pHSA

Table 5 IgG, IgA, IgM, L chain (K), L chain(l), C1q, C4 and C3a)on various peptides prepared from purified HBsAg par ticles a) Each antibody (Behringwerke AG) was used for detections of determinants

6 Table 5 IgG, IgA, IgM, L chain (K), L chain(l), C1q, C4 and C3a)on various peptides prepared from purified HBsAg par ticles a) Each antibody (Behringwerke AG) was used for detections of determinants of immunoglobulins and complements. b) Case shows Kd of molecular weights of peptides after SDS-PAGE. Fig. 6 Western-blotting pattern of HBV-peptides from case 1. From left, marker protein, IgG determinant, IgA determinant, IgM deter minant, L chain(k) determinant and L chain(l) determinant were shown. (Fig. 6-a) From left, marker protein, C1q, C4 and C3 determinant were shown. (Fig. 6-b)

9 1. Machida A, Kishimoto S, Ohnuma H, Miyamoto H, Baba K, Nakamura T and Miyakawa Y: A hepa titis B surface antigen polypeptide (P31) with the receptor for polymerized human as well as chimpan zee albumins. Gastroenterology (1983) 85, Machida A, Kishimoto S, Ohnuma H, Baba K, Ito Y, Miyamoto H, Funatsu G, Oda K, Usuda S, Togami S, Nakamura T, Miyakawa Y and Mayumi M: A polypeptide containing 55 amino acid re sidues coded by the pre-s region of heptitis B virus deoxyribonucleic acid bears the receptor for polymerized human as well as chimpanzee albumins. Gastroenterology (1984) 86, Okamoto H, Imai M, Usuda S, Tanaka E, Tachibana K, Mishiro S, Machida A, Nakamura T, Miyakawa Y and Mayumi M: Hemoagglutination assay of polypeptide coded by the pre-s region of hepatitis B virus DNA with monoclonal antibody: Correlation of pre-s polypeptide with the receptor for polymerized human serum albumin in serums containing hepatitis B antigens. J Immunol (1985) 134, Magnius LO and Espmark JA: New specificities in Australian antigen positive sera distinct from the Le Bouvier determinants. J Immunol (1972) 109, Stevens CE, Neurath AR, Szmuness W, Beasley RP and Ikram H: Correlations between HBe Ag/ anti-hbe, HBsAg titer, HBs Ag-associated albumin binding sites and Infectivity; Viral Hepatitis, Vyas, Cohen, Schmid eds, Franklin Institute Press, Philadelphia (1978) pp Pontisso P, Alberti A, Bortolotti F and Realdi G: Virus-associated receptors for polymerized human serum albumin in acute and in chronic hepatitis B virus infection. Gastroenterology (1983) 84,

10 7. Tsuji T, Yamamoto I, Tsuchiya M and Nagashima H: Enzyme-linked immunosorbent assay for detec tion of receptors for polymerized human serum albumin on hepatitis B surface antigen particles. J Im munol Methods (1984) 73, Pharmacia Japan K. K.: Manuals of affinity chromatography of Pharmacia. Pharmacia Japan K. K., Tokyo (1982) pp Laemmli UK: Cleavage of structual proteins during the assembly of the head of bacteriophage T4. Na ture (1970) 227, Towbin H, Staehelin T and Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA (1979) 76, Sanchez Y, Ionescu-Matiu I, Hollinger FB, Melnick JL and Dreesman GR: Antigenic relationship of a hepatitis B surface antigen-derived polypeptide and human serum albumin. J Gen Virol (1980) 48, Gold JWM, Shin JWK, Purcell RH and Gerin JL: Characterization of antibodies to the structural polypeptides of HBsAg: Evidence for subtype-specific determinants. J Immunol (1976) 117, Ohnuma H, Takahashi K, Kishimoto S, Machida A, Imai M, Mishiro S, Usuda S, Oda K, Naka mura T, Miyakawa Y and Mayumi M: Large hepatitis B surface antigen polypeptides of Dane parti cles with the receptor for polymerized human serum albumin. Gastroenterology (1986) 90, Tsuji T, Takahashi K, Naito K and Nagashima H: Detection of serum blocking factors and antibodies to the albumin receptor on HBsAg particles in healthy persons and patients with liver diseases. Acta Med Okayama (1984) 38, Schifferli JA, Bartolotti SR and Peters DK: Inhibition of immune precipitation by complement. Clin Exp Immunol (1980) 42, Hongo K, Tanaka Y, Sayama K, Kozono H, Takeda J, Nakano Y, Kinoshita T and Inoue K: Inhibi tion of immune precipitation by complement. J Immunol (1984) 133,

Immunochemical study of constituent peptides of HBs-antigen particles in sera from hepatitis B patients Masahiko SAWAHARA The First Department of Internal Medicine, Okayama University Medical School,

11 Immunochemical study of constituent peptides of HBs-antigen particles in sera from hepatitis B patients Masahiko SAWAHARA The First Department of Internal Medicine, Okayama University Medical School, Okayama 700, Japan (Director: Prof. H. Nagashima) To examine the characteristics of hepatitis B virus (HBV) in sera from hepatitis B patients in various stages of disease, the protein volume percent of each constituent peptide of HBV was investigated, and both the HBV-pAR activity and HBsAg activity of each peptide were examined. The relationship between HBV, immunoglobulins and complements was also investigated. The constituent peptides were divided into a few bands of more than 94Kdal., 3 bands of 94 `67Kdal. and 12 bands of 67, 52, 49, 43, 39, 35, 31, 27, 22, 19, 15 and 8Kdal.. The samples of HBeAg positive sera showed increased 22, 27, 31, 35 and 67Kdal. bands, and the samples of anti-hbe positive sera showed de creased 22 and 27Kdal. bands and increased 15 and 43Kdal. bands. The samples of sera from asymptomatic carriers showed an increased 43Kdal. band, and that from patients with chronic active hepatitis showed an increased 15Kdal. band. HBV-pAR consisted of 67, 52, 43, 35, 31 and 8Kdal. peptides, and the determinants of HBsAg consisted of 67, 52, 49, 43, 39, 35, 31, 27 and 22Kdal. peptides. HBV-peptides of 67, 52 and 22Kdal. bound with immunoglobulins and complements in Western-blotting.

Fig.1 Procedures of the spot tests for the detection of serum HBV-DNA

Key words: HBV DNA, HBV carrier, HBeAg Fig.1 Procedures of the spot tests for the detection of serum HBV-DNA 昭和61年2月20日 87 Table 1 Comparison of HBeAg/anti-HBe Fig.2 Comparison of the sensitivity of the