Sadakazu Usuda

Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product.

An ELISA was developed for serological determination of the six genotypes of hepatitis B virus (HBV) designated A, B, C, D, E, and F. Monoclonal antibodies were raised against genotype-specific epitopes in the preS2-region product, and labeled with horseradish peroxidase. Hepatitis B surface antigen (HBsAg) in sera was captured by immobilized antibodies against the common determinant, and evaluated for reactivity with genotype-specific monoclonal antibodies labeled with the enzyme. Serological genotyping was in complete accord with genotypes determined by S-gene sequences in a panel of 68 sera containing HBV/HBsAg of different genotypes. Of 514 sera with HBsAg from Japan, 507 (98.6%) were genotyped serologically: genotype A was identified in 24 (4.7%); B in 196 (38.1%); C in 282 (54.9%); D in 2 (0.4%); and F in 3 (0.6%). There were no sera containing HBV of genotype E. Likewise, 425 of 446 (95.3%) sera with HBsAg from Brazil, China, India, Indonesia, Kenya, Korea, Nepal, Papua New Guinea, the Philippines, and Thailand were classified into A (25.6%), B (24.2%), C (33.9%), and D (11.7%) genotypes; there were no sera with HBsAg of genotype E or F among them. Some sera unclassifiable by ELISA revealed mixed infection with HBV of distinct genotypes, or contained HBsAg deprived of genotype-specific epitopes by point mutations. The ELISA would be useful for large-scale surveys, because it allows serological detection of HBV genotypes without sequencing nucleotides.

Determination of hepatitis B virus genotype G by polymerase chain reaction with hemi-nested primers

Hepatitis B virus (HBV) has been classified into six genotypes designated A-F by sequence divergence in the entire genome exceeding 8%. Very recently, the seventh genotype was reported and named genotype G. HBV genotype G is distinct from genomes of the other six genotypes in that it possesses an insertion of 36 nucleotides in the core gene, and has been found so far in France and the United States. A method for determining HBV genotype G was developed by polymerase chain reaction (PCR) with primers deduced from the 36-nucleotide (nt) insertion in five isolates of HBV genotype G the sequences of which have been deposited in DNA databases. The validity of this method, for specifically detecting HBV genotype G, was verified on a panel consisting of 142 HBV isolates of six major genotypes and four of genotype G. A total of 540 sera containing HBV in Japan covering symptom free carriers and patients with a spectrum of chronic liver disease were tested by this method, but not a single HBV genotype G sample was found. A possible method for serological determination of hepatitis B surface antigen of genotype G is suggested, without amplification or sequencing nucleotides, which would expand epidemiological and clinical researches on HBV genotype G.

Frequent coinfection with hepatitis B virus strains of distinct genotypes detected by hybridization with type-specific probes immobilized on a solid-phase support

A genotype-specific probes assay (GSPA) was developed for distinguishing the seven genotypes (A-G) of hepatitis B virus (HBV). Nucleotide (nt) sequences corresponding to preS1 region were amplified by PCR with a primer labeled with biotin, and delivered to eight wells on which complementary sequences specific to one or other genotype had been immobilized. Thereafter, hybridization of HBV DNA sequences amplified from the test serum was detected by colorimetry. When 256 sera from HBV carriers in Bangladesh, Cameroon, Japan, South Africa, USA and Uzbekistan were subjected to GSPA, genotypes were concordant with those of ELISA with monoclonal antibodies to epitopes on preS2-region products in 242 (94.6%) of them; 8 sera (3.1%) were not genotypeable by either method. Cloning analysis confirmed the presence of two distinct HBV genotypes in the seven selected sera with coinfection. There were 7 (2.7%) sera with discordant genotyping results between GSPA and ELISA. When HBV DNA clones propagated from these sera were sequenced and analyzed phylogenetically, the genotypes determined by GSPA were verified. Coinfection with HBV strains of two distinct genotypes was identified by GSPA in 28 (10.9%) sera, while it was suggested by ELISA in only 2 (0.8%) sera. The GSPA method would be particularly useful for detecting the coinfection with distinct HBV genotypes of any clinical relevance, which seems to be more frequent than reported previously.

A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies

Monoclonal antibodies were raised against the common (a) as well as subtypic determinants (d, y, w and r) of hepatitis B surface antigen (HBsAg). They were applied to subtyping HBsAg by sandwiching it between antibody against a fixed on a solid-phase support and antibody against one or other of d, y, w and r, linked to horseradish peroxidase. The assay was applied to evaluate antigenic specificities of the NIH and Japanese panels composed of 44 sera containing HBsAg particles of various subtypes. HBsAg particles of a hybrid subtype, adyr, were sandwiched between monoclonal antibody against d and that against y, thereby indicating that they possessed both d and y determinants on the selfsame particle. The expression of d and y determinants on hybrid HBsAg particles was much less than that on ordinary particles of adw, adr, ayw or ayr subtype.

A solid-phase enzyme immunoassay for the determination of IgM and IgG antibodies against translation products of pre-S1 and pre-S2 regions of hepatitis B virus

The envelope of hepatitis B virus is coded for by pre-S1, pre-S2 regions and the S gene. A method was developed to determine antibody to the product of pre-S1 region (anti-pre-S1) and antibody to the product of pre-S2 region (anti-pre-S2), either of IgM or IgG class, by a solid-phase enzyme immunoassay. For the determination of anti-pre-S1, tubular particles containing translation products of pre-S1, pre-S2 regions and the S gene were broken into constituent envelope polypeptides and immobilized on a solid support. Serums were absorbed with spherical particles containing translation products of pre-S2 region and the S gene, obtained from plasma positive for hepatitis B e antigen (HBeAg) and deprived of particles carrying pre-S1 product by an affinity column. They were then tested for the binding with tubular polypeptides fixed on a solid support, and the bound antibody representing anti-pre-S1 was detected by monoclonal antibody to human IgM/mu or IgG/gamma labeled with horseradish peroxidase. For the determination of anti-pre-S2, test serums were absorbed with spherical particles containing the product of the S gene, obtained from plasma positive for antibody to HBeAg and deprived of particles bearing pre-S2 product by an affinity column. They were then tested for the binding with polypeptides, fixed on a solid support, composed of products of pre-S2 region and the S gene. The assay was applied to the determination of anti-pre-S1 and anti-pre-S2 of IgM or IgG class in asymptomatic carriers and in persons who had recovered from infection with hepatitis B virus.

Large Hepatitis B Surface Antigen Polypeptides of Dane Particles With the Receptor for Polymerized Human Serum Albumin

Large hepatitis B surface antigen polypeptides with apparent molecular sizes of 39,000 and 43,000 daltons (P39 and P43) were liberated from a purified preparation of Dane particles of subtype adr. They were tested for reactivity with monoclonal antibodies raised against three synthetic oligopeptides representing fundamental sequences of the pre-S region in deoxyribonucleic acid of hepatitis B virus (subtype adr), as well as with monoclonal antibody against the major surface antigen polypeptide (P22) coded for by the S gene. Both P39 and its glycosylated form P43 bound to all four monoclonal antibodies, thereby indicating that they were coded for by the sequence of 1200 nucleotides, from the second ATG codon in the pre-S region to the stop codon of the S gene. Both P39 and P43 bound to polymerized human and chimpanzee albumins, but not to polymerized albumin from species without susceptibility to hepatitis B virus. Due to their presence in Dane particles and the expression of a polyalbumin receptor, the immune responses against P39 and P43 may have significance in infection with hepatitis B virus and its immunoprophylaxis.

Immunoglobulin a antibody against hepatitis B core antigen in the acute and persistent infection with hepatitis B virus

Antibody to hepatitis B core antigen of immunoglobulin A class was determined in the serum of patients infected with hepatitis B virus by a sandwich-type solid-phase radioimmunoassay with monoclonal antibodies. The antibody, as defined by a sample to normal ratio greater than 2.1, was detected in all of 39 patients with acute hepatitis, with titers varying widely depending on the time of blood sampling. In persons with persistent infection, the antibody was detected in only 2 (4%) of 46 asymptomatic carriers of the virus, contrasting with the positivity in as many as 15 (41%) of 37 patients with chronic persistent hepatitis, in 45 (94%) of 48 patients with chronic active hepatitis, and in 40 (87%) of 46 patients with liver cirrhosis with or without hepatocellular carcinoma. The mean +/- SE titer of antibody in chronic persistent hepatitis (3.8 +/- 0.9) was significantly lower than those in chronic active hepatitis (13.8 +/- 3.2) and cirrhosis with or without carcinoma (25.6 +/- 6.1) (p less than 0.001). Based on the results obtained, the antibody may reflect hepatic injury in the persistent hepatitis B virus infection.

Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein

Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a.

A two-site sandwich radioimmunoassay of β2-microglobulin with monoclonal antibodies

Among 21 batches of monoclonal antibodies raised against beta 2-microglobulin (anti-beta 2m), 6 reacted with soluble beta 2m, as well as with beta 2m present in association with HLA heavy chains on the surface of leukocytes. The remaining 15 anti-beta 2m antibodies bound with soluble beta 2m, but failed to react with beta 2m on the cell surface. No monoclonal anti-beta 2m antibodies revealed precipitin lines when they were tested against beta 2m in immunodiffusion. When 2 anti-beta 2m antibodies with different specificities were mixed together, however, they developed a precipitin line against beta 2m. Based on these observations, there was only 1 epitope on beta 2m that was available for the binding with a monoclonal anti-beta 2m antibody with either specificity. This allowed the development of a solid-phase radioimmunoassay in which beta 2m in test specimens was sandwiched between immobilized anti-beta 2m of 1 specificity and radiolabeled anti-beta 2m of a heterologous specificity in a single step. Monoclonal antibodies with at least 2 different specificities would be required for developing a sandwich-type immunoassay of polypeptides, such as beta 2m, that do not display 2 or more epitopes of the same specificity.

Reactivity of Sera from Japanese Patients with Type 2 Autoimmune Hepatitis to Peptides Derived from Host Genes, Cytochrome HLD8-2 and GOR

We investigated whether the clinical linkage between type 2 autoimmune hepatitis (AIH), hepatitis C virus (HCV) infection, and anti-GOR antibody is related to the amino acid sequence homology found between the target protein for anti-liver/ kidney microsome (LKM1) antibody and those for anti-HCV and anti-GOR. In our present study, the putative LKM1 antigen-associated short linear sequence on the HLD8-2 protein was not recognized by a human monospecific polyclonal anti-GOR antibody. Similarly, despite considerable homology in several regions between HLD8-2 and HCV, we found no cross-reactive antibody in our anti-HCV-positive type 2 AIH patients. In addition, only 1 of 12 patients with type 2 AIH had antibody directed to the central antigenic domain of HLD8-2. These results suggest that molecular mimicry between HLD8-2, HCV, and GOR does not explain the high association of anti-GOR and anti-HCV with type 2 AIH. It is also suggested that the target epitope(s) for anti-LKM1 in Japanese patients may be different from those for European patients.