A new artificial antigen of the hepatitis E virus.

An artificial antigen composed of 12 small antigenic regions derived from the ORF2 and ORF3 HEV proteins was designed. The gene encoding for this artificial antigen was assembled from synthetic oligonucleotides by a new method called Restriction Enzyme-Assisted Ligation (REAL). The diagnostic relevance of this second generation HEV mosaic protein (HEV MA-II) was demonstrated by testing this antigen against a panel of 142 well defined anti-HEV positive and anti-HEV negative serum samples. The data obtained in this study support the substantial diagnostic potential of this HEV mosaic antigen.

[Diagnostic advantages of the test system "DS-EIA-HBsAg-0.01" for detection of HBV surface antigen].

The new highly sensitive test system "DS-EIA-HBsAg-0.01" (Priority Certificate No. 2006129019 of August 10, 2006) in detecting hepatitis B surface antigen (HBsAg) was assessed. The sensitivity of the test was estimated using the federal standards sample HBsAg 42-28-311-06, panels' samples Boston Biomedica Inc. (West Bridgewater, Mass, USA) and ZeptoMetrix Corp. (Buffalo, NY, USA). The findings have indicated that "DS-EIA-HBsAg-0.01" is equally effective in detecting different subtypes of HBsAg during a seroconversion period earlier than alternative assays. Along with its high analytical and diagnostic sensitivity, the system shows a high diagnostic specificity.

Enzyme potentiated radioimmunoassay (EPRIA): a sensitive third-generation test for the detection of hepatitis B surface antigen.

A sensitive, specific immunoassay for detection of hepatitis B surface antigen (HBsAg) is described. The assay combined enzyme-linked immunosorbent assay and solid-phase radioimmunoassay and is termed enzyme potentiated radioimmunoassay (EPRIA). HBsAg was quantitated by enzymatic conversion of L[14C]glutamic acid to 14CO2 and gamma-aminobutyric acid by glutamate decarboxylase (GDC) conjugated wih goat anti-HGs IgG. Conjugation of IgG and GDC was by a thiol-disulfide bond exchange reaction after reacting N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) with each reagent. A positive/negative ratio of 2.2 was established as significant by examination of 40 normal sera negative for HBsAg. This value was the mean cpm plus 3 standard deviations. By an identical statistical analysis of sensitivity, EPRIA was found to be approximately 100-fold more sensitive than Ausria II (Abbott Laboratories, North Chicago, IL).

Characteristics of chemically aggregated IgM in an immunoglobulin class-specific immune complex assay.

Immune complexes have been implicated in the pathogenesis of a number of infectious diseases. The predominant immunoglobulin class associated with circulating immune complexes is IgG, although immune complexes containing IgM have been described. The role of IgM immune complexes in disease pathogenesis has been difficult to characterize due to the lack of a reliable in vitro model. Immunoglobulins aggregated with bis-diazotized benzidine (BDB) are known to function as model immune complexes. We have developed an IgM immune complex using BDB-aggregated IgM which can be used as a reference in a conglutinin-based immune complex assay. Using this assay system, humans and chimpanzees with acute hepatitis A were found to have circulating immune complexes that contained IgM as the predominant antibody.

Purification of acute phase anti-hepatitis A virus (HAV) IgM and development of an IgM solid-phase radioimmunoassay for the detection of HAV.

A simple two-step procedure for the purification of IgM from acute phase hepatitis A infected chimpanzee serum has been developed. The procedure involves the precipitation of the euglobulin fraction with boric acid and the fractionation of the resuspended material on Sephacryl S-300 (Pharmacia Fine Chemicals, Piscataway, NJ). The purified IgM was used to develop a solid-phase radioimmunoassay (SPRIA) in which IgM was used as capture antibody and iodinated IgM was used as a probe. This assay was compared with a conventional IgG-based SPRIA. The IgM-based assay resulted in a superior response (P/N) for the detection of antigen in crude preparations; however, endpoint analyses demonstrated similar sensitivities.

Seroepidemiology of hepatitis B virus infection in the United States. 1976 to 1980.

The prevalence of hepatitis B virus (HBV) infection was determined using sera from persons participating in the second National Health and Nutrition Examination Survey, conducted from 1976 to 1980. Of 14,488 scientifically selected participants aged 12 to 74, 821 had evidence of past or present infection with HBV. In the white population, the weighted estimate of hepatitis B infection was 3.2 percent (95 percent confidence interval, 3.1 to 4.2). A steady increase with age was seen; by ages 65 to 74, the prevalence was 6.9 percent (95 percent confidence interval, 5.2 to 8.5). In the black population, the overall weighted estimate of prevalence was 13.7 percent (95 percent confidence interval, 11.6 to 15.8). In this racial group, there was a dramatic increase with age, with the oldest age groups having a prevalence of 39.6 percent (95 percent confidence interval, 29.1 to 50.0). In both racial groups, there was a low prevalence of infection in young children that began to rise between ages 12 and 18. In a multivariate analysis of factors associated with infection, there was an interaction of race with age; therefore, the odds ratio for race is presented for four ages. This ratio ranged from 3.0 (95 percent confidence interval, 1.8 to 4.2) for a 15-year-old to 8.2 (95 percent confidence interval, 6.5 to 10.3) for a 70-year-old. These relative odds estimates were not substantially affected by adjustment for the available information on risk factors for HBV infection. The results of this study in a representative sample of the United States population show that adult black Americans are at high risk for hepatitis B infection. Other independent predictors of HBV positivity include male sex; residing in the South, Northeast, or West; residing in a city of 250,000 or more people; serving in the armed forces; living below the poverty level; and having a positive treponemal test for syphilis. These data suggest that the immunization practices for controlling this disease should be re-examined.

A small open reading frame of the hepatitis delta virus antigenomic RNA encodes a protein that elicits antibodies in some infected patients.

A small open reading frame (ORF) was found in the hepatitis delta virus (HDV) antigenomic RNA encoding a short peptide that shares structural similarity with a region of the hepatitis B virus terminal protein. Analysis of all published HDV genome sequences indicates a high degree of conservation for the small ORF. This ORF is located at the 3'-terminal region of the gene encoding the hepatitis delta antigen (HDAg). We speculated that a peptide encoded by this ORF can be represented as the C-terminal domain of a new protein called HDAg'. This protein contains almost the entire sequence represented in the small form of HDAg and a peptide as an additional 'extension' sequence at the C-terminus. Two long synthetic peptides representing the two different types of peptides encoded by the small ORF were synthesized. These peptides were used for the development of an immunoassay for the detection of antibody to the HDAg' specific domain in sera of patients with HDV infection. Among 162 serum samples analyzed, 13 were found to be positive for an antibody reactive with these synthetic peptides. These antibodies were identified in patients with HDV infections and were not found in patients infected with hepatitis B virus, hepatitis C virus, or non-A,non-B,non-C virus. Thus, these data support the identification and existence of a new antigen encoded by the antigenomic RNA of the HDV.

Characterization of the antibody reactivity to synthetic peptides from different parts of the hepatitis C virus genome.

Infection by hepatitis C virus (HCV)*, the aetiologic agent responsible for the majority of non-A-non-B posttransfusion hepatitis, is detected by assaying for antibodies against structural and nonstructural recombinant proteins or synthetic peptides. The aim of this study was to characterize the antibody reactivity of selected sera against antigenic peptides spanning immunodominant regions of the core, NS4 and NS5 HCV proteins. Reactivity to synthetic peptides was determined by enzyme immunoassay (EIA) for 11 selected sera from blood donors (good responders), for 27 selected sera from hemodialysis patients (poor responders), all positive for HCV antibodies (tested by different second and third-generation assays), and for 7 negative sera. Some peptides from the core and the NS4 region were widely recognized by the tested sera. Sera not reactive with core, NS4, or NS5 region by some immunoblot assays exhibited reactivity against peptides from these proteins. Autoimmune reactivity associated with HCV infection was evaluated by using a synthetic peptide derived from the GOR peptide; 8/11 HCV-positive sera were found reactive against this peptide. No correlation was found between reactivity to any of the peptides tested and the presence of HCV RNA in the serum or with HCV genotype. The EIA reactivity of peptides from the core region suggested a multideterminant antigenic structure, where reactivity of each epitope may be differentially affected by neighboring amino acids depending on individual sera. This situation was particularly evidenced in selected sera from poor responder specimens where a more restricted antibody response to core peptides was observed. Reactivity of sera from HCV-infected patients with synthetic peptides from the core, NS4, and NS5 regions indicated the presence of multiple linear epitopes (particularly in the core region) that may be used in a mixture for immunodiagnosis; however, the length and exact position of the synthetic peptides must be chosen carefully.

Prevalence of hepatitis B virus infection in Tonga: identifying high risk groups for immunization with hepatitis B vaccine.

A serological survey for evidence of hepatitis B virus (HBV) infection was conducted in the Kingdom of Tonga as the first step in developing a strategy for an immunization programme. There were 414 individuals from the general population plus 137 pregnant women included in the survey. HBsAg was found in 20% of the general population and 88% had one or more serologic markers of HBV infection. In the 5-9 year age group, 80% of the children had one or more markers of HBV, and in the 10-19 year age group, the prevalence was 96.4%, indicating that most transmission of HBV in the Tongans studied occurs in the young. Of the pregnant women studied, 15% were positive for HBsAg, and 57% of those positive for HBsAg were also positive for HBeAg. Evidence of delta virus infection was not found in any of 82 HBsAg positive sera tested. Surveillance data suggested that significant serious sequelae to HBV infection (cirrhosis and primary hepatocellular carcinoma) also occur in Tonga. Immunization of infants and children is the most effective strategy for reducing or eliminating HBV infection and its sequelae in developing countries like Tonga.

Serologic markers for hepatitis B among Marshallese accidentally exposed to fallout radiation in 1954.

At least one serologic marker of prior hepatitis B infection (hepatitis B surface antigen, antibody to surface antigen, or antibody to core antigen) was found in 91.7% of 314 Marshallese tested. The prevalence of hepatitis B surface antigenemia (3.3%) in a subpopulation that had resided on Rongelap Atoll at the time of accidental exposure to radioactive fallout from a thermonuclear test in 1954 did not differ significantly from the prevalence in a selected unexposed population (10.5%).