Comparative analysis of serological markers of chronic delta infection: HDV-RNA, serum HDAg and anti-HD IgM

To study the concordance, sensitivity and specificity of HDV-RNA determination by molecular hybridization, serum HDAg by immunoblot and anti-HD IgM by commercial enzyme immunoassay as compared to intrahepatic HDAg detection by an immunoperoxidase method, a statistical analysis was applied to the results of serum sample and liver biopsy determinations in 50 patients with chronic delta hepatitis (38 positive to tissue HDAg and 12 negative).Of the 38 patients with hepatic HDAg, HDV-RNA was found in 31 (82%), serum HDAg by immunoblot in 27 (71%) and anti-HD IgM in 33 (87%). Among the 12 patients without hepatic HDAg, one was found with serum HDAg using the immunoblot technique, two (17%) had HDV-RNA, and 7 (58%) had anti-HD IgM. Serum HDAg determination by immunoblot was the most specific test, followed by HDV-RNA analysis. The least specific was the anti-HD IgM technique. The anti-HD IgM test was the most sensitive, followed by HDV-RNA and serum HDAg. The concordance with intrahepatic HDAg detection was highest for HDV-RNA determination, followed by HDAg in serum. The least degree of concordance was found with anti-HD IgM determination. These results suggest that the determination of HDV-RNA by the hybridization method can be of great value for the diagnosis and monitoring of chronic delta hepatitis.     

Sensitive enzyme immunoassay for the detection of delta antigen and anti-delta, using serum as the delta antigen source

A sensitive enzyme-linked immunosorbent assay (ELISA, EIA) was developed for the detection of delta antigen in serum treated with Tween 20. The serum delta antigen so derived was used in an ELISA for anti-delta. Both tests were specific and more sensitive than radioimmunoassay (RIA) when applied to testing parenteral drug abusers. It is concluded that the different sources of delta-antigen used may account for the different sensitivities noted, and that delta antigenaemia in acute infection may be more frequently detectable than was first thought, amounting to 71% of those with delta infection in this study and that these sera are a convenient alternative source of antigen.     

The localization of delta antigen in the hepatocytes in chronic delta hepatitis

The results of immunohistochemical and electron microscopic examinations of liver biopsies from 47 patients with chronic delta hepatitis are presented. Delta antigen (HDAg) was detected by immunoperoxidase method in the liver of 35 (74.4%) patients. HDAg was demonstrated in hepatocyte nucleus alone in 22 (62.9%) patients, simultaneously in the nucleus and the cytoplasm in 9 (25.7%), in the cytoplasm alone in 4 (11.4%). In nuclear localization, delta antigen may fill the entire nucleoplasm or only the peripheral zone of the nucleus. When delta antigen fills the entire nucleus, hepatocytes undergo more marked pathological changes than those in which HDAg is localized in the nuclear periphery or those where no delta antigen was found.     

Evaluation of a commercial anti-delta EIA kit for detection of antibodies to hepatitis delta virus

Anti-hepatitis delta virus (anti-HDV) antibodies were measured by solid phase IgG and IgM capture radioimmunoassays (RIA) as well as by a competitive binding enzyme immunoassay (EIA) in both acute and chronic HDV infections. EIA anti-delta test measures total delta antibody without discriminating IgM from IgG anti-delta. Low titer IgG antibodies were detected by both techniques with equal sensitivity. High titer IgG antibodies reached the end point sooner with EIA than with RIA (10(-3) versus greater than 10(-6)). When IgM anti-HDV was present without accompanying IgG anti-HDV, EIA failed to identify the antibody. Presence of high titer rheumatoid factor in the serum and lipemic samples produced false-positive results by EIA. Usage of undiluted serum samples for EIA probably exaggerates the factors contributing to false-positive reaction.     

Heterogeneity of hepatitis delta antigen

Hepatitis delta antigen (HDAg) is the only known protein encoded by the hepatitis delta virus (HDV). Two HDAg species of different sizes have been detected in the sera and livers of the infected humans, chimpanzees, and woodchucks, even though only one RNA species was previously identified in most of the HDV strains. To study HDAg heterogeneity, we took advantage of the fact that a single base mutation at nucleotide 1015 (C to U), which results in an amber termination codon in the HDAg open reading frame (ORF), eliminates a unique Ncol restriction enzyme site. We screened various HDV cDNA clones and detected sequence heterogeneity of the HDAg-coding region on the basis of the presence or absence of the Ncol site. Five delta hepatitis patients were examined. In every patient, two types of HDAg-coding sequence were detected at nucleotide 1015: one which contains a C and results in an ORF encoding a delta antigen of 214 amino acids, and the other which possesses a U and results in an amber termination codon and a truncated HDAg species of 195 amino acids. The in vitro translation products of these two ORFs comigrated with the two HDAg species from the patient's plasma on SDS polyacrylamide gels. Polymerase chain reaction (PCR) amplification of the HDV RNA from some patients' sera and subsequent sequencing showed several additional mutations in the HDAg-coding region. These mutations are independent of the C or U nucleotide change at the site of the amber termination codon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of IgM antibodies to delta antigen after coinfection and superinfection with the delta virus

A sensitive microtitre radioimmunoassay was developed for detection of IgM antibodies to delta antigen. The assay was based on the selective binding of IgM from test sera to antihuman IgM (u-chain specific) fixed to wells of a microtitre plate, and utilized delta antigen extracted from the liver of an experimentally infected chimpanzee. This test proved to be useful in distinguishing between coinfection and superinfection with the hepatitis delta virus (HDV). Transient anti-delta IgM responses were observed in patients coinfected with HDV, while prolonged elevated IgM levels were found in HBsAg carriers with chronic liver disease superinfected with HDV. Two distinct serological patterns were observed in both coinfection and superinfection. In coinfection, only 50% of patients with detectable anti-delta IgM went on to develop a long-lasting antibody response. Following superinfection with HDV either stationary or fluctuating levels of IgM antibody were demonstrated. In patients with fluctuating antibody levels, the presence or absence of IgM antibody related to the level of viral replication.     

Anti-HBc IgM and anti-delta screening by EIA method

The clinical value of an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-HBc IgM was evaluated by testing 202 sera from acute viral hepatitis B (AVHB), hepatitis B (HB), chronic hepatitis (CAH), chronic liver disease (CLD), cirrhosis, primary hepatoma, HBsAg carrier, acute viral hepatitis A (AVHA), hepatitis A (HA), non-A, non-B (NANB) hepatitis and miscellaneous conditions other than hepatic disease, and 19 additional various hepatic disease cases were examined for anti-delta. In clinical situations the accurate diagnosis of HB is not always possible and the differential diagnosis seems to be very important especially in making decisions of treatment and estimation of prognosis. In overall cases the highest positive rate of anti-HBc IgM was found in AVHB as shown as 74.3% (26/35) comparing to other conditions in which the positive rate was extremely low (2.1%). The anti-HBc IgM appeared to be highly specific to AVHB (83.9%) as compared to the other. The positive rate of HBsAg was high in AVHB, CAH and HBsAg carrier (100.0%) followed by CLD, cirrhosis and HB (up to 70.8%). The ALT activities and ALPalb fractions were significantly high in AVHB (p < 0.005). The correlation between the positivity of anti-HBc IgM and highly abnormal ALT appeared be high. AVHB was confined mostly to 10-20 age group and the male to female ratio was about 6 to 1. Subgroup of AVHB II with positive anti-HBc IgM appeared to have a greater chance being positive for HBsAg and ALPalb. The S/N ratio of anti-HBc IgM was as high as 20 which was unique to AVHB.(ABSTRACT TRUNCATED AT 250 WORDS)     

The IgM antibody responses to the core antigen of hepatitis B virus

Little is known about the immunoglobulin class of antibodies to HBcAg. In the present study sera containing anti-HBc were fractionated by sucrose density-gradient centrifugation, and all serum fractions were tested against HBcAg by immunoelectro-osmophoresis. In addition selected fractions were examined by complement fixation test, immune adherence hemagglutination and immune electron microscopy. Anti-HBc activity in IgG serum fractions was demonstrated by all four techniques used, but HBcAg-specific IgM was detected only by immunoelectro-osmophoresis and by immune electron microscopy. In acute hepatitis B, HBcAg-specific IgM was detected for up to eight weeks after the onset of jaundice. It was also found transiently in two patients who developed chronic hepatitis B without an icteric episode and in one out of thirteen patients with HBsAg-positive chronic liver disease, but in none of eight healthy HBsAg carriers. The results suggested that HBc Agspecific IgM is formed transiently in response to primary HBV infection but is generally undetectable in established HBsAg carriers.     

The detection of hepatitis B surface antigen by radioimmunoassay.

A comparative study was performed to assess the sensitivity and specificity of counterimmunoelectrophoresis (CIEP) and radioimmunoassay (RIA) for the detection of hepatitis B surface antigen. The 8,823 sera examined included selected reference panels and sera collected from populations with low, moderate and high rates of chronic antigen carriage. Overall, hepatitis B surface antigen was detected in 265 sera by CIEP and in 376 by RIA. As well as detecting 46.4% additional positives, the RIA test detected all CIEP-positive sera; i.e., there were no false negative results. However, 150 sera (1.8% of the total tested) gave a positive result by RIA which was not repeatable on retesting. The explanation for this phenomenon appeared to lie in inadequate washing of the antibody-coated tubes.     

Intrinsic disorder and oligomerization of the hepatitis delta virus antigen

The 195 amino acid basic protein (δAg) of hepatitis delta virus (HDV) is essential for replication of the HDV RNA genome. Numerous properties have been mapped to full-length δAg and attempts made to link these to secondary, tertiary and quaternary structures. Here, for the full-size δAg, extensive intrinsic disorder was predicted using PONDR-FIT, a meta-predictor of intrinsic disorder, and evidenced by circular dichroism measurements. Most δAg amino acids are in disordered configurations with no more than 30% adopting an α-helical structure. In addition, dynamic light scattering studies indicated that purified δAg assembled into structures of as large as dodecamers. Cross-linking followed by denaturing polyacrylamide gel electrophoresis revealed hexamers to octamers for this purified δAg and at least this size for δAg found in virus-like particles. Oligomers of purified δAg were resistant to elevated NaCl and urea concentrations, and bound without specificity to RNA and single- and double-stranded DNAs.     

血清中丙型肝炎NS3抗原ELISA检测方法的建立和初步应用

目的 评价血清中丙型肝炎病毒(HCV)游离NS3抗原的酶联免疫吸附(ELISA)检测方法的特异性和灵敏度,初步探讨该方法在临床应用中的意义.方法 对77例正常人血清标本,173例抗-HCV阳性标本和3708例抗-HCV阴性的其他类型肝炎血清标本检测HCV游离NS3抗原;对部分HCV NS3抗原阳性标本进行验证,包括HCV RNA测定、中和试验和免疫斑点试验;对11例患者的25份系列血清标本进行了HCV游离NS3抗原、HCV RNA和HCV抗体的联合检测,并结合临床资料综合分析.结果 3708例抗-HCV阴性的其他类型肝炎血清标本中有48例为HCV NS3抗原阳性,其中3030例单纯乙型肝炎和445例其他类型肝炎血清标本中分别有44例和4例为HCV NS3抗原阳性;173例HCV抗体阳性标本中有42例为HCV NS3抗原阳性;77例正常人血清标本的HCV NS3抗原检测结果均为阴性;15例HCV NS3抗原阳性标本中有9例为HCV RNA阳性;23例HCV NS3抗原阳性标本的中和率和免疫斑点试验的阳性率分别为87.0%和69.6%;25份系列血清标本的检测结果显示其HCV NS3抗原的吸光度值与时间呈负相关,并有2例HCV NS3抗原阳性标本随着血清中HCV NS3抗原的吸光度值下降,其HCV抗体转阳.结论 血清中HCV游离NS3抗原的ELISA检测方法有较好的特异性和敏感度,在发展中国家应用此方法进行HCV感染的早期诊断有一定的临床意义和推广价值.     

Evaluation of five hepatitis delta virus marker assays for detection of antigen and antibody.

Five commercially available assays for hepatitis delta (HD) virus markers were compared for sensitivity, specificity, and reproducibility: three assays for antibody (anti-HD), provided by Diagnostics Pasteur, Organon Teknika, and Abbott Laboratories, and two assays for antigen (HD Ag), from Pasteur and Organon Teknika. The assay from Organon Teknika is the less sensitive assay for anti-HD detection. Although the sensitivities of the Pasteur and Abbott assays for anti-HD detection are similar, the use of Abbott reagents is hampered by the lack of specificity when HD Ag is present. The greater sensitivity for HD Ag detection is obtained with Organon assay.     

Diagnostic significance of IgM antibody to hepatitis delta virus in fulminant hepatitis B

The prevalence of hepatitis delta virus (HDV) infection was studied in 25 adult patients with fulminant hepatitis who were admitted consecutively to our unit from February, 1986, to September, 1988. Enzyme and radioimmunoassays were used for the detection of serological markers of HAV, HBV, and HDV (HDAg, IgM anti-HD, total [IgG] anti-HD) infections. Two hundred twenty-nine serum samples (three to 19 samples/patient) were tested for serological markers of HDV infection. Of the 25 patients, 17 (68%) were HBsAg-positive, and the remaining eight (32%) were HBsAg-negative on admission to the hospital. All patients were seropositive for IgM anti-HBc. Serological markers of HDV infection were detected in 13 (52%) of the 25 patients. In particular, HDV infection was observed in nine (53%) of the 17 HBsAg-positive and in four (50%) of the eight HBsAg-negative patients with type B fulminant hepatitis. Survival was 16.7% for patients with hepatitis B and 57.8% for patients with B and D coinfection. Coinfections were responsible for fulminant hepatitis in 100% of drug addicts and 40% in patients who were not drug addicts. All patients with HBV/HDV coinfections became seropositive for IgM anti-HD. The results show that HDV infection has a significant role (52%) in type B fulminant hepatitis in an area with a moderate prevalence of HBV infections, that it should be tested in cases with early clearance of HBsAg, and that it does not seem to be accompanied by a high fatality rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hybrid EIA test: A specific and sensitive assay for the detection of woodchuck antibody to hepatitis surface antigen (anti-WHs)

‘Ausria II’ polystyrene beads (Abbott Labs, N. Chicago) are reacted with wood-chuck serum positive for WHsAg in a dilution predetermined by titration. This modified bead is used in a blocking assay to detect the presence of antibody to the surface antigen of woodchuck hepatitis virus (anti-WHs). Serum containing wood-chuck anti-WHs and commercial horseradish peroxidase (HRP) labeled anti-HBs are sequentially added. A drop in optical density at 492 nm of 50% or more due to the blocking of HRP conjugated anti-HBs by anti-WHs compared with a control (negative woodchuck serum) is a measure of anti-WHs. The ease and simplicity of converting readily available ‘Ausria II’ beads to specific reagents for detecting anti-WHs should be welcomed by investigators studying WHV. The method described is both sensitive and reproducible.     

Enzyme-linked immunosorbent assay for detection of antibody to the hepatitis B surface antigen-associated delta antigen.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for specific detection of antibody to the hepatitis B surface antigen-associated delta antigen. The sensitivity of ELISA was intermediate between that of previously described immunofluorescence and radioimmunological assays for anti-delta. Performance of ELISA was simple and required only ordinary and inexpensive laboratory equipment.     

Histone H1e interacts with small hepatitis delta antigen and affects hepatitis delta virus replication

Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg (SHDAg) is required for HDV RNA replication, while the large form of HDAg (LHDAg) is required for viral assembly. Using tandem affinity purification method combined with mass spectrometry, we found that linker histone H1e bound to SHDAg. The binding domain of SHDAg to histone H1e was mapped to the N-terminal 67 amino acids. Oligomerization of SHDAg was required for its interaction with histone H1e. LHDAg barely bound to histone H1e and was masked at N-terminus. The binding domain of histone H1e to SHDAg was mapped to its central globular domain. HDV replication was inhibited by N- or C-terminal deletion mutants of histone H1e and was rescued by wild-type histone H1e. We conclude that histone H1e plays a significant role in HDV replication through forming protein complex with SHDAg.     

The detection of specific IgM to infectious bronchitis virus in chicken serum using an elisa

An ELISA is reported for the detection of specific IgM to infectious bronchitis virus (IBV) in serum samples from IBV-infected chickens. Both IgM and IgG antibodies to a heterologous, as well as to a homologous, strain could be detected by the ELISA. The IgM response was both rapid and transient, suggesting that its detection could be used as a means of identifying a recent IBV infection. Because IgG appeared to compete successfully with IgM for sites on the viral antigen, it was found necessary to separate IgM from IgG prior to testing in the ELISA.     

The detection and definition of IgM alloantibodies in the presence of IgM autoantibodies using flowPRA beads

We have developed a flow cytometry-based screening method using FlowPRA (One Lambda) human leukocyte antigen (HLA) class I panel beads and FlowPRA (One Lambda) HLA class I specificity beads for the detection and definition of immunoglobulin (Ig)M HLA-specific antibodies in the presence of IgM autoantibodies. Forty-six autoantibody-positive patients who were on the waiting list for a renal transplant (56 sera) were tested in parallel with FlowPRA (One Lambda) HLA class I beads and FlowPRA (One Lambda) control beads. Sera that were positive for IgM HLA class I antibodies were subsequently tested with FlowPRA HLA class I specificity beads to determine the HLA specificities. Thirteen of the 46 patients were positive for IgM HLA class I-specific antibodies. Eleven of the 13 had previous failed transplants and 2 were awaiting a primary transplant. For 9 of the 13 positive patients, IgM HLA class I specificities were defined. We have demonstrated the presence of IgM HLA-specific antibodies in patients with IgM autoantibodies. This study demonstrates the value of FlowPRA HLA class I panel and specificity beads for the detection and definition of IgM HLA class I-specific antibodies.     

Elimination of hepatitis delta virus infection after loss of hepatitis B surface antigen in patients with chronic delta hepatitis

The aim of this study was to evaluate whether patients with chronic hepatitis delta virus (HDV) infection treated with alpha interferon and subsequent loss of hepatitis B surface antigen (HBsAg) eliminate HDV. HDV RNA was detected in 26 of 28 patients with chronic delta hepatitis using the polymerase chain reaction. Seventeen patients in whom HDV RNA was detected were treated with alpha interferon; in 65%, HDV RNA remained detectable during treatment or reappeared after stopping therapy whereas in three patients HDV RNA remained absent (17.5%). HDV RNA became and remained undetectable in serum and liver of two of these three patients who lost HBsAg from serum and in one patient who was intermittently HBsAg negative during therapy. After loss of HBsAg, hepatitis B virus (HBV) DNA was still detectable in the liver, but not HBV RNA, indicating absent or very low HBV replication. Three patients were lost to follow up (17.5%). Two nontreated patients with chronic HDV infection also lost HBsAg during follow up; HDV RNA also became undetectable in their serum. Thus, HDV replication does not persist after the loss of HBsAg. Clearance of HBsAg may be a useful guide to when therapy can be stopped. © 1994 Wiley-Liss, Inc.