U. only 34 were positive and 83 were negative. Western blot analyses and additional experiments strongly indicated that these 83 discordant sera were bad for anti-HEV IgM. Furthermore, among the 117 sera, 5 reacted with both the immunodominant and truncated polypeptides, with similar optical densities at 450 nm. However, their reactivity was demonstrated to result from nonspecific binding. Together, the data indicate that the poor reactivity of a truncated ORF2 polypeptide can be used to exclude nonspecific binding in the detection of anti-HEV IgM. Hepatitis E disease (HEV), transmitted from the fecal-oral route, is the main cause of acute self-limiting hepatitis in many developing countries (1, 2, 19). Recently, HEV illness and the disease hepatitis E have also gained attention in industrialized countries (3, 14). HEV is definitely a single-stranded positive-sense RNA disease having a genome of approximately 7.2 kb. The viral genome consists of three open reading frames (ORFs). You will find four major genotypes of HEV recognized, yet there is only a single serotype (19). In China, most sporadic instances are caused by genotype 4 (7, 13). Clinically, hepatitis E is not distinguishable from acute hepatitis caused by other viruses. Therefore, the analysis of hepatitis E depends upon laboratory evidence of recent infection. Detection of HEV Acetazolamide RNA in serum or feces by reverse transcription (RT) and nested or real-time PCR is definitely direct evidence of acute infection; however, in addition to the expensiveness of the assays, viremia usually is present at the early acute phase and subsides soon after the apparent symptoms. Thus, the approach of detecting HEV RNA is not a practical way to clinically diagnose hepatitis Acetazolamide E. Detection of IgM antibody against HEV (anti-HEV IgM) in serum can diagnose acute HEV infection. Commercial enzyme-linked immunosorbent assay (ELISA) packages for IgM are available; these assays were established based on recombinant antigens and/or synthetic polypeptides derived from ORF2 and/or ORF3 sequences (4, 6, 26, 28). However, false-positive results occurred when the available kits were used to detect anti-HEV IgM (5, 8, 9, 16, 18). Consequently, a more specific assay for anti-HEV IgM that does not sacrifice sensitivity would be important for diagnosing hepatitis E. The polypeptide encoded by ORF2 forms the principal (probably only) structural protein of HEV. It has been documented the C-terminal portion of the ORF2 polypeptide covers the immunodominant epitopes (17, 20), which are located in amino acids (aa) Acetazolamide 459 to 607 (29). A polypeptide in which several aa residues are truncated from either terminus does not react with the anti-HEV induced by natural infection in humans and experimental illness in nonhuman primates (29). Based on these observations, we assumed the truncated ORF2 polypeptide could serve as a nonreactive polypeptide to rule out false-positive results in the detection of anti-HEV IgM. Here we present data to support our assumption that false-positive results may be excluded by use of the truncated polypeptide. MATERIALS AND METHODS Serum samples. In total, 313 serum samples were included in the present study and were divided into three organizations. One group comprised the sera from 159 healthy persons who have been bad for anti-HEV IgM, as recognized having a Genelabs kit (Genelabs Diagnostics Pte. Ltd., Singapore Technology Park, Singapore), and bad for IgM antibodies against hepatitis A disease and hepatitis B disease core antigen and for IgG antibody against hepatitis C disease. The second group was composed of samples from 37 acute hepatitis E individuals who have been diagnosed from the detection of HEV RNA (7). The third group included 117 sera which were positive for anti-HEV IgM, repeatedly recognized having a Genelabs kit. Tal1 All sera were stored at ?70C. This study was authorized by the institutional review boards of the Nanjing Drum Tower Hospital. Cloning and manifestation of HEV ORF2 fragment. Total RNA was extracted from 200 l serum from a hepatitis E patient with positive anti-HEV IgM, using Trizol (Invitrogen, CA). The ORF2 fragment was cloned by RT-PCR. The cloning primers were U1stF and U1stR as the outer pair and U2ndF.