Rapid diagnosis of echovirus type 33 meningitis by specific IgM detection using an enzyme linked immunosorbent assay (ELISA)

During an outbreak of meningitis in France (in the Lyon area), from June to October 1982, serum and stool samples were collected from 227 patients. An enzyme-linked immunosorbent assay (ELISA) for titrating IgG and IgM antibodies anti-echovirus type 33 was developed and compared with the virus isolation technique, and with the titration of neutralizing antibodies. In 39 patients excreting echovirus 33 in faeces, the ELISA test allowed a positive serodiagnosis in 85% of the cases by detection of specific IgM (64% of the cases) and by seroconversion (21%). Compared with the neutralization (Nt) test, ELISA was found to be more sensitive. The antibody titres in ELISA were over 50 times higher and detected earlier than the neutralizing antibodies. This early immune response allowed a rapid diagnosis by specific IgM detection in the acute sera collected within 8 days after the appearance of the clinical symptoms in more than 50% of the 97 patients examined, whereas the Nt test allowed a positive serodiagnosis in only 32% of the patients. The use of a caesium chloride purified antigen insured the specificity of the reactions.     

Varying abilities of recombinant polypeptides from different regions of hepatitis E virus ORF2 and ORF3 to detect anti‐HEV immunoglobulin M

Following infection with hepatitis E virus (HEV), anti‐HEV immunoglobulin (Ig) M is thought to develop before anti‐HEV IgG and to be a better marker for differentiating between the acute and convalescent phases of infection. In order to select polypeptides for improved detection of anti‐HEV IgM, six and three overlapping polypeptides from open reading frames (ORFs) 2 and 3, respectively, of HEV genotypes 1 and 4 were expressed as fusion proteins in Escherichia coli. The reactivities of the polypeptides with anti‐HEV IgM were evaluated using immunoblotting and enzyme immunoassays (EIAs). The data indicated that polypeptides from the N‐terminus of ORF3 and middle region of ORF2 were weakly or not reactive with anti‐HEV IgM, while those from the remaining regions of ORF2 and ORF3 contained reactive epitopes. Anti‐HEV IgM against the N‐ or C‐terminus of ORF2 appeared earlier and disappeared faster than that against polypeptides from the C‐terminus of ORF3, based on serum samples from rhesus monkeys infected experimentally, and from patients infected naturally, with HEV. The N‐ and C‐terminal polypeptides from ORF2 complemented one another in detecting anti‐HEV IgM and EIA sensitivity was improved significantly with a combination of these polypeptides. The reactivities of ORF2 polypeptides from genotypes 1 and 4 were similar but that of ORF3 differed with sera from monkeys infected by the two genotypes. Thus, a combination of N‐ and C‐terminal polypeptides of ORF2 from one genotype may be effective in EIAs to detect anti‐HEV IgM. J. Med. Virol. 81:1052–1061, 2009. © 2009 Wiley‐Liss, Inc.     

Development of an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay for detection of equine and swine IgM antibodies to vesicular stomatitis virus

An immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MC-ELISA) was developed for the detection of primary infection of vesicular stomatitis virus (VSV) in equine and swine sera. The test was based on the use of biotinylated sheep antibodies against equine or swine IgM molecules bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of antigens prepared from the New Jersey and the Indiana VSV serotypes (VSV-NJ and VSV-IN, respectively) and mouse polyclonal antibodies against VSV-NJ and VSV-IN. The MC-ELISA was compared to a competitive ELISA (C-ELISA) and the standard microtiter serum neutralization (MTSN) assay by testing serum samples from horses and pigs experimentally infected with VSV-NJ or VSV-IN. The MC-ELISA detected specific homologous IgM antibodies from equine and swine sera as early as 5 and 4 days postinfection (DPI), respectively, and as late as 35 DPI. The MTSN test also detected antibodies as early as 5 DPI and as late as 160 DPI. In a similar fashion, the C-ELISA detected antibodies from 6 to 7 DPI and as late as 160 DPI. These results demonstrated that the MC-ELISA is a useful test for serodiagnosis of primary VSV infection in horses and pigs.     

Rapid diagnosis of acute Epstein-Barr virus infection by an indirect enzyme-linked immunosorbent assay for specific immunoglobulin M (IgM) antibody without rheumatoid factor and specific IgG interference.

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of Epstein-Barr virus-specific immunoglobulin M (IgM) antibody was developed with commercial reagents. Sera containing rheumatoid factor (RF) (as little as 0.5 IU/ml) coupled with specific IgG resulted in false-positives in the ELISA. This interference was eliminated by the use of anti-human IgG antibodies to remove RF and IgG. Thus, pathogen-specific IgG complexes to which IgM-RF could be bound during the subsequent test were inhibited, and competition between specific IgG and IgM was also prevented. Of the 1,672 serum specimens tested, 353 were found to be Epstein-Barr virus IgM antibody positive by indirect immunofluorescence (IF). Compared with the IF test, the ELISA showed 96.6% sensitivity, 99.7% specificity, and 99% accuracy. Further evidence indicated that most of the 12 ELISA false-negatives were IF false-positives. There was a linear correlation between mean ELISA values and increasing IF titers (r = 0.96). However, the IF test has the disadvantages that it lacks automated reading and requires considerable technical expertise, both of which restrict the range of laboratories performing the test. The indirect ELISA has the advantages that it is simple and rapid and can be automated. All the reagents used in this assay are commercially available, have been prestandardized, and are stable.     

Establishment of an enzyme‐linked immunosorbent assay system for determining soluble CD96 and its application in the measurement of sCD96 in patients with viral hepatitis B and hepatic cirrhosis

CD96, previously named T cell activation increased late expression (Tactile), is a transmembrane molecule that functions as an activated receptor on natural killer cells. It is well known that many transmembrane molecules have soluble forms, which were either shed from the cell surface or spliced at mRNA level. In many cases, the levels of soluble forms in the circulation could be used as biomarkers of lymphocyte activation in bacterial or virus infection, tumour, transplantation and autoimmue disease. To investigate whether CD96 could be released into the sera and the possible biological fuction of soluble hCD96 (sCD96), we generated and characterized five clones of anti‐hCD96 mouse monoclonal antibodies (mAb) and developed a sandwich enzyme‐linked immunosorbent assay (ELISA) system based on two anti‐hCD96 mAbs with different epitope specificities. Using this ELISA system, sCD96 in serum samples from 99 healthy individuals could be detected. Furthermore, we found that the level of sCD96 in serum samples from patients with chronic viral hepatitis B or classes B and C of hepatic cirrhosis classified using the Child–Pugh score was much higher (P < 0·001 versus healthy individuals; P = 0·006 versus healthy individuals respectively) than that from healthy individuals (0·98 ng/ml). Our study demonstrates for the first time that sCD96 existed in sera, and suggestes that sCD96 may be used as a serous marker for some diseases such as chronic viral hepatitis B infection or hepatic cirrhosis in classes B and C. The level of sCD96 in patients' serum may have some relationship with a chronic inflammatory reaction.     

Non‐neutralizing epitopes induce robust hepatitis C virus (HCV)‐specific antibody‐dependent CD56+ natural killer cell responses in chronic HCV‐infected patients

Natural killer (NK) cell‐mediated antibody‐dependent cellular cytotoxicity (NK‐ADCC) is of considerable interest in viral infection. However, little is known about NK‐ADCC responses in chronic hepatitis C virus (HCV) infection. In this study, impaired non‐specific antibody‐dependent CD56⁺ NK cell responses were observed in chronic HCV infection, as shown by decreased degranulation (extracellular CD107a expression) and interferon (IFN)‐γ production in response to antibody‐bound P815 cells. A peptide pool composed of epitopes recognized by anti‐HCV‐E1/E2 antibodies could induce pronounced HCV‐specific antibody‐dependent NK cell responses in sera from approximately half the chronic HCV carriers. Additionally, HCV‐specific epitopes with the capacity to induce robust NK‐ADCC activity were identified. Five linear NK‐ADCC epitopes (aa211‐aa217, aa384‐aa391, aa464‐aa475, aa544‐aa551 and aa648‐aa659 of the HCV envelope) were identified and do not overlap with putative linear neutralizing epitopes. This study revealed the dysfunctional characteristics of antibody‐dependent CD56⁺ NK cell responses in chronic HCV carriers. The key non‐neutralizing NK‐ADCC epitopes identified in this study may act as new targets for immunological intervention.