Diagnosis of Hepatitis C Virus Infection Using a Novel Mouse Monoclonal Antibody to Detect Serum HCV E1/E2 Antigens.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of liver disease worldwide and in Egypt. The aim of this study was to detect HCV E1/E2 antigens using a novel mouse monoclonal antibody (mAb) designated (7G9) as a diagnostic and alternative approach for HCV detection. METHODS: The detection of HCV-E1/E2 antigens in 138 patients positive for HCV infection tested by RT-PCR and 25 healthy individuals negative for HCV as control group was done by an optimized in-house ELISA and DotELISA (based on the molecular mimicry of E2 to immunoglobulins). RESULTS: The mAb (7G9) was found to be IgM (heavy-chain)/kappa (light-chain) and characterization by western blot revealed two bands at 63 kDa for E2 and 31 kDa for E1. ELISA peptide mapping showed high reactivity with peptide derived from HCV E1 (a.a. 315 - 323) and low reactivity to peptides derived from HCV E2 (a.a. 517 - 531) and HCV E2 (a.a. 412 - 419). The mAb (7G9) showed no reactivity with HBV Ag, S. typhi or B. Abortus Ag proving high specificity. AUC for HCV-E1/E2 detection was 0.96 for all HCV patients with sensitivity 87% (119/137), specificity 88% (22/25) and efficiency 87%. The HCV-E1/E2 antigens detection by Dot-ELISA showed 76.8% sensitivity, 88% specificity and the efficiency of the assay was 78.5%. Furthermore, no correlation was found between serum HCV viral load and HCV E1/E2 antigens detection. CONCLUSIONS: The ELISA and Dot-ELISA based on the monoclonal antibody (7G9) are reliable, rapid, easy and economic diagnostic assays for HCV infection.

Discovery and phylogenetic analysis of novel members of class b enterotoxigenic Escherichia coli adhesive fimbriae.

Enterotoxigenic Escherichia coli (ETEC) is recognized to be a common cause of acute watery diarrhea in children from developing countries. Colonization factors (CFAs) have been identified predominantly in ETEC isolates secreting heat-stable enterotoxin (ST) or cosecreting ST with a heat-labile toxin (LT). We hypothesized that LT-only-secreting ETEC produces unique colonization factors not previously described in ST and LTST-secreting ETEC. A set of degenerate primers based on nucleotide sequence similarities between the major structural genes of CS20 (csnA), CS18 (fotA), CS12 (cswA), and porcine antigen 987 (fasA) was developed and used to screen a collection of 266 LT-secreting ETEC isolates in which no known CFA was detected. PCR-amplified products of different molecular masses were obtained from 49 (18.4%) isolates. Nucleotide sequence analysis of the PCR amplicons followed by GenBank nucleotide BLASTn analysis revealed five novel DNA sequences; translated amino acid BLASTx analysis confirmed sequence similarity to class 1b major structural proteins encoded by csnA, fotA, and fasA. Strains expressing the novel CFAs were phylotyped and analyzed using multilocus sequence typing (MLST; Achtman scheme), and the types detected were compared to those of a collection of archived global E. coli strains. In conclusion, application of the degenerate primer sets to ETEC isolates from surveillance studies increased the total number of ETEC isolates with detectable CFAs by almost 20%. Additionally, MLST analysis suggests that for many CFAs, there may be a requirement for certain genetic backgrounds to acquire and maintain plasmids carrying genes encoding CFAs.

Design and validation of a multiplex polymerase chain reaction for the identification of enterotoxigenic Escherichia coli and associated colonization factor antigens.

Development of a genetic tool for the detection of genes encoding enterotoxins and colonization factors would greatly enhance enterotoxigenic Escherichia coli (ETEC) surveillance. Oligonucleotide primers were designed to amplify genes encoding human ST, porcine ST, LT and the structural genes of colonization factor antigen (CFA)/I, CS1 to CS8, CS12 to CS15, CS17 to CS22, and PCFO71. Screening 89 ETEC isolates phenotypically expressing a known CFA showed that, without exception, the multiplex polymerase chain reaction (mPCR) detected the structural gene of the expressed CFA, in addition to CS21 in 22.5% of isolates. Silent genes such as cssB (CS6) were also detected in 9.0%. Additionally, we screened 71 CFA phenotypically negative isolates and detected a CFA in more than 50% of tested isolates. In conclusion, we have designed a simple 4-step mPCR for the rapid detection of ETEC virulence factors. The assay is rapid, reproducible, relatively inexpensive, and has the potential to be field applicable.