Serodiagnosis of melioidosis by a competitive enzyme-linked immunosorbent assay using a lipopolysaccharide-specific monoclonal antibody.

Burkholderia pseudomallei is the causative agent of melioidosis, a severe and potentially fatal infectious disease in humans known to be endemic in Southeast Asia and northern Australia. The infection is also increasingly recognized in various animal species with a potential to spread to humans. With the potential as a biological warfare agent, specific serodiagnosis of melioidosis for surveillance in large populations at risk, humans or animals, would be highly valuable. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) using a lipopolysaccharide-specific monoclonal antibody was developed. The assay provides high specificity, based on a previously described monoclonal antibody to a specific epitope on the lipopolysaccharide (LPS) of B. pseudomallei. The assay sensitivity of 96.0% and specificity of 100% were achieved at a cutoff value of 50% inhibition in human culture-proven melioidosis cases. An optimal cutoff value of 65% inhibition for sera from a melioidosis endemic area was obtained by ROC analysis and resulted in an assay specificity of 86.2%, while maintaining assay sensitivity of 92.0%. A potential application of the assay in the serodiagnosis of melioidosis in animal species was also evaluated usina dolphin sera with satisfactory results.

Hepatitis C virus nonstructural 3 protein: recombinant NS3 protein of the Thai isolates as an antigen in a diagnostic assay.

Nonstructural 3 (NS3) protein of hepatitis C virus (HCV) is one of the antigens commonly used in diagnostic assays for antibody to hepatitis C virus. However, immune response to the NS3 protein from one genotype may not cross-react with that from other genotypes. In the development of an anti-HCV assay, the NS3 genes from genotypes 1 and 3 commonly found in Thailand were amplified and cloned into a bacterial expression system. These recombinant NS3 proteins were immunogenic and reacted with plasma samples of Thai patients infected with various HCV genotypes. Interestingly, the NS3 proteins from the Thai genotypes could react with 3 plasma samples from HCV infected Thai blood donors, which could not bind to the NS3.1 protein in the commercial HCV immunoblot kit using antigen from HCV genotype 1. This finding supports our prior observation that the appropriate HCV antigens used in a diagnostic assay should be derived from the virus genotypes commonly found in that geographical region.

The development of a reverse transcription polymerase chain reaction system for detection of estrogen receptor in breast cancer.

Estrogen receptor (ER) is widely used as an indicator of prognosis and response to endocrine treatment of primary breast cancer. ER phenotypes detected by conventional assays may not reflect their capability on binding specifically to estrogen response elements of target DNA. The reverse transcription polymerase chain reaction (RT-PCR) has been shown to offer the sensitive and specific method for measuring the ER mRNA in breast cancer. The ER-mutant gene which is positively detected in breast tumor by biochemical or immunocytochemical assays may be negatively shown by RT-PCR or vice versa. PCR technique for examining expression of ER mRNA may therefore provide a good screening method for detection of functioning ER that will affect the selection of appropriate treatment and prognosis of breast cancer patients. We have developed RT-PCR assay using b2-microglobulin as internal control for detection and relative-quantitation of ER mRNA in breast cancer tissue. Preliminary results show that the developed assay provides a sensitive and specific method for detection of ER expression in breast tumor. Also, the assay procedure is simple, rapid, non- expensive and required very small amount of breast cancer tissues.

Detection of hepatitis C virus RNA by polymerase chain reaction: Design of amplification primers.

New sets of primers for amplification of hepatitis C virus (HCV) RNA were designed from the conserved regions of American and Japanese isolates of HCV. Primers set A amplified parts of the 5’-untranslated and core gene regions, whereas set B amplified parts of the core and envelope gene regions. PCR amplification were carried out from HCV RNA isolated from sera of 13 Thai patients with antibody to HCV, using these newly developed primers. HCV RNA was detectable in 8 patients (61.5%). Of those PCR positive samples, only 4 patients (50%) were positive with primer set A, whereas 7 patients (87.5%) were positive with primer set B. Interestingly, 4 patients were tested positive with only set B, 3 patients with both sets A and B, and just one patient with set A only. This result suggests that PCR assay as a diagnostic tool for HCV may need to be carried out with more than one primer set. The relatively low percentage of PCR positivity of the HCV from Thai patients using primer set A, which was previously identified as the most conserved region of the HCV genome, also indicates that the sequence of the Thai isolates of the virus may be different from those of the American and Japanese strains.