Retrospective study on the diagnostic value of IgG ELISA, dot immunoassay and indirect hemagglutination in septicemic melioidosis.

Three serological methods for diagnosis of melioidosis were compared with the culture method currently used as the "gold standard". The diagnostic values of the serological methods were evaluated retrospectively in 306 patients residing in an endemic area. The enzyme-linked immunosorbent assay (ELISA), using affinity purified antigen for detecting specific IgG antibody, showed a slightly higher specificity (86.0%) than the dot immunoassay (DOT) (84.0%) and both were superior to indirect hemagglutination (IHA) (72.0%). The sensitivity of DOT (96.4%) and ELISA (85.7%) were considerably higher than that of IHA (50.0%). The primary benefit of the high negative predictive value of both ELISA (96.4%) and DOT (99.0%) in an area of high prevalence is the ability to rule out most of the non-melioidosis patients.

Antigenic heterogeneity of lipopolysaccharide among Burkholderia pseudomallei clinical isolates.

Burkholderia pseudomallei (BP) causes melioidosis, a potentially fatal human infection in the tropics. Clinical isolates from different geographical locations have similar morphological and biochemical characteristics. Although BP has been reported to possess 2 types of lipopolysaccharide (LPS) differing in the chemical structure of their O-polysaccharide (O-PS) component, earlier report demonstrated that the clinical strains exhibited identical LPS moieties. Recently, we reported antigenic similarity between the pathogenic (Ara-) and nonpathogenic (Ara+) biotypes. However, a few clinical isolates showed atypical SDS-PAGE profiles. In this study, LPS from 739 BP isolated from patients and animals in different geographical areas were extracted by proteinase K digestion method. Their SDS-PAGE profiles and their immunoreactivities with patients' sera and monoclonal antibody (MAb) to LPS were analyzed. The isolates showed 3 LPS patterns differing in the number and electrical mobility of bands in silver-stained gel. A majority of BP (711) isolates exhibited identical typical ladder pattern, 21 isolates showed atypical ladder pattern and 7 isolates did not exhibit ladder appearance. However, all LPS preparations exhibited similar endotoxic activity as determined by Limulus amebocyte lysate assay. On the other hand, there were no immunological cross reactivity between typical and atypical LPS, as judged from Western blot against homologous and heterologous sera from melioidosis patients from whom the typical and atypical LPS were isolated. Nevertheless, a Western blot profile of the typical LPS showed some variations when probed with MAb against BP LPS (9D5). Heat-killed bacteria from all LPS groups could similarly activate mouse macrophage cell line to produce nitric oxide (NO) and inducible NO synthase (iNOS).

Studies on the 52 kDa antigen of Salmonella typhi: physicochemical stability, purification by affinity chromatography and immunochemical specificity.

The 52 kDa specific protein antigen of Salmonella typhi, as identified by monoclonal antibodies (Ekpo et al. 1990) has been studied with respect to its physicochemical stability, purification by affinity chromatography and immunochemical specificity. It was found that the 52 kDa protein was degraded into smaller antigenic fragments of MW 30-51 kDa when treated with acetone, ethanol, sodium thiocyanate, 0.3M sodium chloride and Veronal and Tris buffers. The exact chemical nature of the degradation of the protein under these conditions is not known but digestion by conventional proteases and dissociation of the non-covalent subunit type have been ruled out. It is proposed that the degradation may be the result of yet unidentified enzyme(s) which become activated by various physical or chemical treatments. Affinity chromatography using a specific monoclonal antibody has been carried out in an attempt to purify the 52 kDa protein. The binding of S. typhi protein to the column was saturable at 65.6 microgram protein/ml gel. The amount of S. typhi protein adsorbed on the column was 0.51% of the total sonicated cell protein. SDS-PAGE of the immunoadsorbent purified protein revealed bands at Mr 15-58 kDa, indicating that the protein obtained had been severely degraded. However, Western blot of the purified protein stained with a specific monoclonal antibody and with rabbit polyclonal antibody against S. typhi showed striking similarity, indicating that the protein obtained was close to immunochemical purity. The 52 kDa protein purified by affinity adsorbent was used as an antigen for the detection of specific IgM in sera of patients. It was shown that sera of patients infected with S. typhi as well as those infected with other bacteria, contained specific IgM against the 52 kDa protein. Thus, it appears that the 52 kDa protein contains species specific as well as cross-reacting epitopes. The possible development of specific diagnosis of S. typhi based on the present experimental results in discussed.