Yuwaporn Ruangkunaporn

Studies on immunodiagnosis of human paragonimiasis and specific antigen of Paragonimus heterotremus

Adult Paragonimus heterotremus were recovered from the lungs and pleural cavity of cats orally infected with metacercariae. The worms were ground and extracted with distilled water. The soluble crude antigen (CA) contained about 40% proteins which could be fractionated by gel filtration on Sephadex G-200 into three profiles namely the F1, F2 and F3. The CA and its Sephadex profiles were used in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to P. heterotremus in three groups of patients, i.e. patients whose sputum and/or faeces revealed P. heterotremus eggs (group 1), patients with other parasitic infections (group 2), bacterial proven tuberculosis patients (group 3) and healthy, parasite-free controls (group 4). The sensitivity and specificity of the assay when the F1 was used as the antigen were 100%. Western blot analysis revealed that specific antigen of P. heterotremus was a non-protein component of Mr35 kDa.

Specific monoclonal antibodies to Opisthorchis viverrini

A Balb/c mouse was immunized with a crude soluble antigen of Opisthorchis viverrini adult worms (OVAA) over a period of 7 months. Spleen cells from the immune mouse were fused with Sp2/0 myeloma cells. Among the 264 tissue culture wells containing the fused cells, cells of 96 wells (36%) produced antibodies to the immunizing agent. Antibodies produced by cells in several wells reacted with antigens from other species of parasite. Cells of 17 wells produced antibodies specific only to OVAA, thus cells from three representative wells were cloned by limiting dilution. Hybrids obtained produced antibodies which could be classified according to their tissue specificities into three groups. The first group of antibodies reacted strongly to the worm integument and weakly with the muscles while those belonging to the second group reacted only to muscles of the worms. The monoclonal antibodies of the third group gave a positive reaction to both muscles and tegument.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody. International Journal for Parasitology 22: 527-531. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting Opisthorchis viverrini antigen in faecal extracts of four groups of individuals. These were 24 patients with O. viverrini infection only (group 1), 31 patients with O. viverrini and other parasitic infections (group 2), 141 patients with other parasitic infections (group 3) and 21 normal, parasite-free individuals (group 4). The first antibody used in the ELISA was polyclonal immunoglobulin G prepared from the serum of a rabbit previously immunized with crude extract of O. viverrini. The second antibody was monoclonal antibody specific to an antigen located in the worm tegument and muscular tissue. Sensitivity of the assay was 31% while specificity was 100%. Considerations for improving the sensitivity are discussed.

Serum antibody responses in opisthorchiasis.

We evaluated an enzyme-linked immunosorbent assay using crude parasite homogenates as a diagnostic test for Opisthorchis viverrini infection in humans. Serum antibody (Ab) responses to O. viverrini adult worm homogenate (AWH) and metacercaria homogenate (MH) were studied in 83 infected residents of an opisthorchiasis-endemic area in Thailand. Elevated levels of Ab persisted for over 1 year following curative treatment with praziquantel, and cross-reactivity to O. viverrini AWH and MH antigens was observed in sera from individuals with other parasitic infections. Serum Ab to crude AWH and MH are therefore unsuitable for immunodiagnosis since they may be non-specific and would not differentiate between ongoing and past infection.

Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection.

Advanced third-stage larvae of G. spinigerum were obtained from two separate sources, namely from cysts in the livers of naturally infected eels (L3E) and from experimentally infected mice (L3M). Morphology of the L3E was studied microscopically. The larvae were homogenized in distilled water, 1% Triton X-100 or 1% sodium deoxycholate containing protease inhibitors. Protein compositions of the three crude extracts were compared, on the same weight basis, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining while their antigenicities were studied by Western blot analysis using serum of a patient with parasitologically confirmed gnathostomiasis. Distilled water was found to be the best extraction solution in solubilizing proteins especially the diagnostic antigen, namely the 24,000 (24 kDa) mol. wt component from the larvae. The L3E and L3M contained relatively equal amounts of the 24 kDa antigen. This diagnostic component was anatomically located in the body fluid, oesophagus and intestine of the larva.

Rapid detection of V. cholerae 01

Abstract Monoclonal antibodies directed against group specific antigen A of Vibio cholerae 01 were obtained through hybridoma technology which involved fusions of Sp 2/0 myeloma cells with splenocytes of Balb/c mice immunized with whole cell lysates of V. cholerae 01. Specificity and crossreactivity of the monoclonal antibodies were determined against whole cell lysates prepared from 38 strains of V. cholerae 01, six strains of V. cholerae non-01, five strains of other vibrios, 45 strains of enterobacteria and Entamoeba histolytica by indirect and dot-blot enzyme-linked immunosorbent assay (ELISA). The monoclonal antibodies (MAb) reacted specifically to the whole cell lysates of the V. cholerae serogroup 01 and did not react to the antigens prepared from other organisms. The MAb were used in a dot-blot ELISA for detecting V. cholerae 01 antigen in 335 seafood specimens and in stools and rectal swabs of patients with acute watery diarrhoea. The dotblot ELISA performed on rectal swab specimens enriched in alkaline peptone water gave 96.0% sensitivity, 99.3% specificity, 94.7% positive predictive value, 99.5% negative predictive value and 98.9% efficacy, respectively, when compared to the conventional culture method. The two methods showed excellent agreement beyond chance, on the basis of a k coefficient value of 94.7%. No V. cholerae 01 was isolated from the 335 seafood samples and the dot-blot ELISA for V. cholerae 01 antigen was also negative, consistent with a high specificity of the assay. The dot-blot ELISA is easy to perform, relatively inexpensive, highly sensitive and specific. It permits multisample analysis at a single time, requires no special equipment and does not pose any disposal problem (compared with the culture method). Most of all, diagnosis of cholera cases could be made accurately within 1–3 h (the dot-blot ELISA takes 1 h, while the culture method takes 2 days). The method is recommended for rapid detection of V. cholerae 01 in contaminated foods, in environmental samples and in stools of diarrhoeic patients.

Immunogenicity of two f0ormulations of oral cholera vaccines in Thai volunteers

A formulation of oral vaccine consisting of Vibrio cholerae lipopolysaccharides (LPS), cell-bound haemagglutinin (CHA) and procholeragenoid (P), namely vaccine A, was compared with another formulation, vaccine B, prepared from killed whole vibrios plus procholeragenoid on their immunogenicity and reactogenicity in Thai male volunteers. Volunteers were randomly allocated into three groups. The first two groups received orally three doses of vaccines A and B, respectively at 14-day intervals. Volunteers in group 3 were controls and received orally 100 ml 5% (w/v) NaHCO3 also at 14-day intervals. Serum samples were collected from all volunteers before each immunization. Intestinal lavage was performed 3 to 7 days before the first dose of vaccine or placebo and 7, 21 and 45 days after the last dose. Serum vibriocidal antibodies were determined and class-specific, antigen-specific antibodies of all serum and lavage samples were assessed by indirect enzyme-linked immunosorbent assay (ELISA) using purified LPS, CHA and cholera toxin (CT) as antigens. Diarrhoea occurred in 10 and 40% of the vaccinees ingesting the vaccines A and B, respectively. The immunogenicity of the vaccine B in terms of seroconversion for vibriocidal antibodies and anti-LPS was higher than the vaccine A. Both vaccines had equal immunogenicity concerning serum anti-CT, while the vaccine A was slightly better than the vaccine B on serum anti-CHA response. The immunogenicity of the two vaccines in evoking intestinal responses was different from the systemic one.(ABSTRACT TRUNCATED AT 250 WORDS)