[Detection of somatic antigens in Mycobacterium avium subspecies paratuberculosis]

Johne's disease or Paratuberculosis as a chronic granulomatosis enteritis in ruminants will be caused by Mycobacerium avium subsp . Paratuberculosis . Detecting whole bacterial cell wall antigens would be helpful in potential applications for diagnosis, vaccine production, and elucidation of pathogenesis . To determine secreted somatic cell antigens of Mycobacterium avium subspecies Paratuberculosis . Standard strain [III-V] of Mycobacterium avium subspecies Paratuberculosis DNA was extracted from the cultured and gene analysis was done using PCR to confirm bacterial purity . On the other hand, protein concentrations in both media and cell entracts were determined . Furthermore, all proteins pattern were shown by SDS -PAGE . Electrophoretic findings showed some somatic antigens in the range of 19 - 100 KDa. These purified somatic antigens can be used for further study and potential application in vaccine production

[Preparation of neuraminidase specific antiserum from H9N2 subtype of avian influenza virus]

In this research, affinity chromatography have been developed and standardized for production of Neuraminidase antigen of influenza virus for preparation of monospecific antiserum in rabbits. Avian influenza Virus stocks [A/chicken/Iran/259/1998/[H9N2]] were propagated in the allantoic cavities of 10-day old embryonated chicken eggs. The harvested suspension was concentrated by polyethylenglycol 6000. Concentrated samples were layered onto sucrose gradient [30-60%]. Both hemagglutinin and neuraminidase were solubilized from purified viruses with Triton X-100, across 30% sucrose gradient. NA was isolated from HA and other viral proteins by affinity chromatography on N- [paminophenyl] oxamic acid. Fractions that had high NA-activity and did not show HA activity were pooled and analyzed by neuraminidase inhibition and SDS-PAGE. For preparation of antisera, rabbits were immunized by purified NA and Freund's adjuvant at three weeks interval, and sera collected 7 days after boosting. In SDS-PAGE no viral protein band detected except for single band in the position of NA. NA activity of purified protein was 3.8 x 104 NA units. Enzymatic activity of Neuraminidase purified by this procedure decrease sharply above 48°C. The purified neuraminidase was producing a significant antibody response in agar gel precipitation. No reaction was observed with neuraminidase specific antiserum and H9-HA of the same virus. According to virtual purity and enzymatic activity of purified neuraminidase and highest avidity and specificity of antiserum, it was speculated that optimized protocol can be directly applied to produce antigen and antiserum from all subtypes of virus and can be easily used in commercial diagnostic tests

Designing an ELISA technique for H. pylori antibody detection using water extracted antigens

H.pylori infection stimulates immune responses. These responses at the mucosal level are predominantly of IgA types, while circulating antibodies against this microorganism are predominantly IgG classes. IgM antibodies are rarely found and seem to be non-specific for this bacterium. In this research, water extract antigen, from three strains of H.pylori [isolated from patients with gastritis, duodenal ulcer and normal human] was investigated for the detection of serum IgG antibodies against H.pylori by an indirect ELISA technique. Antibody titers against H.pylori were measured in 72 patients of whom 64 cases were H.pylori positive and 8 cases were H.pylori negative [confirmed by culture and urease tests]. In this test, those titers that were more than 1/6400 indicated the rising of IgG titers and serum positive, being in testee, and the titers, which were equal or less than 1/6400 indicated the serum negative, being in individuals. Our ELISA results indicated that between 64 H.pylori positive individuals, 61 cases were serum positive and between 8 H.pylori negative patients, 5 individuals were serum negative; thus, specificity, sensitivity, positive predictive value [PPV] and negative predictive value [NPV] of the test were, 62.5%, 95.31%, 95.31%, 62.5%, respectively. The high level of sensitivity is because of using 3. different strains for preparing of antigens. But the reasons of low specificity are probably using of semi purified antigen