Development of an enzyme immunoassay using recombinant invariant surface glycoprotein (rISG) 75 for serodiagnosis of bovine trypanosomosis.

Trypanosomosis or surra is caused by the haemoflagellate parasite, Trypanosoma evansi and is an important disease of animals, including domestic and wild herbivores and carnivores, in tropical countries. The invariant surface glycoproteins (ISGs) are blood stream stage specific and are uniformly distributed over the entire surface of the trypanosomes. In the present study, the extracellular domain (ED) region of ISG-75 from T. evansi, consisting of 1320 nt, encoding a polypeptide of 440 amino acids, has been heterologously expressed in Escherichia coli. Further, the immunoreactivity of recombinant ISG-75 (rISG-75) was characterized in immunoblot and ELISA using T. evansi hyper immune sera raised in experimental animals. The protein was found immunoreactive when compared with a panel of antigens (VSG RoTat 1.2 and whole cell lysate) using bovine serum samples from field. The diagnostic potential of rISG-75 was evaluated in ELISA with large number of bovine field serum samples. The optimum sensitivity and specificity were 98.47 and 99.1, respectively. The present finding showed that the expressed protein has potential use in the serodiagnosis of trypanosomosis.

Rapid serodiagnosis of leptospirosis by latex agglutination test and flow-through assay.

PURPOSE: Diagnosis of leptospirosis facilitates patient management and initiation of therapy. The microscopic agglutination test (MAT) is the serological test used in reference laboratories because of its high degree of sensitivity and specificity. But the results are not available quickly for patient management. In the present study, in order to develop a simple, rapid immunodiagnostic assay, one of the outer membrane proteins (OMPs), recombinant LipL41 (rLipL41) has been utilised in latex agglutination test (LAT) and flow-through assay. METHODS: Part of LipL41 gene was expressed in Escherichia coli system and purified. The rLipL41 antigen of pathogenic Leptospira interrogans serovar Icterohaemorrhagiae, which is conserved in all pathogenic Leptospira spp. was used as capture antigen in the LAT and flow-through test. Both tests are very rapid and could be completed within 5 minutes. The sensitivity and specificity of rLipL41 was assessed and evaluated in LAT and flow-through assay in comparison with standard MAT. RESULTS: The sensitivity and specificity of the LAT were 89.70 and 90.45% and flow-through assay were 89.09 and 77.70%, respectively. CONCLUSIONS: The developed LAT and flow-through assays were simple, rapid and economical for the detection of leptospira infection and suitable for large-scale screening of samples in endemic areas without any sophisticated equipment.

Evaluation of commercially available third-generation anti-hepatitis C virus enzyme-linked immunosorbent assay in patients on haemodialysis.

Restricted antibody reactivity to hepatitis C virus (HCV) synthetic peptides has been observed in HCV-infected patients on haemodialysis (HD). The aim of this study was to evaluate third-generation anti-HCV enzyme-linked immunosorbent assay (ELISA) test systems containing either synthetic peptide HCV antigens or recombinant HCV antigens or a combination of synthetic and recombinant antigens in screening of 69 chronic renal failure patients on HD for HCV infection. Seven patients were detected to have antibodies to HCV by the 'recombinant HCV antigens'-containing kits, of which the recombinant immunoblot assay for HCV confirmed four cases. The recombinant kits had a sensitivity of 100% and a specificity of 66%. However, the ELISA kits with only synthetic HCV antigens failed to detect antibodies in any of the cases (zero sensitivity). Hence a recombinant protein containing ELISA test system is ideal for screening of HCV infection in patients on hemodialysis.

Recombinant expression of Toxocara canis excretory-secretory antigen TES-120 in Escherichia coli.

The gene encoding the excretory-secretory antigen TES-120 of dog ascarid worm Toxocara canis was cloned into the bacterium Escherichia coli. The specificity of the recombinant TES-120 antigen produced by the bacterium was investigated. A total of 45 human serum samples from patients infected with differenthelminthes and protozoa, including 8 cases of toxocariasis, were tested against the recombinant antigens in immunoblot assays. The results from the assays revealed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.

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.

Sensitivity of EIA in the diagnosis of tuberculosis using 38-kDa antigen.

Patients, who reported to Government Medical College Hospital, Chandigarh with suspected signs and symptoms of pulmonary tuberculosis, were examined. Their sputum was investigated for the presence of acid-fast bacilli, using the routine Ziehl-Neelsen staining, for 3 consecutive days. The positive cases were referred for treatment and the rest were referred for supplementary tests like x-ray, Mantoux test, FNAC and the presence of IgG antibodies in their serum against recombinant 38-kDa antigen derived from Mycobacterium tuberculosis, using micro-ELISA plates. It was found that 37.8% of the cases who were negative for acid-fast bacilli found to be positive for the presence of antimycobacterial IgG antibodies with an antibody titre greater than the cut off value in addition to one of the above mentioned positive supplementary finding. Hence, EIA for tuberculosis is more sensitive as compared to the routine acid-fast bacilli staining.

Comparative results in detection of HCV antibodies by using a rapid HCV test, ELISA and immunoblot.

Viral hepatitis C is a major problem of post-transfusion hepatitis. The best measure for preventing hepatitis C infection in transfusion is blood donor screening. There are many methods for detecting hepatitis C virus antibodies. ELISA is one of the sensitive screening methods. However it needs equipment, technical skills and it is time consuming. The Genelabs Diagnostics HCV-SPOT is a simple, rapid test for the detection of anti-HCV. This paper reports comparative results in detection of HCV antibodies by using GLD HCV-SPOT, ELISA and GLD/DBL HCV BLOT. One hundred and ninety-two specimens from blood donors patients with chronic hepatitis, cirrhosis, hepatoma and miscellaneous chronic liver diseases were tested by HCV-spot assay and HCV ELISA. The positive specimens were confirmed by the immunoblot assay. Only one of 140 samples negative by HCV-SPOT was positive by ELISA. The sensitivity and specificity of HCV-SPOT assays were 97.6% and 92.6% respectively. The positive predictive value was 78.8%, negative predictive value was 99.3%, accuracy was 93.6%. The rapid HCV-SPOT assay can be used in the management of transplantation graft procedures or emergency blood screening for transfusion. In addition, it can be used in small, local hospitals without any expensive equipment.

Immunodiagnosis of swine trichinellosis.

A rapid, sensitive and specific serologic test has been developed for the diagnosis of swine trichinellosis. The ELISA based test utilizes L1 stichosome antigens recovered as excretory-secretory (ES) products from in vitro cultivated muscle larvae. Field studies conducted with 20,000 commercial swine using crude ES antigen demonstrated that the test could detect 98% of the medically significant infections. The test had a false-positive rate of less than 3%. Because of difficulties in regulating the quality and quantity of ES antigen and the need to continually maintain infected laboratory animals for producing the diagnostic reagent, efforts have been made to clone and express the gene(s) encoding the immunodominant ES antigens. To date a cDNA sequence, designated TsA-12, which codes in part for a 53-kDa ES antigen, has been identified and expressed in bacteria. Results demonstrate that TsA-12 is recognized by immune sera and further suggest that the immunodominant 45-, 48- and 53-kDa ES proteins which share antigenic epitopes are distinct glycoproteins.

A recombinant immunodiagnostic antigen for bovine cysticercosis.

The 70% ammonium sulfate-soluble fraction of the cyst fluid of Taenia hydatigena (designated ThFAS) was previously shown to have potential as an immunodiagnostic reagent for bovine cysticercosis. Western blot analysis indicated that the specific reactivity with antibodies in sera of T. saginata-infected cattle was associated with a 10 kDa component. Rabbit antiserum to ThFAS identified a homologous antigenic protein from the cestode Taenia crassiceps. Consequently, a cDNA expression library was constructed in lambda gt11 using poly A mRNA purified from T. crassiceps metacestodes and screened with rabbit antiserum to ThFAS. One strongly reactive clone (designated lambda TCA-2) produced a 123 kDa beta-galactosidase fusion protein which reacted in Western blot with sera from calves experimentally-infected with T. saginata and did not react with sera from uninfected calves or from cattle infected with Fasciola hepatica or with common gastrointestinal cattle parasites.