Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens
| Autor: | Jeffrey R. Ryan, Anthony M. Smithyman, John M. Stiteler, Samuel K. Martin, Liwang Cui, Scott R Hillier, G. Halli R. Rajasekariah, Lisa P Yi |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
medicine.drug_class
Immunology Leishmania donovani Antibodies Protozoan Antigens Protozoan Enzyme-Linked Immunosorbent Assay Buffers Monoclonal antibody Absorbance Antigen Casein medicine Immunology and Allergy Animals Humans Serologic Tests biology Sodium Dodecyl Sulfate biology.organism_classification medicine.disease Molecular biology Solutions Visceral leishmaniasis Polyclonal antibodies biology.protein Leishmaniasis Visceral Electrophoresis Polyacrylamide Gel Antibody |
| Zdroj: | Journal of immunological methods. 252(1-2) |
| ISSN: | 0022-1759 |
| Popis: | An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 μg/ml) was coated with PBS–methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref−ve sera and higher absorbance with Ref+ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref+ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 μg/ml Ld-ESM mixed in PBS–methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n =22 endemic sera from North Africa were evaluated and resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials. |
| Databáze: | OpenAIRE |
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