Electroimmunotransferblot assay for the detection of mycobacterial antigens in the cerebrospinal fluid for diagnosis of tubercular meningitis.

ABSTRACT

Background: The diagnosis of tubercular meningitis (TBM) is often delayed as it presents initially with non-specific signs and symptoms leading to delay in appropriate therapy. Conventional modes of diagnosis are time-taking and immunodiagnosis has its own pitfalls. Antigen detection assays have been found to be quite promising in this aspect. Aim: In the present study, attempts were made to evaluate the ElectroImmunoTransferBlot (EITB) test for detection of Mycobacterium tuberculosis antigens in CSF. Methods: A total of 46 CSF specimens were collected from 26 clinically suspected cases of TBM and 20 non-TBM cases. The mycobacterial antigens were concentrated by immunoprecipitation and separated based on their molecular weight by SDS-PAGE which were further transferred and immobilized onto a matrix and detected by EITB. Results: In TBM CSF specimens distinct bands of molecular weight 12kDa, 30-32kDa, 71kDa, 86kDa, 96kDa, 110kDa and 120kDa were seen in addition to 50kDa Immunoglobulin (Ig) heavy chain, 25kDa Ig light chain and an indistinct human albumin band at 69kDa. The control group CSF specimens also showed the Ig and albumin bands but showed no cross-reactive antigens. The following proteins 12kDa (7.7%), 30-32kDa (23%), 71kDa (19.2%), 86kDa (77%), 96kDa (57.5), 110kDa (23%) and 120kDa (15.4%) were identified as reactive bands. The results were compared to the reverse passive latex agglutination test. Conclusion: The likelihood of diagnosing TBM as evidenced by detecting at least a single mycobacterium specific band was 88.4% by our protocol for antigen detection in CSF. The specificity of EITB for diagnosing TBM was found to be 100% when the 86kDa antigen was excluded from the analysis. However, the method of diagnosis is labour/reagent intensive and needs substantial validation.