Detection of circulating free and immune-complexed antigen in pulmonary tuberculosis using cocktail of antibodies to mycobacterium tuberculosis excretory secretory antigens by peroxidase enzyme immunoassay.

ABSTRACT

Background: Decreased sensitivity has been a limiting factor of antigen assay for detection of tuberculosis. Assay of more than one antigen may improve sensitivity of an assay. Aim: To develop a simple, rapid and less-expensive serodiagnostic method compared to culture method for Pulmonary Tuberculosis. Method: A cocktail of affinity purified antibodies against Mycobacterium tuberculosis H37Ra antigens (SEVA TB ES-31, ES-43 and EST-6) was explored for detection of circulating free and Immune-Complexed (IC) cocktail antigen by microtitre plate Peroxidase sandwich ELISA. The assay was evaluated in 27 clinical sera of sputum acid fast bacilli (AFB) positive and 10 AFB negative but anti-tuberculosis therapy responded pulmonary tuberculosis patients and 20 normal sera as controls. Results: Assay of cocktail antigen showed marginal improvement in sensitivity compared to assay of ES-31 antigen alone. The assay for circulating free cocktail antigen showed a sensitivity of 77.7% for AFB positive cases and 70% for AFB negative cases compared to assay of ES-31antigen with sensitivity of 74% and 70% respectively. The assay for ICcocktail antigen showed sensitivity of 77.7% for AFB positive and 80% for AFB negative cases compared to assay of ICES- 31 antigen with sensitivity of 77% and 70% respectively. Specificity of antigen assay was found to be 90%. Detection of IC-antigen as adjunct assay improved the sensitivity of detection in AFB-ve but ATT responded cases. Peroxidase enzyme immunoassay of cocktail antigen showed a sensitivity of detection of 0.25 μg/ ml and levels of free and IC cocktail antigens were 1.70 ± 1.04 and 1.13 ± 0.047 μg/ ml in AFB positive patients’ sera. Conclusions: Peroxidase enzyme immunoassay for circulating antigen was found to be a useful serodiagnostic assay and in particular in AFB –ve cases responding to ATT.