Padronização inicial de teste de ELISA indireto para diagnóstico de tuberculose pulmonar

Abstract

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis, which remains a major public health challenge, especially due to the complexity of early and effective diagnosis. Although the infection primarily manifests in the pulmonary form, several important extrapulmonary clinical manifestations are also described. Accurate detection is crucial for implementing effective treatment and preventing transmission. Traditional methods such as smear microscopy and mycobacterial culture are still widely used, but they present significant limitations in terms of sensitivity, response time, and the need for specialized labor. Early diagnosis is essential, as it significantly increases the chances of cure, with success rates reaching up to 85% when treatment is initiated promptly. The use of faster and more sensitive technologies, such as molecular testing, represents a major advance toward disease control, especially in low-income countries still facing financial and accessibility challenges. In this context, the development of more accessible diagnostic alternatives, such as the adaptation of the ELISA test for detecting antibodies against recombinant M. tuberculosis antigens, emerges as a promising strategy. This study aimed to standardize an indirect ELISA protocol using the recombinant proteins PMT64 and MTG3F3, mimetic to M. tuberculosis antigens, to improve the sensitivity and specificity of TB detection. Various preparation conditions were evaluated to develop a diagnostic test that is easy to apply and has high discriminatory power between infected and healthy individuals. Three critical parameters were assessed: blocking solution, serum dilution, and antigen concentration. Bovine serum albumin (BSA) at 3% showed better performance in reducing nonspecific binding. Regarding serum samples, a 1:100 dilution provided the best balance between sensitivity and specificity, although the response may vary depending on individual antibody loads. As for antigen concentration, 100 ng for MTG3F3 and 250 ng for PMT64 were more effective in differentiating positive cases from healthy controls. The results highlight the importance of carefully optimizing each step to ensure assay reliability.