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Mycobacterium cell wall and cell membrane proteins serve as key modulators in the host immune response. Some of these proteins, like MPT63, are known as immunodominant antigens, and others, such as LpqS and LpqH, are being explored as subunit vaccine candidates. However, due to their hydrophobic nature, conventional separation methods are not able to solubilize and isolate these proteins individually, leading to limited research on their systematic separation and immunological characterization.

In this study, we successfully employed a combination of Preparative Isoelectric focusing and Preparatory SDS-PAGE to separate mycobacterial cell wall and membrane proteins into 234 fractions. These fractions were pooled based on the proteins molecular weight, resulting in 24 pooled fractions labelled as Ag1 to Ag24. Immunological analyses on these pooled fractions were carried out in 3 different study groups (subjects with latent tuberculosis infection (LTBI, N=20), tuberculosis-diseased patients (PTB, N=20), and healthy subjects (HC, N=20). In these study groups, Interferon-γ and TNF-α responses against these antigens were analysed using the cytokine ELISA method.

Notably, one pooled fraction (Ag24 - Cell wall high molecular weight proteins pool) induced significantly higher IFN gamma response in LTBI compared to PTB, while also triggering significantly higher TNF-alpha response in PTB compared to LTBI. Additionally, three other pooled fractions (Ag6 - Cell Membrane Medium molecular weight proteins Pool, Ag11 - Cell Membrane high molecular weight proteins Pool, Ag23 - Cell wall high molecular weight proteins pool) induced significantly higher IFN gamma response in LTBI compared to PTB.

Proteomic analysis of the Ag24 fraction identified four proteins (ACN, KatG, Rv2623, and Rv0404). Among them, KatG and Rv0404 were previously reported as immunodominant T-cell antigens. On the other hand, ACN lacks immunological information, and limited information is available on protein Rv2623. In conclusion, extensive immunological studies on these four proteins could enhance their development as tuberculosis prognostic biomarkers, while our systematic 2-dimensional separation method shows promising potential for characterizing cell wall and membrane proteins in other pathogens