Generation of novel polyclonal antibodies against Mycobacterium tuberculosis lipoarabinomannan, EspB, and Mtb8

The unique cell wall of Mycobacterium tuberculosis (Mtb) creates a barrier to hydrophilic drugs, which is crucial for its survival and pathogenicity. However, the immune reactivity elicited by its components remains incompletely understood. We aimed to assess the antibody responses induced by Mtb H37Rv cell wall components and to develop and characterize antigen-specific polyclonal antibodies (pAbs). Rabbits were immunized with these components. Immune serum reactivity was tested against various Mtb antigens. Specific polyclonal antibodies (pAbs) were purified by affinity chromatography. The results showed that immune serum reacted with lipoarabinomannan (LAM), ESAT-6 secretion system-1 (Esx-1) secreted protein B (EspB), and Mtb8, but showed no reactivity with other tested Mtb antigens. LAM-, EspB-, or Mtb8-specific pAbs were subsequently affinity-purified. The affinity-purified LAM pAb, EspB pAb, and Mtb8 pAb each demonstrated high specificity and sensitivity, showing no cross-reactivity with non-target antigens. They recognized antigens in culture supernatants and cells from diverse mycobacterial strains, including both slow-growing mycobacteria (SGM) and rapid-growing mycobacteria (RGM). In a sandwich ELISA using LAM pAb as the capture antibody and biotinylated LAM-specific monoclonal Abs (BJRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, BJRbL52-Bio, or BJRbL76-Bio) as detection antibodies, the assay detected SGM but did not react with RGM species. EspB pAb recognized EspB in both cell lysate and culture supernatant fractions, where full-length and mature EspB are predominantly found, respectively. Mtb8 pAb reacted with monomeric and polymeric forms of Mtb8. In conclusion, we successfully generated novel pAbs against LAM, EspB, and Mtb8, providing promising research tools for investigating these critical molecules.