ABSTRACT
Objective To prepare a novel monoclonal antibody (mAb) that specifically against Mycobacterium tuberculosis culture filtrate protein 10 (CFP-10).Methods The BALB/c mice were immunized by a peptide with 14 amino acids (aa residues 53 to 66) of CFP-10,and then the splenocytes of mice were fused with myeloma cell line SP2/0.The resultant fused cells were subjected to screening culture,enzyme linked immunosorbent assay (ELISA) assay and subcloning by limited dilution to establish hybridoma cell lines of stable secreting anti-the peptide of CFP-10 antibody.The antibody was purified,and its isotypes were analyzed.Then,the antibody was further evaluated by Western blotting,immunoprecipitation and ELISA in 38 culture supernatant samples of Mycobacterium tuberculosis,20 culture supernatant samples of non-Mycobacterium tuberculosis,32 samples of tuberculous pleural effusion,24 samples of non-tuberculous pleural effusion,and 20 serum samples from healthy controls.Results The isotype of the mAb against the specific peptide of CFP-10 was an IgG1 with κ chain,and it was applicable for Western blotting and immunoprecipitation analysis.ELISA quantitative test showed that the sensitivity and specificity for diagnosis of Mycobacterium tuberculosis were 78.6％ (55/70) and 92.2％ (59/64),respectively.Conclusion The mAb generated against the specific peptide of CFP-10 is high in sensitivity and specificity,and it might be used in the early diagnosis of tuberculosis.
ABSTRACT
Background & objectives: The immune responses to different antigens of Mycobacterium tuberculosis H37Rv vary from patient to patient with tuberculosis (TB). Therefore, significant difference might be documented between the H37Rv with long histories of passages and recent clinical isolates of M. tuberculosis. In the present study, immune response of TB patients and healthy controls against 39 clinical M. tuberculosis isolates was correlated with laboratory strain H37Rv. Methods: The antibody response was studied coating whole cell extracts and culture filtrate proteins of M. tuberculosis isolates and laboratory strain H37Rv by enzyme linked immunosorbent assay (ELISA). Lymphoproliferation was studied by incorporation of tritiated thymidine and cytokines (IFN-γ and IL-4) by using commercially available kits. Results: Sero-reactivity to whole cell extract (WCE) of 11 clinical isolates was higher with pooled serum and individual's serum from tuberculosis patients showed significant reactivity (P<0.05) to ten of these isolates using ELISA. Of the WCE of 39 clinical isolates, 10 were found to be potent inducer of lymphoproliferation as well as cytokine secretion (P<0.05) in peripheral blood mononuclear cells from PPD+ healthy controls. Six culture filtrate proteins (CFPs) from these selected clinical isolates were also better inducers of antibody and T-cell response. Interpretation & conclusion: Overall, our results revealed that the clinical isolates belonging to prevalent genotypes; CAS1_Del (ST-26), East African-Indian (ST-11) and Beijing family (ST-1) induced better antibody and T cell responses compared to H37Rv laboratory strain. Further studies need to be done to purify and identify the dominant protein (s) using whole cell extract and culture filtrates from these immunologically relevant clinical M. tuberculosis isolates, which will be worthwhile to find out pathogenic factors, potential diagnostic markers and protective molecules for tuberculosis.