Development of monoclonal antibodies reacting against mycobacterial 65 kDa heat shock protein by using recombinant truncated products.

A mycobacterial 65 kDa molecule is a member of the GroEL heat shock protein family. We developed mAbs reacting against recombinant 65 kDa protein by using a gene (pTB12) which encodes this protein. Three mAbs (B20, B97 and B167) reacted selectively with 65 kDa proteins of Mycobacterium tuberculosis, BCG and Mycobacterium leprae, although B20 and B167 may weakly react with a 15 kDa molecule of mammalian cells. One (B108) was obviously cross-reactive between mycobacterial 65 kDa and the mammalian intracytoplasmic protein. We also developed deletion mutants of pTB12. The localization of these mAb-defined epitopes was determined by using truncated proteins of the Mycobacterium tuberculosis 65 kDa molecule produced in E. coli. Immunohistochemical analysis showed that B20, B97 and B167 mAbs could detect this antigen in experimental granulomas induced by injection of BCG in the subcutaneous tissue of rats. These mAbs should be useful for analyzing the immunobiologic roles of mycobacterial 65 kDa molecules.

Relationship between T-cell population in neonatally thymectomized Lewis rats and susceptibility to infection with mycobacterium leprae.

The neonatally thymectomized Lewis rat (NTLR) is highly susceptible to infection with M. leprae. However, a significant percentage of NTLR respond to infection with M. leprae in much the same way as do intact rats, yet show no evidence of residual thymus. To determine whether there was a correlation between the number of remaining T-cells and susceptibility to infection with M. leprae, a direct fluorescent antibody test was performed using a highly specific, absorbed antithymocyte globulin labeled with fluorescein isothiocyanate. Both total circulating white blood cells and T-cells were significantly depressed in all NTLR examined. Although the greatest numbers of M. leprae were found in NTLR from the groups having the lowest percentage of circulating T-cells, these groups also contained NTLR infected with small numbers of M. leprae. The groups containing NTLR with the highest percentages of circulating T-cells also contained animals with both moderate and severe M. leprae infection. The response of cultured splenic lymphocytes from NTLR and normal rats to the T-cell mitogen concanavalin A was investigated to determine whether there was any correlation between T-cell activity and susceptibility to M. leprae infection. The mean stimulation index for normal rats was five to ten times greater than indices for NTLR, but there were no significant differences between NTLR with a well developed, generalized infection and those with a poorly developed infection. it was concluded that since there was no apparent relationship between T-cell depletion and susceptibility to infection with M. leprae, an additional, unknown mechanism was also involved.

New method for concentration and quantitation of Mycobacterium leprae.

A new method of enumerating Mycobacterium leprae has been developed. Suspensions containing the organisms were filtered through a polycarbonate membrane filter (25-mm diameter, 0.4-micronm pore size, 10-micronm thick; Nucleopore) to concentrate the organisms. The membrane was then mounted on a glass slide and stained with a standard acid-fast stain. Finally, the membrane was treated with a small amount of chloroform to fix it to the slide and make it transparent. This method enabled us to detect M. leprae in quantities as small as 4.98 X 10(2) regardless of the total volume of the original material. Comparison with a standard method for enumerating M. leprae showed that both methods gave similar results when the organisms counted by the standard method were present in sufficient quantity for reproducibility. Because the least number of organisms that can be detected with the standard method is 10(4) ml and because the organisms detected with the new method could be concentrated on the polycarbonate filter from a large amount of infected fluid, a substantial number of suspensions were shown by the new method, but not by the standard method, to contain M. leprae.