Characterization and Comparison of Mycobacterial Antigens by Discontinuous Pore Gradient Gel Electrophoresis

Abstract

Concentrated culture filtrates and cell extracts were prepared from selected mycobacteria and analyzed by a multistage polyacrylamide electrophoretic procedure. Various staining procedures were used to detect protein, carbohydrate, nucleic acid, and lipid constituents. Visual examination of the more prominently stained components indicated differences and similarities in each species of Mycobacteria. The majority of the individual components were of small molecular weight; however, there appeared in all culture filtrates and extracts examined a slowly moving protein component of relatively large molecular size. Evidence based on immunodiffusion suggests that it is a common mycobacterial antigen.It has been demonstrated by a number of studies that the mycobacterial cell and its metabolic products are of an exceedingly complex nature. The biology of the tubercle bacillus and the relationship of its complex products within the host have been studied for many years to better understand the pathogenesis of tuberculosis. There have been many physicochemical techniques which have been applied to the separation and isolation of these complex cellular products. It is also important that their chemical composition be known so that the mechanism of tuberculous disease and, consequently, how to combat it may be better understood. Moreover, by employing more refined mycobacterial fractions of known composition, the similarities and differences that exist among the various acid-fast organisms could be more sharply defined and thus contribute to a more satisfactory understanding of mycobacterial relationships.The present study is a direct outgrowth of our long-range investigations concerned with developing and standardizing methods for isolating and purifying mycobacterial antigens and elucidating their possible biological effects. Specifically, it has been one of the major aims of this study to identify and compare mycobacterial antigens by utilizing a modified pore gradient polyacrylamide electrophoresis method. The present report characterizes these biologically active components from various selected strains of mycobacteria utilizing this technique. Antigenic profiles from each of them are obtained and compared, and the possible interrelationships are examined. MATERIALS AND METHODS Source of organisms for culture filtrates and bacterial extracts. Selected mycobacterial species were obtained from the American Type Culture Collection and the culture collection of the Microbiology Department of The George Washington University School of Medicine. These included: Mycobacterium tuberculosis H37Ra, M. tutberculosis H37Rv, M. bovis, M. bovis BCG, M. aviuim, M. kansasii, M.intracellitlare, M. scrofulaceum, M. phlei, M. fortuitum, and M. smegmatis. Cells were grown as surface pellicles on Proskauer-Beck medium at 37 C for 6 to 8 weeks before separation from the culture fluid by Seitz filtration. The culture filtrates were concentrated by pervaporation to one-tenth their original volume. The total volumes obtained were each divided ...

Mycobacterial antigens: a review of their isolation, chemistry, and immunological properties.

“…Augier and Augier-Gibory (9) and Wright and co-workers (202) have introduced two-dimensional procedures for analytic purposes. Affronti et al (5) introduced the use of discontinuous gel gradients in this technique. Finally, Minden and Farr (133) developed a preparative scale method for using gradient acrylamide gel electrophoresis to fractionate mycobacterial antigens.…”

“…Other side chains contain principally mannose shown. If the D-galactose residues are in the furanose form, the linkage should be (1)(2)(3)(4)(5). Reproduced with permission of the Journal ofBiochemistry.…”

Characterization and Comparison of Mycobacterial Antigens by Two-Dimensional Immunoelectrophoresis

“…Although gel diffusion and immunoelectrophoresis have been used for the analysis of mycobacterial antigens (3,14,16,17), they are not sensitive enough for studying all the antigenic moieties of a crude or multicomponent antigen mixture. For example, acrylamide pore gradient electrophoresis of a mycobacterial cell extract preparation results in the detection of between 40 and 60 bands which stain with proteinspecific dyes (1); yet in immunoelectrophoresis only 11 to 20 bands appear after reaction with 564 on August 27, 2020 by guest http://iai.asm.org/ Downloaded from homologous antiserum (L. F. Affronti and G. L. Wright, Jr., 1968, Amer. Rev.…”

Characterization and Comparison of Mycobacterial Antigens by Two-Dimensional Polyacrylamide Gel Electrophoresis

“…Because of the greater resolving power of the 2-D electrophoretic procedure, it was thought that this system might be more sensitive for the taxonomic characterization of mycobacteria than the step-gel, multistage technique described in the preceding paper (2). Furthermore, several physicochemical properties of the crude cell preparation can be measured from the 2-D electropherograms, such as: (i) complexity of the biologic mixture (cell extracts); (ii) estimation of relative molecular weight of the separated components; and (iii) determination of the optimal pore size (gel concentration) for the subsequent isolation of specific components (antigens).…”