Characterization and Comparison of Mycobacterial Antigens by Two-Dimensional Immunoelectrophoresis

Abstract

Two-dimensional immunoelectrophoresis (2D-IEP), in which a complex of antigens is subjected to electrophoresis first through an agarose matrix in one direction and secondly through an antiserum-agarose matrix at right angles to the first direction, was evaluated as a tool for analysis of mycobacterial antigens. Cell extracts from four species of mycobacteria, Mycobacterium tuberculosis (four strains), M. bovis strain BCG, M. scrofulaceum, and M. phlei, were assayed by 2D-IEP with four anti-mycobacterial antisera. Besides displaying the precipitin curves in a more easily interpreted format than did conventional immunoelectrophoresis (IEP), 2D-IEP offered greater sensitivity in terms of numbers of precipitin curves when like reactions were compared with IEP patterns. As many as 60 immunoprecipitates were observed on 2D-IEP slides compared to 18 on comparable IEP plates. Technical reproducibility of patterns from run to run was excellent. Other parameters, such as the influence of using different batches of antigen on the pattern, are discussed. Each of the cell extract antigens gave a unique pattern of precipitin peaks which could be easily differentiated from the patterns given by the other mycobacterial cell extracts when reacted with any of the antisera in 2D-IEP. Since both the species and strains of mycobacteria could be easily and reproducibly differentiated solely on the basis of two-dimensional immunoelectrophoretic patterns obtained with any of the antisera employed in this study, it may be possible, by using IEP, to differentiate and identify all species and strains of mycobacteria with one standard, highly sensitive antiserum, rather than a battery of antisera.

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

“…The complexity of the immunoprecipitate pattern obtained on crossed immunoelectrophoresis of cytoplasmic fractions is comparable 1285 VOL. 13,1976 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from 1286 SMYTH, FRIEDMAN-KIEN, AND SALTON with those for similar soluble antigens from P. aeruginosa (55 immunoprecipitates), Mycobacterium species (40 to 60 immunoprecipitates), Candida albicans (78 immunoprecipitates), and Micrococcus lysodeikticus (>70 immunoprecipitates) (2,12,21,42; P. Owen, personal communication).…”

“…The antigenic complexity of several microorganisms has been revealed in recent years by crossed immunoelectrophoresis (2,12,21,24,37,42,50,51,53). The method is superior in sensitivity and resolution to immunodiffusion by the Ouchterlony technique and immunoelectrophoresis as introduced by Grabar and Williams (55).…”

Comparison of antigens in sonic and pressure cell extracts of Mycobacterium tuberculosis

“…However, because they are autolytic products and subject to continuous enzymatic alteration and degradation during incubation, the antigenic composition of culture filtrate preparations can vary markedly, not only between strains of tubercle bacilli but also between identical cultures of the same strain (3, 21, 26). Preparations of mycobacterial cytoplasmic extracts, in contrast, have been reported to be uniform in composition (3,6,21,22,(26)(27)(28). In other studies, bacillary extracts have been demonstrated to be suitable materials for immunological study.…”

Cross‐Reactions between Mycobacteria

“…immunoelectrophoresis (7) and crossed immunoelectrophoresis (1 I), efforts have been made by several groups to define a reference system for the study of mycobacterial antigens. Roberts et al (11) suggested that a single sensitive antiserum against M . tuberculosis be used in the analysis of the antigen pattern of all species and strains of mycobacteria in crossed immunoelectrophoresis.…”

“…Drawing of the same pattern as shown in A. Due to lack of space three of the lines have not been numbered: no 11. (dotted) just beneath no.…”