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

Abstract

Comparisons were made of the yield, chemical content, and biological activity of filtrates and extracts obtained by sonic and pressure cell disruption of bacilli from 4-and 8-week-old Proskauer and Beck cultures of the H37Rv strain (TMC no. 102) of Mycobacterium tuberculosis. The culture filtrates were dialyzed, freeze-dried, reconstituted in saline, and sterilized by membrane filtration. The viable bacilli were washed and resuspended in distilled water and subsequently disrupted either by sonication in the cold for 15 or 30 min or by treatment at 20,000 or 40,000 lb/in2 in a pressure cell. The resulting extracts were clarified by centrifugation, concentrated, and sterilized by filtration. All preparations were adjusted to contain 10 mg of solids (dry weight)/ml and were analyzed quantitatively for protein, deoxyribonucleic acid, ribonucleic acid, polysaccharide, and lipid content. Separation patterns obtained by gradient acrylamide gel electrophoresis, as well as by one-and two-dimensional immunoelectrophoresis, provided the basis for qualitative comparisons of the culture filtrates and cell extracts. Three-point dose-response curves also were used to compare the preparations for skin test reactivity in BCG-vaccinated guinea pigs. It was concluded that, although there were no consistent differences in chemical content or biological activity between the preparations, a 15-min sonic treatment appeared to be the most suitable method for preparation of bacillary extracts based on yield of active components and ease of preparation.

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

“…Kim et al (111, 129) related antigen production to the medium used for culture as well as to the age and pH of the culture at the time of harvest. Age-related variation in cell extracts also has been described by others (64,107,187,196). Incubation temperature has been reported to affect quantitatively but not qualitatively the antigen content of bacillary extracts and culture filtrates (124).…”

“…Whole cells have been disrupted by a variety of physical techniques, and whole organisms have been extracted in many ways. As reviewed by Janicki and his co-workers (107), mycobacteria have been disrupted by a variety of procedures, including mechanical grinding, pressure, and sonication. In fact, they showed that few antigenic differences could be detected in mycobacterial cell extracts prepared under different conditions.…”

“…Incubation temperature has been reported to affect quantitatively but not qualitatively the antigen content of bacillary extracts and culture filtrates (124). Finally, variation in antigenic composition of culture filtrates grown under closely parallel cultural conditions for identical times also has been observed (107). This poorly understood variability emphasizes the importance of using common reference systems for the identification of antigens being studied in different laboratories and of using single large batches of antigen source materials in individual laboratories.…”

Cross‐reactions between Mycobacteria

“…Crossed immunoeiectrophoresis (CIE) has even greater resolving power and more than forty antigenic components have been demonstrated in various mycobacterial species by this technique, e.g. in M. tttberctilosis [13,22]. M. smegmatis [14], A/, lepraenmritim [3], M. bovis BCG [10.…”

Antibody Response in Rabbits to Mycobacterium bovis BCG

“…In several mycobaeterial species more than forty distinct antigenic components have been identified by this technique, e.g. in Mycobaeteritmi tubercido.sis [17,22], M. smegmatis [18], M. lepraemtirium [6], M. bovis BCG [12,13], and in M. simiae [21]. CIE with intermediate gel is a sensitive method for detecting antibodies in various types of sera and is particularly valuable for characterizing their specificity.…”