INFECTION AND IMMUNITY, Oct. 1978, p. 101-106 0019-9567/78/0022-0101$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 22, No. 1 Printed in U.S.A.
Immunological Method to Differentiate Between Antigens of Tubercle Bacilli, Other Mycobacterial Species, and Non-AcidFast Bacteria H. RODNEY FERGUSON,t J. KENNETH McCLATCHY, THOMAS R. SHARPTON, AND PERCY MINDEN* Departments of Medicine and Clinical Laboratories, National Jewish Hospital and Research Center, Denver, Colorado 80206 Received for publication 11 July 1978
Sera from rabbits immunized with sonicates of Mycobacterium bovis BCG were passed through an immunoadsorbent made of a soluble BCG extract to make partially purified antibodies to BCG. These antibodies were in turn used to prepare an immunoadsorbent through which the BCG extract was passed. The partially purified antigenic material was radiolabeled and subjected to electrophoresis in acrylamide gels. One of the radiolabeled fractions isolated (BCG-O) was found to bind to antibodies to BCG and H37Rv, but not to antibodies in sera from rabbits immunized with other mycobacterial species or Nocardia asteroides. The reaction between BCG-C and the partially purified antibodies to BCG was inhibited by small amounts of different BCG antigens. Cultures obtained from 25 patients with tuberculous diseases, other bacterial cultures, and various bacterial extracts were tested for their capacity to inhibit this reaction. Each of 13 mycobacteria identified as M. tuberculosis inhibited this reaction. Equivalent numbers of 12 strains of mycobacteria other than M. tuberculosis and high concentrations of other bacterial extracts did not inhibit, indicating that determinants of BCG present in M. tuberculosis were not detected in the other mycobacteria or in non-acid-fast bacteria. The use of sequential purification procedures could be of potential clinical value in quickly differentiating between M. tuberculosis and a variety of other mycobacteria. Many attempts have been made to study immunological reactions between mycobacterial antigens and antibodies (6, 10, 14). It has been difficult to compare and evaluate the methods used because tubercle bacilli consist of many antigenic components, some of which are shared with taxonomically unrelated bacteria (6,18,19). Furthermore, sera from animals and humans immunized with tubercle bacilli contain antibody populations that vary, depending on the test systems, in the way they react with mycobacterial antigens (3, 6, 7, 14, 24). A sensitive and specific method to measure the binding between defined mycobacterial antigens and antibodies would be desirable. It could be used for the precise study of immunological reactions involving mycobacteria and to detect small amounts of mycobacterial antigens. Specific, characterized antigens could also be of value in the study of cellular immunological reactions involving mycobacteria. In the present investigation, components of t Present address: Department of Veterinary Medicine, Colorado State University, Ft. Collins, CO 80523. 101
Mycobacterium bovis strain BCG were isolated by a series of immunochemical techniques. Antibodies specific for these components were similarly prepared. Small amounts of antibodies and mycobacterial antigens were then detected by way of a radioimmunoassay. With this system, it was possible to distinguish antigens from M. tuberculosis from those of non-acid-fast and other species of mycobacteria. MATERIALS AND METHODS Bacteria. The following bacteria obtained from the culture collection of this institution were grown as previously described (19): M. bovis strain BCG (Glaxo); M. tuberculosis strain H37Rv; M. kansasii; M. intracellulare; Nocardia asteroides; Staphylococcus aureus (strain Cowan 1); S. aureus (Wood strain); Listeria monocytogenes; Streptococcus pyogenes; Pseudomonas sp.; Salmonella typhimurium; Klebsiella pneumoniae; Proteus vulgaris; Propionibacterium acnes (Corynebacterium parvum); Escherichia coli; Haemophilus influenza; Brucella abortus; and Bordetella pertussis. Mycobacteria were also isolated from sputa from patients on the Tuberculosis Service at this institution. Sputa were processed by the N-acetyl-L-cysteine +
102
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FERGUSON ET AL.
NaOH technique (11), plated on 7H11 agar (4), and incubated at 370C in a 5% CO2 atmosphere for 3 weeks. Acid-fast bacteria were identified by standard biochemical methods (11). Portions of isolates were subcultured in 7H9 broth for 10 to 14 days at 370C, and colony-forming units (CFU) were determined by plate counts on 7H11 agar. Before use in the experiments described below, mycobacteria were heated at 80'C for 30 min. Antigens. Soluble extracts of BCG were prepared as previously described (14, 19). Briefly, heat-killed BCG cells were disrupted by sonic treatment, and the resulting supernatant fluids are referred to as BCG-S. This material has previously been referred to as BCGSS (14, 17). Extracts of other bacteria listed above were similarly prepared. Bovine serum albumin (BSA) was obtained from Armour Pharmaceuticals, Chicago, Ill. Serum and serum components. Sonicates of heat-killed bacteria were suspended in incomplete Freund adjuvant and injected into New Zealand white rabbits as previously described (19). Some animals were injected with BSA in incomplete Freund adjuvant. Antisera are referred to as anti-BCG, antiH37Rv, etc. Immunoglobulin G (IgG) was isolated by DE-52 chromatography of normal rabbit sera (NRS) and of some immune sera, using a 0.0175 M, pH 7.0 phosphate buffer (23). They are referred to as NRS IgG and anti-BCG IgG, respectively. Purity of IgG preparations was confirmed by immunoelectrophoresis, and protein content was determined by the method of Lowry et al. (12), using rabbit IgG as the standard. Immunoadsorbents. Immunoadsorbents were prepared by coupling BCG-S or immunoglobulins to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) (13). The washed immunoadsorbents were transferred to 25mm-diameter glass columns, and samples were applied. Phosphate-buffered saline, pH 7.0, was passed through the columns, and unbound substances were collected until no protein was detected with a spectrophotometer at 280 nm. Bound substances were then eluted with a 0.85% NaCl, pH 3.0 reagent and identified spectrophotometrically at 280 nm. Eluates were collected directly into borate buffer, pH 8.0 (at onetenth the volume of the eluate), to neutralize the pH quickly. When eluates consisted of BCG components, they were concentrated by Amicon filtration using a UM-10 filter. When they were immunoglobulins, they were concentrated by Amicon filtration using an XM100 filter. Measurement of antibodies. BCG-S, components of BCG-S, and BSA antigens were labeled with '25I as previously described (19). ['25I]BCG-S and other BCG components were diluted so that the cpm of 0.1 ml was 10,000 to 15,000. The nitrogen content of radiolabeled antigens was determined by an automated micro-Kjeldahl method (8). The binding capacities of antisera to radiolabeled antigens were tested by reacting dilutions of antisera or immunoglobulins with radiolabeled antigens. Dilutions of antisera and immunoglobulins were in 1:5 NRS in borate buffer and in 2 mg of NRS IgG per ml, respectively. Radiolabeled antigen-antibody complexes were then precipitated with anti-rabbit IgG prepared in goats. Results are
expressed as counts per minute in resulting precipitates or as percentage in the precipitates of trichloroacetic acid-precipitable counts added. In some experiments, the dilutions of antisera that precipitated 33% of radiolabeled antigens were determined. Details of the procedures and calculations involved have been described (15, 16). In some experiments, 0.1 ml of IgG preparation was preincubated overnight with 0.1 ml of borate buffer, unlabeled bacterial extracts, or suspensions of unfractionated, heat-killed bacteria. ['25I]BCG was then added, and binding capacities of sera were determined. Details of inhibition procedures have been described (15). Analytic gradient polyacrylamide electrophoresis. Nonlinear gradient polyacrylamide electrophoresis was carried out by using a system of gels with segments of 3.75, 4.75, 7.0, and 12.0% gel concentration (14, 25). Radiolabeled antigens with a radioactivity of 107 cpm per 0.3 ml were separated by electrophoresis as described previously (2). Upon completion of electrophoresis, gels were cut transversely with a gel cutter (Hoeffer Scientific Instruments, San Francisco, Calif.) into uniform disks 1.0 mm in width. Radioactivity of each disk was determined, and selected disks were incubated with 0.25 ml of deionized water at 40C overnight to elute radioactive components.
RESULTS
Preparation of purified anti-BCG (antiBCG-P). A 50-ml immunoadsorbent was prepared by coupling BCG-S to activated Sepharose in a ratio of 800 ,ug of nitrogen of BCG-S to 1 ml of activated Sepharose. Anti-BCG IgG was passed through a column containing this immunoadsorbent, and bound antibodies were eluted with the pH 3.0 reagent. This partially purified immunoglobulin (anti-BCG-P) was concentrated to 2.0 mg/ml, and its capacity to bind ['25I]BCG-S was compared with that of an equivalent concentration of unpurified anti-BCG IgG (Fig. 1). The 33% end point for the purified 100 W 80 a.
w
_
60;
~ ~ ~ 1:130
Z 40
Z
N
1:25
Vol. 22, No. 1 Printed in U.S.A.
Immunological Method to Differentiate Between Antigens of Tubercle Bacilli, Other Mycobacterial Species, and Non-AcidFast Bacteria H. RODNEY FERGUSON,t J. KENNETH McCLATCHY, THOMAS R. SHARPTON, AND PERCY MINDEN* Departments of Medicine and Clinical Laboratories, National Jewish Hospital and Research Center, Denver, Colorado 80206 Received for publication 11 July 1978
Sera from rabbits immunized with sonicates of Mycobacterium bovis BCG were passed through an immunoadsorbent made of a soluble BCG extract to make partially purified antibodies to BCG. These antibodies were in turn used to prepare an immunoadsorbent through which the BCG extract was passed. The partially purified antigenic material was radiolabeled and subjected to electrophoresis in acrylamide gels. One of the radiolabeled fractions isolated (BCG-O) was found to bind to antibodies to BCG and H37Rv, but not to antibodies in sera from rabbits immunized with other mycobacterial species or Nocardia asteroides. The reaction between BCG-C and the partially purified antibodies to BCG was inhibited by small amounts of different BCG antigens. Cultures obtained from 25 patients with tuberculous diseases, other bacterial cultures, and various bacterial extracts were tested for their capacity to inhibit this reaction. Each of 13 mycobacteria identified as M. tuberculosis inhibited this reaction. Equivalent numbers of 12 strains of mycobacteria other than M. tuberculosis and high concentrations of other bacterial extracts did not inhibit, indicating that determinants of BCG present in M. tuberculosis were not detected in the other mycobacteria or in non-acid-fast bacteria. The use of sequential purification procedures could be of potential clinical value in quickly differentiating between M. tuberculosis and a variety of other mycobacteria. Many attempts have been made to study immunological reactions between mycobacterial antigens and antibodies (6, 10, 14). It has been difficult to compare and evaluate the methods used because tubercle bacilli consist of many antigenic components, some of which are shared with taxonomically unrelated bacteria (6,18,19). Furthermore, sera from animals and humans immunized with tubercle bacilli contain antibody populations that vary, depending on the test systems, in the way they react with mycobacterial antigens (3, 6, 7, 14, 24). A sensitive and specific method to measure the binding between defined mycobacterial antigens and antibodies would be desirable. It could be used for the precise study of immunological reactions involving mycobacteria and to detect small amounts of mycobacterial antigens. Specific, characterized antigens could also be of value in the study of cellular immunological reactions involving mycobacteria. In the present investigation, components of t Present address: Department of Veterinary Medicine, Colorado State University, Ft. Collins, CO 80523. 101
Mycobacterium bovis strain BCG were isolated by a series of immunochemical techniques. Antibodies specific for these components were similarly prepared. Small amounts of antibodies and mycobacterial antigens were then detected by way of a radioimmunoassay. With this system, it was possible to distinguish antigens from M. tuberculosis from those of non-acid-fast and other species of mycobacteria. MATERIALS AND METHODS Bacteria. The following bacteria obtained from the culture collection of this institution were grown as previously described (19): M. bovis strain BCG (Glaxo); M. tuberculosis strain H37Rv; M. kansasii; M. intracellulare; Nocardia asteroides; Staphylococcus aureus (strain Cowan 1); S. aureus (Wood strain); Listeria monocytogenes; Streptococcus pyogenes; Pseudomonas sp.; Salmonella typhimurium; Klebsiella pneumoniae; Proteus vulgaris; Propionibacterium acnes (Corynebacterium parvum); Escherichia coli; Haemophilus influenza; Brucella abortus; and Bordetella pertussis. Mycobacteria were also isolated from sputa from patients on the Tuberculosis Service at this institution. Sputa were processed by the N-acetyl-L-cysteine +
102
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FERGUSON ET AL.
NaOH technique (11), plated on 7H11 agar (4), and incubated at 370C in a 5% CO2 atmosphere for 3 weeks. Acid-fast bacteria were identified by standard biochemical methods (11). Portions of isolates were subcultured in 7H9 broth for 10 to 14 days at 370C, and colony-forming units (CFU) were determined by plate counts on 7H11 agar. Before use in the experiments described below, mycobacteria were heated at 80'C for 30 min. Antigens. Soluble extracts of BCG were prepared as previously described (14, 19). Briefly, heat-killed BCG cells were disrupted by sonic treatment, and the resulting supernatant fluids are referred to as BCG-S. This material has previously been referred to as BCGSS (14, 17). Extracts of other bacteria listed above were similarly prepared. Bovine serum albumin (BSA) was obtained from Armour Pharmaceuticals, Chicago, Ill. Serum and serum components. Sonicates of heat-killed bacteria were suspended in incomplete Freund adjuvant and injected into New Zealand white rabbits as previously described (19). Some animals were injected with BSA in incomplete Freund adjuvant. Antisera are referred to as anti-BCG, antiH37Rv, etc. Immunoglobulin G (IgG) was isolated by DE-52 chromatography of normal rabbit sera (NRS) and of some immune sera, using a 0.0175 M, pH 7.0 phosphate buffer (23). They are referred to as NRS IgG and anti-BCG IgG, respectively. Purity of IgG preparations was confirmed by immunoelectrophoresis, and protein content was determined by the method of Lowry et al. (12), using rabbit IgG as the standard. Immunoadsorbents. Immunoadsorbents were prepared by coupling BCG-S or immunoglobulins to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) (13). The washed immunoadsorbents were transferred to 25mm-diameter glass columns, and samples were applied. Phosphate-buffered saline, pH 7.0, was passed through the columns, and unbound substances were collected until no protein was detected with a spectrophotometer at 280 nm. Bound substances were then eluted with a 0.85% NaCl, pH 3.0 reagent and identified spectrophotometrically at 280 nm. Eluates were collected directly into borate buffer, pH 8.0 (at onetenth the volume of the eluate), to neutralize the pH quickly. When eluates consisted of BCG components, they were concentrated by Amicon filtration using a UM-10 filter. When they were immunoglobulins, they were concentrated by Amicon filtration using an XM100 filter. Measurement of antibodies. BCG-S, components of BCG-S, and BSA antigens were labeled with '25I as previously described (19). ['25I]BCG-S and other BCG components were diluted so that the cpm of 0.1 ml was 10,000 to 15,000. The nitrogen content of radiolabeled antigens was determined by an automated micro-Kjeldahl method (8). The binding capacities of antisera to radiolabeled antigens were tested by reacting dilutions of antisera or immunoglobulins with radiolabeled antigens. Dilutions of antisera and immunoglobulins were in 1:5 NRS in borate buffer and in 2 mg of NRS IgG per ml, respectively. Radiolabeled antigen-antibody complexes were then precipitated with anti-rabbit IgG prepared in goats. Results are
expressed as counts per minute in resulting precipitates or as percentage in the precipitates of trichloroacetic acid-precipitable counts added. In some experiments, the dilutions of antisera that precipitated 33% of radiolabeled antigens were determined. Details of the procedures and calculations involved have been described (15, 16). In some experiments, 0.1 ml of IgG preparation was preincubated overnight with 0.1 ml of borate buffer, unlabeled bacterial extracts, or suspensions of unfractionated, heat-killed bacteria. ['25I]BCG was then added, and binding capacities of sera were determined. Details of inhibition procedures have been described (15). Analytic gradient polyacrylamide electrophoresis. Nonlinear gradient polyacrylamide electrophoresis was carried out by using a system of gels with segments of 3.75, 4.75, 7.0, and 12.0% gel concentration (14, 25). Radiolabeled antigens with a radioactivity of 107 cpm per 0.3 ml were separated by electrophoresis as described previously (2). Upon completion of electrophoresis, gels were cut transversely with a gel cutter (Hoeffer Scientific Instruments, San Francisco, Calif.) into uniform disks 1.0 mm in width. Radioactivity of each disk was determined, and selected disks were incubated with 0.25 ml of deionized water at 40C overnight to elute radioactive components.
RESULTS
Preparation of purified anti-BCG (antiBCG-P). A 50-ml immunoadsorbent was prepared by coupling BCG-S to activated Sepharose in a ratio of 800 ,ug of nitrogen of BCG-S to 1 ml of activated Sepharose. Anti-BCG IgG was passed through a column containing this immunoadsorbent, and bound antibodies were eluted with the pH 3.0 reagent. This partially purified immunoglobulin (anti-BCG-P) was concentrated to 2.0 mg/ml, and its capacity to bind ['25I]BCG-S was compared with that of an equivalent concentration of unpurified anti-BCG IgG (Fig. 1). The 33% end point for the purified 100 W 80 a.
w
_
60;
~ ~ ~ 1:130
Z 40
Z
N
1:25
