Vol. 58, No. 5

INFECTION AND IMMUNITY, May 1990, p. 1445-1449 0019-9567/90/051445-05$02.00/0 Copyright © 1990, American Society for Microbiology

Production, Characterization, and Species Specificity of Monoclonal Antibodies to Mycobacterium avium Complex Protein Antigens DAVID A. ROUSE, SHELDON L. MORRIS, ARTHUR B. KARPAS, PETER G. PROBST, AND S. D. CHAPARAS* Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 Received 16 October 1989/Accepted 13 February 1990

The incidence of Mycobacterium avium-Mycobacterium intracellulare complex infections has increased in years primarily because a significant proportion of acquired immunodeficiency syndrome patients develop disseminated M. avium complex disease. In an effort to develop new tools to study these infections, we have produced eight monoclonal antibodies directed against M. avium. Western blot (immunoblot) specificity analysis and protease sensitivity assays indicate that four of these antibodies recognize M. avium-specific protein epitopes and two react with M. avium complex-specific peptide determinants. These monoclonal antibodies may be useful clinically in the diagnosis of M. avium complex disease and in the laboratory for isolation and characterization of native and recombinant M. avium complex antigens. recent

The Mycobacterium avium-Mycobacterium intracellulare complex (MAC) bacilli are among the most frequent bacterial isolates from individuals with acquired immunodeficiency syndrome (AIDS) (6, 14, 20). About 50% of AIDS patients have been reported to be infected with MAC in some regions of the United States (5). In contrast to nonimmunocompromised hosts where the clinical manifestations are usually pulmonary in nature, MAC bacteria often cause a severe disseminated disease in people afflicted with AIDS. Detection of MAC infections is difficult and is often not determined until autopsy (23). Recent hospital-based studies have indicated that at least 10% of AIDS patients may also have tuberculosis. Chemoprophylactic regimens against tuberculosis are recommended for persons who are seropositive for human immunodeficiency virus and have a positive tuberculin test (18). Because of antigenic cross-reactivity between mycobacterial species, positive tuberculin tests are not always indicative of tuberculosis infection and may result from exposure to nontuberculous mycobacteria (2, 3, 4). The differentiation between MAC and Mycobacterium tuberculosis infections is of clinical importance because M. tuberculosis can be treated with standard antituberculosis drugs, while MAC strains are often resistant to these and other antibiotics. Preventive treatment of human immunodeficiency virusseropositive persons suspected of being infected with MAC may abort the severe clinical complications of MAC-disseminated disease if the patient develops AIDS. We report here the production and characterization of eight anti-M. avium monoclonal antibodies (MAbs) which react with MAC protein-associated epitopes. Our results suggest that four of these MAbs recognize M. avium epitopes and two are specific for MAC epitopes. These antibodies may be especially useful for the diagnosis of MAC infections, for purification and characterization of native mycobacterial antigens, and for screening MAC Xgtll expression libraries (15). Immunoreactive peptides derived from MAb-reactive recombinant mycobacterial proteins being expressed in these gene libraries will be evaluated as specific skin test and serodiagnostic reagents and as components of acellular vaccines. *

MATERIALS AND METHODS

Preparation of mycobacterial antigens. Mycobacterial strains were obtained from the Trudeau Mycobacterial Culture Collection (Saranac Lake, N.Y.), currently housed at the American Type Culture Collection (Rockville, Md.). Additionally, 10 M. avium strains were isolated from 10 different AIDS patients and the serovars were determined at the Centers for Disease Control. Most of the mycobacterial antigen preparations used were sonicates of actively proliferating mycobacteria grown in Long's synthetic medium at 37°C for various periods of incubation, depending upon the rate of growth of the strain. At late-log phase, the cells were harvested by centrifugation, washed in deionized water, sonicated, and centrifuged as previously described (2). To prepare M. avium serovar 4 antigens for immunization, the sonicate supernatant was precipitated with an equal volume of saturated ammonium sulfate at 4°C for 16 h. The precipitate was centrifuged at 10,000 x g at 4°C and suspended in a minimal volume of phosphate-buffered saline. The preparations were then dialyzed against several changes of phosphate-buffered saline at 4°C for 16 h. Protein concentrations of mycobacterial sonicates and ammonium sulfate precipitates were determined by using the Bio-Rad protein assay system (Bio-Rad Laboratories, Richmond, Calif.). Production of MAbs. BALB/c mice were injected intraperitoneally 3 times in 1 week with 8.8 ,ug per injection of ammonium sulfate precipitate of sonicate from a M. avium serovar 4 strain. This M. avium strain was isolated from an AIDS patient with MAC disease. Intermittent bleedings of these animals indicated that serum M. avium antibody levels remained high for several months. After 5 months, when specific antibody levels had fallen, the mice (spleen donors) were given an intravenous booster with 1.2 ,ug of the same M. avium sonicate. Cell fusions were performed 3 days later by the method of Kohler and Milstein (11), with the following modifications. Nonsecreting mouse myeloma cells Sp2/ 0-Agl4 and BALB/c spleen cells were washed separately in Dulbecco modified Eagle medium, combined in a ratio of 1:2 (1 x 108 spleen cells), packed, and drained. Two milliliters of 50% polyethylene glycol 1000 in Dulbecco modified Eagle medium was added at 37°C with stirring. The suspension was repacked at room temperature and diluted to 24 ml with stirring at 37°C, all over a period of 5 min. The fusion

Corresponding author. 1445

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mixture was centrifuged, decanted, and suspended in 50 ml of Dulbecco modified Eagle medium containing 20% fetal bovine serum and other supplements (also known as super medium), as well as hypoxanthine-aminopterin-thymidine selection mixture. Dilutions of this cell mixture were added (100 ,ul per well) to 12 plates containing 50 ,ul of endothelial cell growth supplement (150 ,ug/ml) in super medium plus hypoxanthine-aminopterin-thymidine. Plates were incubated for 10 days at 37°C at 5 to 6% CO2 and 95 to 100% humidity. After 10 days of incubation, growing colonies were counted and fed with 100 ,ul of super medium per well. Feeding was repeated again between days 13 and 15 of incubation. Wells were observed for colonial growth, and when colonies filled one-third to one-half of the well bottom, 300 ,ul of medium was removed for assay. From day 15 to day 17, selected wells were harvested and cells were cloned by limiting dilution in super medium to 1 cell per well or 0.3 cell per well. Residual cells in original wells were expanded to allow freezing of at least 3 vials at 5 x 106 cells per ml in 10% dimethyl sulfoxide in fetal bovine serum. Cells subsequently cloned by limiting dilution were similarly assayed, expanded, and frozen. Of the 1,152 original wells, 720 contained colonies. Ninety-six of these colonies produced antibody, and 82 of these were selected for further study. Screening of hybridomas. One hundred and fifty micrograms of M. avium serovar 4 ammonium sulfate precipitate was electrophoresed through 10% acrylamide preparative gels by using the Mini-Protean II system (Bio-Rad Laboratories). Separated proteins were then transferred to nitrocellulose. The resulting filters were blocked for at least 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline. The blocked Western blots (immunoblots) were incubated with hybridoma supernatants for 1 to 2 h at room temperature by using the Miniblotter 28 apparatus (Immunetics, Cambridge, Mass.). After washing with Tris-buffered saline-0.05% Tween 20, the filters were incubated with anti-mouse immunoglobulin G (whole molecule)-alkaline phosphatase conjugate (Sigma Chemical Co., St. Louis, Mo.). Following several more washes with Tris-buffered saline-Tween 20, the blots were developed with the Bio-Rad alkaline phosphatase detection system. Isotyping and species specificity testing of the M. avium MAbs. Determination of immunoglobulin class and subclass was accomplished with an enzyme-linked immunosorbent assay screening-isotype kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). Costar Serocluster enzyme immunoassay microdilution plates (Costar, Cambridge, Mass.) were coated with 1 ,ug of M. avium (1 ,ug in a 60-,ul volume per well) diluted in a 1:4 solution (vol/vol) of Dulbecco phosphate-buffered saline for 3 h at room temperature. Wells were blocked with a 1% solution of bovine serum albumin in Dulbecco phosphate-buffered saline for 2 h at room temperature and were washed with a solution of 0.1% Brij 35 in Dulbecco phosphate-buffered saline. The remaining protocols were described in the isotyping kit instructions, and reagents used were those supplied in the kit. The species specificity of the eight monoclonal antibodies was evaluated by Western blot analysis. Mycobacterial sonicates were loaded onto 10% acrylamide gels (15 ,ug per lane) and electrophoresed at 120 volts for approximately 2 h (Mini-Protean II gel system). These sonicates were then transferred to nitrocellulose, incubated with the appropriate antibody, and developed with the alkaline phosphatase blot detection system as described earlier.

TABLE 1. MAbs against M. avium Antigen

MAb

Isotype Isotype

~~~~~~~~detected (kDa)

3917 Fl 3918 D12 3954 B12 3989 Cl 4004 F3 4045 H12 4055 E6 4064 E6

IgG2b IgG3 IgG3 IgGl IgGl IgGl IgGl IgGl

34 27 33 30 20

MAb

25/16 35 36

RESULTS BALB/c mice were initially immunized with an ammonium sulfate precipitate of a M. avium serovar 4 sonicate. This M. avium strain was isolated from a patient with AIDS. Thus far, eight different MAbs from these fusions have been characterized. The isotype of each antibody and the molecular mass of the antigen possessing the epitope which reacts with that antibody are listed in Table 1. Because some of these antibodies reacted with similar-size mycobacterial antigens, the Miniblotter 28 apparatus was used to compare MAb binding to antigens on the same preparative nitrocellulose blot. This comparative Western blot analysis indicated that each of the antibodies shown in Table 1 detects different M. avium antigens. Furthermore, none of these MAbs binds to M. avium sonicates that have been pretreated with proteinase K. Sensitivity to protease suggests that each of the M. avium antibodies recognizes a protein-associated

epitope. The species specificity of the eight MAbs was also evaluated by Western blot analysis. Sonicates from 13 different species of mycobacteria were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred to nitrocellulose filters. Following incubation with the appropriate MAb, antibody binding was detected with the alkaline phosphatase blot development system. The results of the specificity studies are shown in Fig. 1 through

4 and are summarized in Table 2. Four of the MAbs appear to bind only M. avium antigens (Fig. 1A through D). Antibodies 3954 B12, 3989 Cl, 4055 E6, and 4064 E6 recognize, respectively, 33-, 30-, 35-, and 36-kilodalton (kDa) M. avium proteins. While this study suggests that 3954 B12, 3989 Cl, 4055 E6, and 4064 E6 detect M. avium-specific epitopes, TABLE 2. Specificity of M. avium MAbsa Bacterial sonicate

M. avium

3917 3918 3954 3989 4004 4045 4055 4064 E6 F3 H12 E6 Fl D12 B12 Cl ++

M. bovis (BCG) . M. chelonei M. fortuitum M. intracellualare M. kansasii. M. marinum M. phlei M. scrofulaceum M. smegmatis M. tuberculosis M. vaccae M. xenopi

Escherichia coli a +

++

++

++

++

..

. ++

++

-

-

-

-

-

-

-

++

-

-

-

-

-

++

++

++

++

-

-

-

-

-

-

+

++

-

++

-++ -++ ++ -+ -++

-.-.-

-

-

and + +, Presence of antibody binding; -, absence of antibody binding.

MAbs TO M. AVIUM COMPLEX PROTEIN ANTIGENS

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A

2 3

4 5 6

7 8 9101112 13

1

B

2 3 4 5 6

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7 8 910111213

9766-

9766-

4246-

27- qp 17-

27 -

1 2 3 4 5 6 7 8 9 10 111213

c

D

1

2

3 4 5 6 7 8 9 10 11 12 13

9797-

66-

66-

42-

42-

27-

27-

FIG. 1. Western blot analysis of four MAbs recognizing M. avium protein antigens. The antibodies 3954 B12 (A), 3989 Cl (B), 4055 E6 (C), and 4064 E6 (D) were incubated with identical Western blots containing 13 different mycobacterial sonicates. The sonicates were loaded in the following lanes: 1, M. avium; 2, M. bovis (BCG); 3, M. chelonei; 4, M. fortuitum; 5, M. intracellulare; 6, M. kansasii; 7, M. marinum; 8, M. phlei; 9, M. scrofulaceum; 10, M. smegmatis; 11, M. tuberculosis; 12, M. vaccae; and 13, M. xenopi.

further studies with a larger number of mycobacterial sonicates will be required to ascertain absolute specificity. To evaluate the reactivity of these MAbs against different M. avium isolates, the antibodies were blotted against sonicates generated from 10 M. avium isolates. Nine of the M. avium isolates were derived from patients with AIDS. These included serovar 3b, 4, 4b, 8, and 20a strains and an untypeable strain. The remaining M. avium isolate was a Weybridge strain culture. The species-specific monoclonal antibodies, 3954 B12, 3989 Cl, 4055 E6, and 4064 E6, strongly react with all of the M. avium strains tested (data not shown). Two of the M. avium MAbs are apparently specific for the MAC (Fig. 2). Antibody 3917 Fl binds a 34-kDa protein only in M. avium and M. intracellulare sonicates, while 4004 F3 reacts with a 20-kDa MAC antigen. Both of these MAbs also react strongly with the 10 other M. avium sonicates described above. A

1

2 3 4

5

6 7 8 910111213

The MAb 3918 D12 reacts with a 27-kDa protein found only in the M. avium-M. intracellulare-Mycobacterium scrofulaceum complex sonicates. This antibody consistently reacts strongly with a M. scrofulaceum antigen. The MAb 3918 D12 recognized the same 27-kDa protein in each of our M. avium sonicates. In contrast to the specific reactivities of the MAbs described above, 4045 H12 (Fig. 4) reacted with 11 of the 13 mycobacterial sonicates tested. Only Mycobacterium chelonei and Mycobacterium fortuitum sonicates were not recognized by this antibody. The pattern of reactivity for MAb 4045 H12 is also quite different. MAb 4045 H12 reacts intensely with 25- and 16-kDa M. avium and M. intracellulare proteins. The Mycobacterium marinum reaction profile is unique, since 40- and 27-kDa M. marinum proteins possess determinants recognized by MAb 4045 H12. However, 4045 H12 recognizes only a 25-kDa antigen in Mycobacterium bovis, Mycobacterium kansasii, M. scrofulaceum, and B

1 2 3 4

5 6 7 8 9 10 11 12 13

97-

9766-

42-

42-

27-

27-

17-

66-

FIG. 2. Western blot analysis of two MAbs recognizing MAC protein antigens. The antibodies 3917 Fl (A) and 4004 F3 (B) were incubated with Western blots containing 13 different mycobacterial sonicates as described in the legend to Fig. 1.

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2 3 4

6 7 8

9 10

11 12 13

976642-

27 17

--

FIG. 3. Western blot analysis of a MAb reacting with a M. avium-M. intracellulare-M. scrofulaceum protein antigen. The antibody 3918 D12 was incubated with Westem blots containing 13 different mycobacterial sonicates as described in the legend to Fig. 1. M. tuberculosis sonicates and recognizes only

a

14-kDa

peptide in Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium vaccae, and Mycobacterium xenopi sonicates. DISCUSSION a

MAbs to M. tuberculosis and M. leprae, which react with wide spectrum of immunogenic components, have been

prepared (9; H. D. Engers and Workshop Participants, Letter, Infect. Immun. 48:603-605, 1985; H. D. Engers and Workshop Participants, Letter, Infect. Immun. 51:718-720, 1986). Six M. leprae antibodies and two M. tuberculosis

antibodies which recognize species-specific antigenic determinants have been described (8; H. D. Engers et al., Letter, 1986). The increased importance of M. avium disease has heightened interest in the generation of MAbs to the MAC. The production of MAbs that recognize serovar-specific MAC glycopeptidolipids has been reported (12, 17). However, only Abe et al. have described a MAb that reacts with MAC protein antigens. This antibody detected a speciesspecific epitope on a 27-kDa M. avium protein (1). We have recently prepared several MAbs which bind to proteinassociated epitopes of the MAC. The characterization of four M. intracellulare-reactive MAbs, two of which apparently react with MAC-specific epitopes on 40- and 43-kDa proteins, has been described previously (16). In this report, we describe the production and characterization of eight MAbs to M. avium determinants. Six of these MAbs may be especially important because of their apparent specificity for M. avium and MAC epitopes. The four antibodies which 1

66

2

3

4

5

6

7

8

9

10

11

12

13

--

42 27 m

- -

m

17

FIG. 4. Western blot analysis of a cross-reactive anti-M. avium MAb. The specificity to 4045 H12 was evaluated as described in the legend to Fig. 1.

recognize presumed M. avium-specific epitopes are 3954 B12, 3989 Cl, 4055 E6, and 4064 E6, which react with 33-, 30-, 35-, and 36-kDa M. avium proteins, respectively. The antibodies 3917 Fl and 4004 F3 react only with MAC antigens: MAb 3917 Fl reacts with a 34-kDa protein and 4004 F3 binds to a 20-kDa peptide. MAbs which recognize protein antigens have facilitated the isolation and characterization of mycobacterial immunogens. For instance, native M. tuberculosis antigens have been purified by immunoaffinity chromatography and their immunoreactivity has been evaluated (10, 21). Worsaae et al. have shown that three affinity-purified tuberculosis proteins induce positive skin test reactions in vivo and positive blastogenic responses in vitro (21). Protein-reactive antibodies also allow for the identification of bacteriophages in gene libraries which express recombinant mycobacterial antigens in Escherichia coli. Studies of recombinant M. tuberculosis and Mycobacterium leprae proteins and their genes have greatly expanded our understanding of the immunogenicity of the tubercle and leprae bacilli (7, 13, 19, 22). We have recently described the isolation of four Xgtll clones which express recombinant M. intracellulare proteins by using anti-M. intracellulare MAbs (16). We are confident that the anti-M. avium MAbs characterized in this report will also be useful for screening mycobacterial expression libraries. A recombinant bacteriophage isolated from a M. avium Agtll library that expresses an antigen recognized by the antibody 3954 B12 has already been identified (D. Rouse and S. Morris, unpublished results). Evaluation of these recombinant MAC proteins will increase our knowledge of the basic biology of MAC antigens. Furthermore, peptides derived from these recombinant mycobacterial proteins have potential clinical utility in a specific skin test or as serodiagnostic reagents for M. avium disease and as acellular vaccine

components. LITERATURE CITED 1. Abe, C., H. Saito, H. Tomoika, and Y. Fukasawa. 1989. Production of a monoclonal antibody specific for Mycobacterium avium and the immunological activity of the affinity-purified antigen. Infect. Immun. 57:1095-1099. 2. Chaparas, S. D. 1981. Antigenic relationships among mycobacterial species studied by modified rocket and crossed immunoelectrophoresis. Rev. Infect. Dis. 3:934-943. 3. Chaparas, S. D. 1984. Immunologically based diagnostic test with tuberculin and other mycobacterial antigens, p. 196-220. In G. P. Kubica and L. G. Wayne (ed.). The Mycobacteria. Marcel Dekker, Inc., New York. 4. Chaparas, S. D., T. Brown, and I. Hyman. 1978. Antigenic relationships among species of Mycobacterium studied by fused rocket immunoelectrophoresis. Int. J. Syst. Bacteriol. 28:547560. 5. Collins, F. M. 1988. AIDS-related mycobacterial disease. Springer Semin. Immunopathol. 10:375-391. 6. Hawkins, C. C., J. W. M. Gold, E. Whimbery, T. E. Kiehn, P. Brannon, R. Cammarata, A. E. Brown, and D. Armstrong. 1986. Mycobacterium avium complex infectious patients with the acquired immunodeficiency syndrome. Ann. Intern. Med. 105: 184-188. 7. Husson, R. N., and R. A. Young. 1987. Genes for the major protein antigens of Mycobacterium tuberculosis: the etiologic agents of tuberculosis and leprosy share an immunodominant antigen. Proc. Natl. Acad. Sci. USA 84:1679-1683. 8. Ivanyi, J., K. Sharp, P. Jackett, and G. Bothamley. 1988. Immunological study of the defined constituents of Mycobacteria. Springer Semin. Immunopathol. 10:279-300. 9. Kadival, G. C., and S. D. Chaparas. 1987. Production, characterization and species specificity of five monoclonal antibodies to Mycobacterium tuberculosis. J. Clin. Microbiol. 25:76-80.

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10. Kadival, G. V., S. D. Chaparas, and D. Hussong. 1987. Characterization of serologic and cell-mediated reactivity of a 38 kDa antigen isolated from Mycobacterium tuberculosis. J. Immunol. 139:2447-2451. 11. Kohler, G., and C. Milstein. 1975. Continuous culture of fused cells secreting antibody of predetermined specificity. Nature (London) 256:495-497. 12. Kolk, A. H. J., R. Evers, D. G. Groothuis, H. Gilis, and S. Kuijpei. 1989. Production and characterization of monoclonal antibodies against specific serotypes of Mycobacterium avium

MAbs TO M. AVIUM COMPLEX PROTEIN ANTIGENS

17.

18.

and the Mycobacterium avium-Mycobacterium intracellulareMycobacterium scrofulaceum complex. Infect. Immun. 57: 2514-2521. 13. Lu, M. C., M. H. Lein, R. E. Becher, H. C. Heine, A. M. Buggs, D. Lipovesk, R. Gupta, P. W. Robbin, C. M. Grossinsky, C. S. Hubbard, and R. A. Young. 1987. Genes for immunodominant protein antigens are highly homologous in Mycobacterium tuberculosis, Mycobacterium africanum and the vaccine strain Mycobacterium bovis BCG. Infect. Immun. 55:2378-2382. 14. Machen, A. M., J. A. Kovars, V. Gill, G. D. Roberts, J. Ames, C. H. Pack, S. Strans, H. C. Lane, J. E. Parillo, A. S. Fauci, and H. Masur. 1983. Bacteremia due to Mycobacterium aviumintracellulare in the acquired immunodeficiency syndrome. Ann. Intern. Med. 79:782-785. 15. Morris, S. L., D. A. Rouse, D. Hussong, and S. D. Chaparas. 1988. Isolation and characterization of a recombinant Agtll bacteriophage which expresses an immunoreactive Mycobacterium intracellulare protein in Escherichia coli. Infect. Immun. 56:3026-3031. 16. Morris, S. L., D. A. Rouse, D. Hussong, and S. D. Chaparas.

19. 20. 21.

22.

23.

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1990. Isolation and characterization of recombinant Xgtll bacteriophages expressing four different Mycobacterium intracellulare antigens. Infect. Immun. 58:17-20. Nishimori, K., H. Yugi, M. Naiki, T. Sugimara, Y. Tanaka, I. Nonomura, Y. Yokomizo, and S. Kubo. 1987. Production and characterization of serovar-specific monoclonal antibodies to serovars 4, 8 and 9 of Mycobacterium intracellulare. Infect. Immun. 55:711-715. Selwyn, P. A., D. Hartel, V. A. Lewis, E. E. Schoenbaum, S. H. Vermund, R. S. Klein, A. T. Walker, and G. H. Freiland. 1989. A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection. N. Engl. J. Med. 320:545-550. Shinnick, T. M. 1987. The 65-kilodalton antigen of Mycobacterium tuberculosis. J. Bacteriol. 169:1080-1088. Wallace, J. M., and J. B. Hannah. 1988. Mycobacterium avium complex infection in patients with the acquired immunodeficiency syndrome. Chest 93:926-932. Worsaae, A., L. Ljungquist, K. Haslov, I. Heron, and J. Bennedsen. 1987. Allergenic and blastogenic reactivity of three antigens from Mycobacterium tuberculosis in sensitized guinea pigs. Infect. Immun. 55:2922-2927. Young, D., R. Lathigia, R. Hendrix, D. Sweetser, and R. A. Young. 1988. Stress proteins are immune targets in leprosy and tuberculosis. Proc. Natl. Acad. Sci. USA 85:4267-4270. Young, L. S., C. B. Inderlied, 0. G. Berlin, and M. S. Gottlieb. 1986. Mycobacterial infections in AIDS patients, with an emphasis on the Mycobacterium avium complex. Rev. Infect. Dis. 8:1024-1033.

Production, characterization, and species specificity of monoclonal antibodies to Mycobacterium avium complex protein antigens.

The incidence of Mycobacterium avium-Mycobacterium intracellulare complex infections has increased in recent years primarily because a significant pro...
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