Antibodies to Mycobacterium leprae - Infection and Immunity

Vol. 37, No. 1

INFECTION AND IMMUNITY, July 1982, p. 172-178 0019-9567/82/070172-07$02.00/0

Production and Partial Characterization of Monoclonal Antibodies to Mycobacterium leprae THOMAS P.

GILLIS'*

AND THOMAS M. BUCHANAN2

Department of Microbiology, Marshall University School of Medicine, Huntington, West Virginia 257011; and Immunology Research Laboratory, Seattle Public Health Hospital, Departments of Medicine and Pathobiology, University of Washington, Seattle, Washington 981952 Received 5 February 1982/Accepted 16 March 1982

Monoclonal antibodies to Mycobacterium leprae were produced by the fusion of BALB/c splenocytes and lymph node cells to BALB/c myeloma (NSI/1) cells. Eleven monoclonal antibodies were characterized as to their reactivity with M. leprae and 18 other mycobacterial species by enzyme-linked immunosorbent assay and immunofluorescence. Two monoclonal antibodies reacted only with M. leprae, and the other nine showed unique patterns of reactivity by enzyme-linked immunosorbent assay. One monoclonal antibody (IIH9) reacted with a 68,000dalton protein present in extracts from M. leprae, M. tuberculosis H37Rv, M. gastri, and M. smegmatis. Potential uses for these antibodies in serological tests and immunochemical analyses are discussed. The antigenic analysis of Mycobacterium leprae has attracted much attention since the demonstration of antimycobacterial antibodies in the serum of leprosy patients (3, 13, 18, 20, 22). Early studies focused on the comparison of antigens from cultivable species of mycobacteria and related genera to antigens of M. leprae extracted from relatively small numbers of bacilli obtained from human sources (1, 6). The availability of larger amounts of armadillo-derived M. leprae through the pioneering work of Kirchheimer and Storrs (10) has made it possible to initiate a systematic investigation of the antigens of M. leprae. Although definitive immunochemical information on M. leprae remains meager, several groups have reported the presence of common and species-specific antigenic determinants associated with M. leprae (4, 7, 8). These studies, however, have not identified the specific molecules that carry the M. leprae antigens of interest. Recent advances in somatic cell hybridization techniques for the production of monoclonal antibodies (11, 12) have made practical the production and use of monoclonal antibodies for antigenic analysis. We applied this technology to the antigenic analysis of M. leprae. We report here the production and partial characterization of 11 monoclonal antibodies prepared by the fusion of NSI/1 myeloma cells to splenocytes and lymph node cells from

BALB/c mice immunized with M. leprae.

MATERIALS AND METHODS Antigen preparation. M. leprae (1010) purified from

infected armadillo liver tissues, was held in acetone overnight at 4°C with gentle shaking. These cells were

172

washed once with acetone and twice with phosphatebuffered saline (PBS). The cells were suspended in 30 ml of 0.2 M lithium acetate solution containing 20 mM EDTA (pH 8.8) and transferred to a 100-ml medium bottle. Glass beads were added, the bottle was sealed, and the extraction was performed on a shaking water bath for 2 h at 45°C. The extract was removed from the bottle and centrifuged for 10 min at 10,000 x g. The resultant pellet contained damaged cells and cell wall material and was referred to as M. leprae 1OKP. The supernatant fluid was removed and centrifuged for 20 min at 30,000 x g. The final supernatant fluid was removed, placed in dialysis tubing, and dialyzed for 24 h at 4°C in deionized H20. The retentate was lyophilized and suspended in a small volume (1 to 2 ml) of PBS. The protein concentration of the extract was determined by the method of Lowry et al. (17) to be 1 mg/ml. This material was referred to as M. leprae 30KS. Antigenic extracts of other mycobacteria were prepared in an identical fashion. Uninfected armadillo liver tissue (2 g) was minced in chilled PBS and thoroughly disrupted in a Potter-S homogenizer at 4°C. Clumps were allowed to settle, and the homogenate was centrifuged at 30,000 x g. for 20 min at 4°C. The resulting supernatant fluid was divided into equal portions and stored at -20°C; it was referred to as armadillo, liver homogenate (ALH). Further dilution of ALH for use in the enzyme-linked immunosorbent assay (ELISA) was done in a sodium carbonate buffer (see below). Immunofluorescence studies were performed on an M. leprae sonicated antigen preparation. The antigen was prepared by suspending 2 x 108 M. leprae in 0.5 ml of PBS (pH 7.0) and sonicating this suspension in a water bath sonicator (Branson Instruments Co., Stamford, Conn.) at full power for 5 min at 25°C. The sonicated suspension was diluted to 3 x 106 organisms per ml and coated onto glass slides for immunofluorescence tests. Immunization protocol. The antigen preparation

VOL. 37, 1982

MONOCLONAL ANTIBODIES TO M. LEPRAE

used for immunizations of BALB/c mice was a combination of M. leprae 30KS and 1OKP. Briefly, 100 ,ul of M. leprae 30KS (50 p,g) and 100 p,l of M. leprae 10KP (100 ,ug) were combined and injected intraperitoneally into BALB/c mice (200 ,ul per mouse) on day 1. Boosters with the same antigen were given intraperitoneally on days 7, 13, and 23. Evidence of a serum antibody response to the antigen(s) was monitored by the ELISA described below. ELISA conditions. M. leprae 3OKS was diluted in sodium carbonate buffer (0.05 M, pH 9.6) to a protein concentration of 5 jig/ml. The antigen was added to 96well polystyrene microtiter plates at 50 ,ul of antigen per well and held at 37°C for 3 h. Antigen plates were used immediately or were held at 4°C for up to 2 weeks with no loss of sensitivity. After adsorption of the antigen, to the solid-phase carrier, the fluid-phase antigen was decanted. To each well was added 75 ,il of a PBS-5% bovine serum albumin (BSA) solution to block nonspecific binding in subsequent steps. Blocking took place at 37°C for 45 min. The removal of BSA from each well served as a single washing step to remove weakly associated antigen from the solid phase. To screen fusion experiments for antibody production, culture supernatants (20 to 40 p,l) from 96well plates were transferred to 96-well antigen plates in replicate and held at 37°C for 45 min. The wells were washed three times with PBS-1% BSA, and peroxidase-conjugated anti-mouse immunoglobulin G (IgG) reagent (Cappel Laboratories, Downingtown, Pa.) was added at a dilution of 1:500 in PBS-1% BSA. The plates were held at 37°C for 45 min and then washed three times with PBS. Next, 50 pl of a substrate-dye mixture (0.003% H202 and 0.1-mg/ml O-phenylenediamine) was added to each well and allowed to react at 25C. At the end of 30 min the reaction was stopped by the addition of 8 N H2SO4 (20 ,ul per well). Generally, only the wells that showed positive reactions (dark color) were selected for further characterization and could be visually discriminated from negative or weakly reactive wells. In some instances, the optical density of each well was determined by a filter photometer. By using the ELISA methodology described above, each monoclonal antibody was characterized with respect to its binding to 30KS extracts prepared from 18 species of mycobacteria. Briefly, each mycobacterial extract was tested for optimal antigen coating to ELISA plates with a pooled unabsorbed leprosy serum. The protein concentration of each extract which yielded strong color development in the ELISA (5 to 10 times more than the background) was chosen as the antigen coating concentration to be used for screening all monoclonal antibodies. This concentration for all extracts fell between 5 and 10 jig of protein per ml. Immunofluorescence tests. Individual drops (20 pl) from a sonicated suspension of M. leprae (2 x 108/ml) were placed on 10-circle template slides, allowed to dry, and fixed in acetone for 10 min. Dilutions of each monoclonal antibody were prepared in PBS, placed on antigen slides, and held for 1 to 4 h at 37°C. Slides were washed three times with PBS, and 1 drop of fluorescein-conjugated goat anti-mouse IgG reagent (Cappel Laboratories) at a dilution of 1:20 in PBS was added to each test slide. After a 1-h incubation period at 37°C in a moist chamber, the slides were washed three times in PBS and mounted in phosphate-buffered glycerol (pH 7.4), with p-phenylenediamine (1 mg/ml) added to

173

reduce the fading of fluorescence (9). Slides were observed for fluorescence on a Zeiss fluorescence microscope (Carl Zeiss, West Germany) with halogenquartz incident illumination under optimal excitation (490 nm) and emission (520 nm) conditions for fluorescein. In experiments designed to test the chemical nature of the antigens detected by various monoclonal antibodies, the acetone-fixed M. leprae organisms were treated with trypsin (10 pLg/ml; Sigma Chemical Co., St. Louis, Mo.) in PBS (pH 7.4) for 1 h at 37°C. The enzyme was removed by washing the slides three times in PBS, and immunofluorescence testing was performed as described above. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Immunoblot. A pellet (109 bacilli) of each species of mycobacteria to be tested was suspended in 50 p1 of a 50 mM Tris solubilization buffer (pH 6.8) containing 1% sodium dodecyl sulfate and 1% 2mercaptoethanol. After boiling this suspension for 3 min, insoluble material was removed by centrifugation at 10,000 x g for 5 min, and the supernatant fluid was electrophoresed in a discontinuous system (15) at 50 mA for approximately 4 h. The slab gel consisted of a 5% stacking gel and an 11.5% separating gel. M. leprae proteins and other components resolved on the polyacrylamide gel were then transferred directly to a sheet of nitrocellulose paper (NCP) by electrophoresis in a Tris-glycine-methanol buffer, as described previously (21, 24). After transfer, the NCP was washed for 45 min in PBS-5% BSA and then reacted with monoclonal antibody at a dilution of 1:100 in PBS-5% BSA for 1 h at 37°C. The NCP was washed extensively in PBS containing 0.5% Triton X-100 and then reacted with 125I-labeled Staphylococcus aureus protein A to detect antigen-bound antibodies on the NCP. Test blots were dried and exposed to X-Omat AR film (Eastman Kodak Co., Rochester, N.Y.) at -70"C for 4 to 6 h in an X-ray cassette fitted with a Coronex MRF 32 clear base intensifying screen (DuPont Co., Newtown, Conn.). lodinated molecularweight markers (Bio-Rad Laboratories, Richmond, Calif.) were included on the gels and transferred to the NCP for molecular-weight measurements. Fusion. The BALB/c myeloma cell line NSI/1 was kindly provided by R. C. Nowinski with the permission of C. Milstein. Fusion and cell culture methods were adapted from techniques previously reported (16, 23). Spleens and mesenteric lymph nodes were removed from mice 3 days after the final booster injection. A single cell mixture of splenocytes and lymph node cells was obtained by mincing the tissues extensively and passing the material through a sterile nylon screen. The cells were washed three times in RPMI 1640 medium (10 mM pyruvate) and then combined with NSI/1 myeloma cells at a ratio of 4 to 1. The cell mixture was pelleted at 350 x g for 12 min in the presence of 40o polyethylene glycol (pH 7.0). The pellet was suspended and washed with RPMI 1640 medium and resuspended in complete medium plus 0.1 mM hypoxanthine-0.4 mM aminopterin-0.015 mM thymidine. Cells were dispensed into 96-well microtiter plates at a density of 5 x 105 cells per well. In addition, each well contained a thymocyte feeder layer (4 x 106 cells per well; BALB/c thymocytes) added to the fused cells before plating. The resulting supematant fluid antibody to M. leprae antigen(s) was screened by ELISA 10 days after the fusion. Wells

174

INFECT. IMMUN.

GILLIS AND BUCHANAN

TABLE 1. Antibody reactivities with M. leprae, M. smegmatis, and ALH antigens 10 days after fusion Antibody-positive Antigen wells' M. leprae M. leprae and M. smegmatis M. leprae and ALH ALH a Total number of wells, 384.

No.

%

34 6 5 7

8.8 1.6 1.3

1.8

containing positive reactors were replated at a density of 5 cells per well to establish stable progeny of the original fusion events. These cells were likewise screened for antibody production, and two to four positive wells were formally cloned at a density of 1 cell per well in the presence of thymocytes. Wells containing single clones were scored visually by microscopy and coalesced into a single plate. These clones were tested again for antibody activity, and strong reactors were chosen for further characterization. Cells were then grown in large quantities in vitro for antibody production (spent media) or injected into the peritoneum of pristane-primed BALB/c mice for the production of an ascites tumor from which ascitic fluid (rich in antibody) was harvested. The immunoglobulin class and subclass of the antibody formed were then determined by immunodiffusion precipitation with antibodies specific for a given immunoglobulin class or subclass (Miles Laboratories, Inc., Elkhart, Ind.).

RESULTS

Table 1 shows the number of antibody-producing wells from a total of 384 wells seeded

from a fusion experiment. Each supernatant fluid was tested for antibody reactivity on three antigens to reduce the possibility of isolating and characterizing antibodies to cross-reacting or contaminating antigens present in the antigen mixture used for immunization. The majority of antibodies produced reacted with M. leprae and M. smegmatis or both (40 of 384 wells, or 10.4%; Table 1), and the majority of these reacted only with M. leprae (34 of 384 wells, or 8.8%; Table 1). Antibodies that reacted with both M. Ieprae and M. smegmatis were considered to be directed against cross-reacting antigens present in both species and were not characterized further. Antibodies that reacted with M. leprae and ALH (5 of 384 wells, or 1.3%) or ALH only (7 of 384 wells, or 1.8%) were considered to be elicited from antigenic material of armadillo origin present in the purified M. leprae preparation used for immunizations. Although >90% of the wells produced viable fusion events, the overall frequency of antibody-positive wells was 52 of 384 wells, or 13.5%. This frequency has held constant in other fusion experiments under similar conditions with other multicomponent mycobacterial antigens. After formal clones were established, each clone was tested for immunoreactivity against various species of mycobacteria in the ELISA test. These results are summarized in Table 2. Monoclonal antibodies obtained from either ascitic fluid or concentrated culture supematants were tested by ELISA, and the color development was scored visually from 0 to +4, with 0 indicating no color development and +4 indicat-

TABLE 2. ELISA reactivity of 11 monoclonal antibodies with 19 species of mycobacteria Species

1

2

3

4

5

Clone 6

7

8

9

10

11

(IIIC8) (IIH9) (IVC10) (IVD2) (IIG1) (IVE12) (IIIC9) (IIC8) (IIIE9) (IVD8) (IF5) M. leprae +4 +3 + +2 +4 +4 +2 +4 +3 +4 +4 M. tuberculosis H37Rv NDa ND ND ND ND ND ND ND ND ± M. lepraemurium +2 M. nonchromogenicum +2 +2 +1 M. triviale +3 +1 +3 +3 +1 +3 M. terrae +2 +2 +1 M. flavescens +3 +2 +2 +3 +3 +1 +2 +3 M. gastri +3 +3 +2 +3 +3 +1 +2 +3 M. gordonae +2 +3 +2 +3 +3 +1 +3 +3 M. intracellulare + +2 +1 M. marinum +3 +3 +2 +1 +2 M. vaccae +2 +1 M. diernhoferi +3 +2 +1 M. thamnopheos + +3 M. peregrinum +3 +2 +1 M. bovis BCG + +2 M. smegmatis +3 +1 +3 +3 +3 M. phlei +3 +3 M. duvali +3 a ND, Not done.

VOL. 37, 1982

MONOCLONAL ANTIBODIES TO M. LEPRAE

TABLE 3. Immunoreactivity of 11 monoclonal antibodies to M. lepraea by indirect immunofluorescence Antibody

Immunoglobulin class and subclass

IIG1 IVE12 IVC1O IVD8

IgG2a IgM IgGI IgGI IgGl IgGl IgG2a

11IC8

Reactivity Pretrypsin Posttrypsin

+2 +2 +2 +2 +1 +1

+ +

+ + + +

IIH9 IIIE9 + 11C8 IgG2b IVD2 IgM HIIC9 IgG2b IF5 IgM a M. leprae antigen was a sonicate of live organisms which were subsequently fixed to glass slides with acetone.

ing a color intensity equal to the positive control (pooled leprosy serum). Of the 10 clones which continually demonstrated +2 to +4 reactivity with M. leprae, only two clones (IIIE9 and IVD8) produced monoclonal antibodies which reacted only with M. leprae (Table 2). Clone 11 (IF5) was difficult to passage and was intermittently weakly reactive with M. leprae, but it was retained because of its strong reactivity to M. phlei. Clone 4 (IVD2) reacted with all mycobacteria tested. Other clones produced antibodies which appear to have unique reactive patterns as defined by the ELISA test (Table 2). In contrast to the ELISA reactivity, the immunofluorescence reactivity of some of the monoclonal antibodies was difficult to demonstrate. It was important to gently sonicate the M. leprae substrate before fixation to demonstrate the binding of the monoclonal antibodies. Attempts to use organisms directly without sonication gave highly variable results. Table 3 shows that five of the antibodies, including IIIE9 (specific for M. leprae by ELISA), were weakly reactive or nonreactive with the M. leprae antigen substrate. Other antibodies which were reactive with M. leprae by immunofluorescence were greatly reduced in their binding after treatment of the antigen substrate with trypsin (Table 3). Immunoblotting was implemented to determine the molecule-specific binding of the monoclonal antibodies. Attempts by immunoblotting to detect the molecule which contains the epitope which elicited either antibody IIIE9 or antibody IVD8 failed. In contrast, immunoblotting with antibody IIH9 showed that this monoclonal antibody binds to a molecule with an apparent molecular weight of approximately 68,000 (Fig. 1, lane ML). It was also evident

175

from the immunoblotting results and the ELISA data that this antigenic determinant is common to other mycobacteria, specifically M. tuberculosis H37Rv (Fig. 1, lane MTB), M. gastri (Fig. 1, lane MG), M. smegmatis (Fig. 1, lane MS), M. flavescens (Table 2), and M. gordonae (Table 2). We do not have direct evidence to show that the 68,000-dalton antigen is a protein molecule. However, polyacrylamide gel profiles of identical extracts from the blotted organisms shown in Fig. 1, stained with Coomassie R250, showed numerous proteins along the entire lane, with two to three protein bands in the 68,000-dalton region. In addition, the treatment of M. leprae with trypsin reduced the immunofluorescence associated with antibody IIH9 (Table 3). These data suggest that the 68,000-dalton antigen is proteinaceous; therefore it will be referred to as the M. leprae 68,000-dalton protein. A crude extract of uninfected armadillo liver tissue was blotted to control possible nonspecific binding of monoclonal antibody to non-mycobacterial cross-reactive antigens present in extracts of either purified M. leprae (armadillo derived) or uninfected armadillo liver tissue. Figure 1 shows no binding of antibody IIH9 at either 50 p.g (lane 1, ALH) or 100 jig (lane 6, ALH) of total protein electrophoresed and blotted. DISCUSSION An antigenic analysis of M. leprae is critical to the understanding of the basic immunopathological phenomena associated with leprosy. The simultaneous development of an animal model for leprosy that yields relatively large amounts of purified M. leprae and the work of Milstein and Kohler (11, 12) in the area of somatic cell hybridization for the production of monoclonal antibodies has set the stage for an organized analysis of the antigens of M. leprae on a molecular level. Theoretically, the production of monoclonal antibodies to M. leprae antigens should provide the tools needed to dissect a highly complex antigenic mixture associated with a microorganism which continues to elude attempts at in vitro cultivation. We initiated studies to determine the applicability of using monoclonal antibodies in the antigenic analysis of M. leprae. We report here the successful production and partial characterization of 11 monoclonal antibodies to M. leprae. It is evident from these studies that the production of monoclonal antibodies to M. leprae under the conditions described is possible. Moreover, a variety of monoclonal antibodies were raised which exhibited unique immunoreactivity as defined by the ELISA test. One of

176

GILLIS AND BUCHANAN

INFECT. IMMUN.

binding experiment, it may be possible to detect an M. leprae-specific antibody in the serum of an individual with subclinical leprosy. This ALH ML M TB N iJG Al h H would allow the detection of the specific antibody in the presence of cross-reactive mycobacterial antibodies which may be present as a 94 result of infection with, or environmental expo_ _1 68 sure to, mycobacteria other than M. leprae. In addition, antibodies to BCG elicited by BCG vaccination should not interfere with a serological test of this type because of the demonstrated specificity of the monoclonal antibody for M. 30 leprae. Such an approach would facilitate the early diagnosis and treatment of individuals who might otherwise take years to develop recognizable clinical symptoms. A possible drawback to the competition experiment with monoclonal antibodies from experimental animals may be that M. leprae antigenic determinants that are FIG. 1. Immunoblot of uninfected armadillo liver immunogenic in the animal used for the produchomogenate (ALH), M. leprae (ML), M. tuberculosis tion of the monoclonal antibodies may be less H37Rv (MTB), molecular-weight markers (MWM), M. immunogenic or non-immunogenic in humans. gastri (MG), and M. smegmatis (MS). The blot was Accordingly, certain monoclonal antibodies may reacted with monoclonal antibody 11H9 and 125I-la- be of limited value in a competition assay as beled Staphylococcus aureus protein A. Molecular- described above. However, if other determiweight markers are, from top to bottom, phosphorylnants on a given antigenic molecule are immunoase B (94,000), bovine serum albumin (68,000), and genic in the experimental animal, the monoclocarbonic anhydrase (30,000). nal antibodies elicited would serve as effective reagents for the purification and subsequent these antibodies (IVD2) was elicited from a characterization of that antigen. common antigenic determinant which was exFurther characterization of the antigenic moltracted from all 19 species tested. Many genus- ecule associated with antibody IIIE9 has been specific and broadly cross-reactive antigens of slow since immunoblotting and immunofluoresthe mycobacteria are high-molecular-weight cence reactivity were negative. Antibody IVD8 polysaccharides such as arabinogalactan (6). It was also nonreactive by immunoblot, but demis likely that similar polysaccharides were pres- onstrated immunoreactivity by indirect immunoent in the crude extracts of M. leprae used for fluorescence on sonicated M. leprae. The treatimmunizations and therefore may have elicited ment of the antigen substrate with trypsin after antibody IVD2. Alternatively, antibody IVD2 sonication greatly reduced antibody IVD8 bindmay be directed at a highly conserved region of a ing, suggesting that the molecule which elicited polymorphic protein molecule found in all spe- antibody IVD8 may be proteinaceous and probacies of mycobacteria tested. bly not exposed on the surface of the organism. In contrast to the broadly reactive antibody The fact that other monoclonal antibodies IVD2, monoclonal antibodies IIIE9 and IVD8 (IIC8, IVD2, etc.) were positive for M. leprae by demonstrated exquisite specificity for M. leprae ELISA but negative by immunofluorescence by ELISA. Since the panel of antigens screened emphasizes the need for confirmation of monodid not include all species of mycobacteria (14) clonal antibody immunoreactivity by more than or all serotypes of certain species (5), the definione assay system. It is possible that differences tion of specificity for these antibodies is taken in antigen exposure concentration or lability within the limits of the 19 species of mycobacte- vary significantly between assay systems, which ria. Among the 18 nonreactive species, 14 are may result in conflicting data. saprophytic or nonpathogenic to humans, 3 are A comparison of immunoblotting, immunofluimportant human pathogens (M. tuberculosis, orescence, and ELISA data for monoclonal antiM. intracellulare, and M. marinum), and 1 (M. body IIH9 showed good correlation. From the bovis BCG) is an attenuated bovine and human ELISA data it was predicted that M. smegmatis, pathogen often used for human vaccination. M. gastri, M. gordonae, M. flavescens, and M. Because of their demonstrated specificity, leprae should all blot similarly. Of the species antibodies IIIE9 and IVD8 are potentially im- tested thus far, M. leprae, M. gastri, M. smegportant. By utilizing one or more M. leprae- matis, and M. tuberculosis all contained a specific antibodies in a competition antibody- 68,000-dalton protein which bound antibody 11H9 B[3W1: M

VOL. 37, 1982

MONOCLONAL ANTIBODIES TO M. LEPRAE

177

ACKNOWLEDGMENTS IIH9 after transfer to NCP. Immunofluorescence of trypsin-treated M. leprae antigen subThis work was supported in part by Public Health Service strate with antibody IIH9 further suggested the grant A116290 and contract A192624 from the National Instiof Infectious Diseases, by Federal Health Services Projprotein or protein-associated nature of this epi- tute ect SEA 78-17, by the Immunology of Leprosy (IMMLEP) tope. component of the UNDP/World Bank/WHO Special ProWith regard to antigenic similarities among gramme for Research and Training in Tropical Diseases, by the 19 species tested, it is apparent that 8 of 11 the Rockefeller Foundation Program for Research on Great Diseases, and by the Victor Heiser Foundation monoclonal antibodies reacted with M. Ieprae Neglected Fellowship Program. M. gastri, (M. species 3 other primarily and We thank Susan Dinning for her excellent technical assistgordonae, and M. flavescens). This suggested ance. that these three organisms may be closely related to M. leprae. Previous studies (2, 8, 19) with LITERATURE CITED polyclonal immune sera have focused on the Y. Yoshino, and K. Okamura. F. Minagawa, M., 1. Abe, antigenic similarities between M. leprae and 1972. Studies on the specificity of Mycobacterium leprae. smegmaM. other species, including M. avium, II. Purification and immunological characterization of the soluble antigen in leprosy nodules. Int. J. Lepr. 40:107tis, M. vaccae, M. nonchromogenicum, and M. 117. bovis BCG. 2. Abe, M., F. Mlnagawa, Y. Yoshino, T. Ozawa, K. This discrepancy is largely the result of the Saikawa, and T. Saitz. 1980. Fluorescent leprosy antibody selection process invoked in our monoclonal absorption (FLA-ABS) test for detecting subclinical infection with Mycobacterium leprae. Int. J. Lepr. 48:109-119. antibody preparation and screening procedure. 3. Axelsen, N. H., M. Harboe, 0. Closs, and T. Godal. 1974. Since our initial objective was to screen for BCG antibody profiles in tuberculoid and lepromatous clones which were potentially specific to M. leprosy. Infect. Immun. 9:952-958. leprae, we did not keep any clones which react4. Brennan, P. J., and W. W. Barrow. 1980. Evidence for species-specific lipid antigens in Mycobacterium leprae. ed with M. smegmatis. This step excluded from Int. J. Lepr. 48:382-387. our analysis clones that produce antibodies to 5. Brennan, P. J., and M. B. Goren. 1979. Structural studies common antigens shared between M. smegmatis on the type-specific antigens and lipids of the Mycobacteand M. leprae and therefore revealed the next rium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum serocomplex. J. Biol. Chem. 254: level of cross-reactivity among these 19 species. 4205-4211. Since many of the shared antigens of the myco6. Estrada-Parra, S. 1972. Immunochemical identification of bacteria are high-molecular-weight polymeric a defined antigen of Mycobacterium leprae. Infect. Imand sometimes structural polysaccharides found mun. 5:258-259. 7. GfUls, T. P., M. Abe, W. E. Bullock, 0. Rojas-Espinosa, in the cell envelope, it is possible that the E. Garcia-Ortgoza, P. Draper, W. F. Klrchheimer, and antigen(s) which elicited the cross-reactive T. M. Buchanan. 1981. Comparison of 22 species of a highly represent may antibodies monoclonal mycobacteria by immunodiffusion against an absorbed conserved functional enzyme(s) important in reference leprosy serum. Int. J. Lepr. 49:287-293. 8. Harboe, M., 0. Closs, G. Bjune, G. Kronvall, and N. H. cellular metabolism and physiology. AlternaAxelsen. 1978. Mycobacterium leprae-specific antibodies tively, the monoclonal antibodies may have detected by radioimmunoassay. Scand. J. Immunol. 7: on a single been elicited from different epitopes 111-120. immunodominant polymorphic protein antigen 9. Johnson, G. D., and G. M. de C. Nogueira Araujo. 1981. A simple method of reducing the fading of immunofluoresof M. leprae. Immunoblotting and radioimmunocence during microscopy. J. Immunol. Methods 43:349precipitation experiments with each monoclonal 350. antibody should clarify this point. 10. Kirchheimer, W. F., and E. E. Stom. 1971. Attempts to As more information is accumulated with reestablish the armadillo (Dasypus novemcinctus Linn.) as a model for the study of leprosy. Int. J. Lepr. 39:693-702. spect to the immunochemistry of M. leprae, it Kohler, G., and C. Milstein. 1975. Continuous cultures of should be possible to construct an antigenic and 11. fused cells secreting antibody of predefined specificity. structural map of the surface of the organism. Nature (London) 256:495-497. Some surface antigens may be virulence factors 12. Kohler, G., and C. Milstein. 1976. Derivation of a specific antibody-producing tissue culture and tumor lines by cell as well as physiologically important envelope Eur. J. Immunol. 6:511-521. proteins that are encoded in the genome. Genet- 13. fusion. Kronvall, G., G. BJune, J. Stanford, S. Menzel, and D. transfer to ic engineering experiments designed Samuel. 1975. Mycobacterial antigens in antibody respecific envelope proteins to a new host capable sponse of leprosy patients. Int. J. Lepr. 43:299-304. of rapid growth and synthesis of that protein will 14. Kwapinski, J. B. G., A. Alcasid,ofE. H. Kwapinski, and V.of Nairn. 1972. The immunology cytoplasmic antigens require specific probes to detect the successful mycobacteria. Can. J. Microbiol. 18:1201-1211. and the prodsubsequent gene transfer of gene 15. Laemmll, U. K. 1970. Cleavage of structural proteins uct. Monoclonal antibodies would serve as speduring the assembly of the head of the bacteriophage T4. 227:680-685. cific probes capable of monitoring these kinds of 16. Nature (London) Lostrom, M. E., M. R. Stone, M. Tam, W. N. Burnette, experiments. These experiments could lead to A. Pinter, and R. C. Nowinsld. 1979. Monoclonal antibodthe production of large amounts of well-characies against murine leukemia viruses: identification of six terized antigens suitable for skin test reagents antigenic determinants on the p15 (E) and gp 70 envelope proteins. Virology 98:336-350. and materials for vaccine development.

178

GILLIS AND BUCHANAN

17. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265-275. 18. Myrvang, B., C. M. Feek, and T. Godal. 1974. Antimycobacterial antibodies in sera from patients throughout the clinico-pathological disease spectrum of leprosy. Acta Pathol. Microbiol. Scand. Sect. B 81:615-620. 19. Navalkar, R. G. 1973. Immunologic studies on leprosy. 2. Antigenic studies of Mycobacterium leprae. Z. Tropenmed. Parasitol. 24:66-72. 20. Navalkar, R. G., P. J. Patel, and R. R. Dalvi. 1975. Immunological studies on leprosy: separation and evaluation of the antigens of Mycobacterium leprae. J. Med. Microbiol. 8:319-324. 21. Renart, J., J. Reisner, and G. R. Stark. 1979. Transfer of proteins from gels to diazobenzyloxymethyl-paper and

INFECT. IMMUN. detection with antisera: a method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. U.S.A. 76:3116-3120. 22. Rojas-Espinosa, O., S. Estrada-Parra, E. Serrano-Miranda, A. Saul, and F. Latapi. 1976. Anti-microbacterial antibodies in diffuse lepromatous leprosy detected by counter-immunoelectrophoresis. Int. J. Lepr. 44:448R452. 23. Stone, M. R., and R. C. Nowinski. 1980. Topological mapping of murine leukemia virus proteins by competition-binding assays with monoclonal antibodies. Virology 100:370-381. 24. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. U.S.A. 76:4350-4354.