Vol. 37, No. 1INFECTION AND IMMUNITY, July 1982, p. 172-1780019-9567/82/070172-07$02.00/0

Production and Partial Characterization of MonoclonalAntibodies to Mycobacterium lepraeTHOMAS P. GILLIS'* AND THOMAS M. BUCHANAN2

Department of Microbiology, Marshall University School ofMedicine, Huntington, West Virginia 257011; andImmunology 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 fusionof 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 immunosorbentassay and immunofluorescence. Two monoclonal antibodies reacted only with M.leprae, and the other nine showed unique patterns of reactivity by enzyme-linkedimmunosorbent assay. One monoclonal antibody (IIH9) reacted with a 68,000-dalton protein present in extracts from M. leprae, M. tuberculosis H37Rv, M.gastri, and M. smegmatis. Potential uses for these antibodies in serological testsand immunochemical analyses are discussed.

The antigenic analysis of Mycobacterium le-prae has attracted much attention since thedemonstration of antimycobacterial antibodiesin the serum of leprosy patients (3, 13, 18, 20,22). Early studies focused on the comparison ofantigens from cultivable species of mycobacteriaand related genera to antigens of M. lepraeextracted from relatively small numbers of bacil-li obtained from human sources (1, 6). Theavailability of larger amounts of armadillo-de-rived M. leprae through the pioneering work ofKirchheimer and Storrs (10) has made it possibleto initiate a systematic investigation of the anti-gens of M. leprae. Although definitive immuno-chemical information on M. leprae remains mea-ger, several groups have reported the presenceof common and species-specific antigenic deter-minants associated with M. leprae (4, 7, 8).These studies, however, have not identified thespecific molecules that carry the M. leprae anti-gens of interest. Recent advances in somatic cellhybridization techniques for the production ofmonoclonal antibodies (11, 12) have made prac-tical the production and use of monoclonal anti-bodies for antigenic analysis. We applied thistechnology to the antigenic analysis of M. le-prae. We report here the production and partialcharacterization of 11 monoclonal antibodiesprepared by the fusion of NSI/1 myeloma cellsto splenocytes and lymph node cells fromBALB/c mice immunized with M. leprae.

MATERIALS AND METHODSAntigen preparation. M. leprae (1010) purified from

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

washed once with acetone and twice with phosphate-buffered saline (PBS). The cells were suspended in 30ml of 0.2 M lithium acetate solution containing 20 mMEDTA (pH 8.8) and transferred to a 100-ml mediumbottle. Glass beads were added, the bottle was sealed,and the extraction was performed on a shaking waterbath for 2 h at 45°C. The extract was removed from thebottle and centrifuged for 10 min at 10,000 x g. Theresultant pellet contained damaged cells and cell wallmaterial and was referred to as M. leprae 1OKP. Thesupernatant fluid was removed and centrifuged for 20min at 30,000 x g. The final supernatant fluid wasremoved, placed in dialysis tubing, and dialyzed for 24h at 4°C in deionized H20. The retentate was lyophi-lized and suspended in a small volume (1 to 2 ml) ofPBS. The protein concentration of the extract wasdetermined by the method of Lowry et al. (17) to be 1mg/ml. This material was referred to as M. leprae30KS. Antigenic extracts of other mycobacteria wereprepared in an identical fashion.

Uninfected armadillo liver tissue (2 g) was minced inchilled PBS and thoroughly disrupted in a Potter-Shomogenizer at 4°C. Clumps were allowed to settle,and the homogenate was centrifuged at 30,000 x g. for20 min at 4°C. The resulting supernatant fluid wasdivided into equal portions and stored at -20°C; it wasreferred to as armadillo, liver homogenate (ALH).Further dilution of ALH for use in the enzyme-linkedimmunosorbent assay (ELISA) was done in a sodiumcarbonate buffer (see below).Immunofluorescence studies were performed on an

M. leprae sonicated antigen preparation. The antigenwas prepared by suspending 2 x 108 M. leprae in 0.5ml of PBS (pH 7.0) and sonicating this suspension in awater bath sonicator (Branson Instruments Co., Stam-ford, Conn.) at full power for 5 min at 25°C. Thesonicated suspension was diluted to 3 x 106 organismsper ml and coated onto glass slides for immunofluores-cence tests.Immunization protocol. The antigen preparation

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used for immunizations of BALB/c mice was a combi-nation of M. leprae 30KS and 1OKP. Briefly, 100 ,ul ofM. leprae 30KS (50 p,g) and 100 p,l of M. leprae 10KP(100 ,ug) were combined and injected intraperitoneallyinto BALB/c mice (200 ,ul per mouse) on day 1.Boosters with the same antigen were given intraperito-neally on days 7, 13, and 23. Evidence of a serumantibody response to the antigen(s) was monitored bythe ELISA described below.ELISA conditions. M. leprae 3OKS was diluted in

sodium carbonate buffer (0.05 M, pH 9.6) to a proteinconcentration of 5 jig/ml. The antigen was added to 96-well polystyrene microtiter plates at 50 ,ul of antigenper well and held at 37°C for 3 h. Antigen plates wereused immediately or were held at 4°C for up to 2 weekswith no loss of sensitivity. After adsorption of theantigen, to the solid-phase carrier, the fluid-phaseantigen was decanted. To each well was added 75 ,il ofa PBS-5% bovine serum albumin (BSA) solution toblock nonspecific binding in subsequent steps. Block-ing took place at 37°C for 45 min. The removal of BSAfrom each well served as a single washing step toremove weakly associated antigen from the solidphase. To screen fusion experiments for antibodyproduction, culture supernatants (20 to 40 p,l) from 96-well plates were transferred to 96-well antigen plates inreplicate and held at 37°C for 45 min. The wells werewashed three times with PBS-1% BSA, and peroxi-dase-conjugated anti-mouse immunoglobulin G (IgG)reagent (Cappel Laboratories, Downingtown, Pa.) wasadded at a dilution of 1:500 in PBS-1% BSA. Theplates were held at 37°C for 45 min and then washedthree times with PBS. Next, 50 pl of a substrate-dyemixture (0.003% H202 and 0.1-mg/ml O-phenylenedia-mine) was added to each well and allowed to react at25C. At the end of 30 min the reaction was stopped bythe addition of 8 N H2SO4 (20 ,ul per well). Generally,only the wells that showed positive reactions (darkcolor) were selected for further characterization andcould be visually discriminated from negative or weak-ly reactive wells. In some instances, the optical densi-ty of each well was determined by a filter photometer.By using the ELISA methodology described above,

each monoclonal antibody was characterized withrespect to its binding to 30KS extracts prepared from18 species of mycobacteria. Briefly, each mycobacte-rial extract was tested for optimal antigen coating toELISA plates with a pooled unabsorbed leprosy se-rum. The protein concentration of each extract whichyielded strong color development in the ELISA (5 to10 times more than the background) was chosen as theantigen coating concentration to be used for screeningall monoclonal antibodies. This concentration for allextracts 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 todry, and fixed in acetone for 10 min. Dilutions of eachmonoclonal antibody were prepared in PBS, placed onantigen slides, and held for 1 to 4 h at 37°C. Slides werewashed three times with PBS, and 1 drop of fluoresce-in-conjugated goat anti-mouse IgG reagent (CappelLaboratories) at a dilution of 1:20 in PBS was added toeach test slide. After a 1-h incubation period at 37°C ina moist chamber, the slides were washed three times inPBS and mounted in phosphate-buffered glycerol (pH7.4), with p-phenylenediamine (1 mg/ml) added to

reduce the fading of fluorescence (9). Slides wereobserved for fluorescence on a Zeiss fluorescencemicroscope (Carl Zeiss, West Germany) with halogen-quartz incident illumination under optimal excitation(490 nm) and emission (520 nm) conditions for fluores-cein. In experiments designed to test the chemicalnature of the antigens detected by various monoclonalantibodies, the acetone-fixed M. leprae organismswere treated with trypsin (10 pLg/ml; Sigma ChemicalCo., St. Louis, Mo.) in PBS (pH 7.4) for 1 h at 37°C.The enzyme was removed by washing the slides threetimes in PBS, and immunofluorescence testing wasperformed as described above.Sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis and Immunoblot. A pellet (109 bacilli) of eachspecies of mycobacteria to be tested was suspended in50 p1 of a 50 mM Tris solubilization buffer (pH 6.8)containing 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol. After boiling this suspension for 3min, insoluble material was removed by centrifugationat 10,000 x g for 5 min, and the supernatant fluid waselectrophoresed in a discontinuous system (15) at 50mA for approximately 4 h. The slab gel consisted of a5% stacking gel and an 11.5% separating gel.M. leprae proteins and other components resolved

on the polyacrylamide gel were then transferred di-rectly to a sheet of nitrocellulose paper (NCP) byelectrophoresis in a Tris-glycine-methanol buffer, asdescribed previously (21, 24). After transfer, the NCPwas washed for 45 min in PBS-5% BSA and thenreacted with monoclonal antibody at a dilution of 1:100in PBS-5% BSA for 1 h at 37°C. The NCP was washedextensively in PBS containing 0.5% Triton X-100 andthen reacted with 125I-labeled Staphylococcus aureusprotein A to detect antigen-bound antibodies on theNCP. Test blots were dried and exposed to X-OmatAR film (Eastman Kodak Co., Rochester, N.Y.) at-70"C for 4 to 6 h in an X-ray cassette fitted with aCoronex MRF 32 clear base intensifying screen (Du-Pont Co., Newtown, Conn.). lodinated molecular-weight markers (Bio-Rad Laboratories, Richmond,Calif.) were included on the gels and transferred to theNCP for molecular-weight measurements.

Fusion. The BALB/c myeloma cell line NSI/1 waskindly provided by R. C. Nowinski with the permis-sion of C. Milstein. Fusion and cell culture methodswere adapted from techniques previously reported (16,23). Spleens and mesenteric lymph nodes were re-moved from mice 3 days after the final booster injec-tion. A single cell mixture of splenocytes and lymphnode cells was obtained by mincing the tissues exten-sively and passing the material through a sterile nylonscreen. The cells were washed three times in RPMI1640 medium (10 mM pyruvate) and then combinedwith NSI/1 myeloma cells at a ratio of 4 to 1. The cellmixture was pelleted at 350 x g for 12 min in thepresence of 40o polyethylene glycol (pH 7.0). Thepellet was suspended and washed with RPMI 1640medium and resuspended in complete medium plus 0.1mM hypoxanthine-0.4 mM aminopterin-0.015 mMthymidine. Cells were dispensed into 96-well micro-titer plates at a density of 5 x 105 cells per well. Inaddition, each well contained a thymocyte feeder layer(4 x 106 cells per well; BALB/c thymocytes) added tothe fused cells before plating. The resulting supema-tant fluid antibody to M. leprae antigen(s) wasscreened by ELISA 10 days after the fusion. Wells

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TABLE 1. Antibody reactivities with M. leprae, M.smegmatis, and ALH antigens 10 days after fusion

Antibody-positiveAntigen wells'

No. %

M. leprae 34 8.8M. leprae and M. smegmatis 6 1.6M. leprae and ALH 5 1.3ALH 7 1.8

a Total number of wells, 384.

containing positive reactors were replated at a densityof 5 cells per well to establish stable progeny of theoriginal fusion events. These cells were likewisescreened for antibody production, and two to fourpositive wells were formally cloned at a density of 1cell per well in the presence of thymocytes. Wellscontaining single clones were scored visually by mi-croscopy and coalesced into a single plate. Theseclones were tested again for antibody activity, andstrong reactors were chosen for further characteriza-tion. Cells were then grown in large quantities in vitrofor antibody production (spent media) or injected intothe peritoneum of pristane-primed BALB/c mice forthe production of an ascites tumor from which asciticfluid (rich in antibody) was harvested. The immuno-globulin class and subclass of the antibody formedwere then determined by immunodiffusion precipita-tion with antibodies specific for a given immunoglob-ulin class or subclass (Miles Laboratories, Inc., Elk-hart, Ind.).

RESULTS

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

from a fusion experiment. Each supernatantfluid was tested for antibody reactivity on threeantigens to reduce the possibility of isolating andcharacterizing antibodies to cross-reacting orcontaminating antigens present in the antigenmixture used for immunization. The majority ofantibodies produced reacted with M. leprae andM. smegmatis or both (40 of 384 wells, or 10.4%;Table 1), and the majority of these reacted onlywith M. leprae (34 of 384 wells, or 8.8%; Table1). Antibodies that reacted with both M. Iepraeand M. smegmatis were considered to be direct-ed against cross-reacting antigens present inboth 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 384wells, or 1.8%) were considered to be elicitedfrom antigenic material of armadillo origin pre-sent in the purified M. leprae preparation usedfor immunizations. Although >90% of the wellsproduced viable fusion events, the overall fre-quency of antibody-positive wells was 52 of 384wells, or 13.5%. This frequency has held con-stant in other fusion experiments under similarconditions with other multicomponent mycobac-terial antigens.

After formal clones were established, eachclone was tested for immunoreactivity againstvarious species of mycobacteria in the ELISAtest. These results are summarized in Table 2.Monoclonal antibodies obtained from either as-citic fluid or concentrated culture supematantswere tested by ELISA, and the color develop-ment was scored visually from 0 to +4, with 0indicating no color development and +4 indicat-

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

Species 1 2 3 4 5 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 - - NDM. 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 - - - - - - +3M. duvali - - - +3 -

a ND, Not done.

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TABLE 3. Immunoreactivity of 11 monoclonalantibodies to M. lepraea by indirect

immunofluorescence

Antibody Immunoglobulin Reactivityclass and subclass Pretrypsin Posttrypsin

IIG1 IgG2a +2 +IVE12 IgM +2 +IVC1O IgGI +2 +IVD8 IgGI +2 +11IC8 IgGl +1 +IIH9 IgGl +1 +IIIE9 IgG2a +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 withacetone.

ing a color intensity equal to the positive control(pooled leprosy serum). Of the 10 clones whichcontinually demonstrated +2 to +4 reactivitywith M. leprae, only two clones (IIIE9 andIVD8) produced monoclonal antibodies whichreacted only with M. leprae (Table 2). Clone 11(IF5) was difficult to passage and was intermit-tently weakly reactive with M. leprae, but it wasretained because of its strong reactivity to M.phlei. Clone 4 (IVD2) reacted with all mycobac-teria tested. Other clones produced antibodieswhich appear to have unique reactive patterns asdefined by the ELISA test (Table 2).

In contrast to the ELISA reactivity, the im-munofluorescence reactivity of some of themonoclonal antibodies was difficult to demon-strate. It was important to gently sonicate the M.leprae substrate before fixation to demonstratethe binding of the monoclonal antibodies. At-tempts to use organisms directly without sonica-tion gave highly variable results. Table 3 showsthat five of the antibodies, including IIIE9 (spe-cific for M. leprae by ELISA), were weaklyreactive or nonreactive with the M. leprae anti-gen substrate. Other antibodies which were re-active with M. leprae by immunofluorescencewere greatly reduced in their binding after treat-ment of the antigen substrate with trypsin (Table3).Immunoblotting was implemented to deter-

mine the molecule-specific binding of the mono-clonal antibodies. Attempts by immunoblottingto detect the molecule which contains the epi-tope which elicited either antibody IIIE9 orantibody IVD8 failed. In contrast, immunoblot-ting with antibody IIH9 showed that this mono-clonal antibody binds to a molecule with anapparent molecular weight of approximately68,000 (Fig. 1, lane ML). It was also evident

from the immunoblotting results and the ELISAdata that this antigenic determinant is commonto other mycobacteria, specifically M. tubercu-losis H37Rv (Fig. 1, lane MTB), M. gastri (Fig.1, lane MG), M. smegmatis (Fig. 1, lane MS),M. flavescens (Table 2), and M. gordonae (Ta-ble 2).We do not have direct evidence to show that

the 68,000-dalton antigen is a protein molecule.However, polyacrylamide gel profiles of identi-cal extracts from the blotted organisms shown inFig. 1, stained with Coomassie R250, showednumerous proteins along the entire lane, withtwo to three protein bands in the 68,000-daltonregion. In addition, the treatment of M. lepraewith trypsin reduced the immunofluorescenceassociated with antibody IIH9 (Table 3). Thesedata suggest that the 68,000-dalton antigen isproteinaceous; therefore it will be referred to asthe M. leprae 68,000-dalton protein.A crude extract of uninfected armadillo liver

tissue was blotted to control possible nonspecif-ic binding of monoclonal antibody to non-myco-bacterial cross-reactive antigens present in ex-tracts of either purified M. leprae (armadilloderived) or uninfected armadillo liver tissue.Figure 1 shows no binding of antibody IIH9 ateither 50 p.g (lane 1, ALH) or 100 jig (lane 6,ALH) of total protein electrophoresed and blot-ted.

DISCUSSIONAn antigenic analysis of M. leprae is critical to

the understanding of the basic immunopatholog-ical phenomena associated with leprosy. Thesimultaneous development of an animal modelfor leprosy that yields relatively large amountsof purified M. leprae and the work of Milsteinand Kohler (11, 12) in the area of somatic cellhybridization for the production of monoclonalantibodies has set the stage for an organizedanalysis of the antigens of M. leprae on amolecular level. Theoretically, the production ofmonoclonal antibodies to M. leprae antigensshould provide the tools needed to dissect ahighly complex antigenic mixture associatedwith a microorganism which continues to eludeattempts at in vitro cultivation. We initiatedstudies to determine the applicability of usingmonoclonal antibodies in the antigenic analysisof M. leprae. We report here the successfulproduction and partial characterization of 11monoclonal antibodies to M. leprae.

It is evident from these studies that the pro-duction of monoclonal antibodies to M. lepraeunder the conditions described is possible.Moreover, a variety of monoclonal antibodieswere raised which exhibited unique immunore-activity as defined by the ELISA test. One of

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176 GILLIS AND BUCHANAN

11H9 B[3W1:

ALH ML MTB M N iJG Al hH

94

_ _1 68

30

FIG. 1. Immunoblot of uninfected armadillo liverhomogenate (ALH), M. leprae (ML), M. tuberculosisH37Rv (MTB), molecular-weight markers (MWM), M.gastri (MG), and M. smegmatis (MS). The blot wasreacted with monoclonal antibody 11H9 and 125I-la-beled Staphylococcus aureus protein A. Molecular-weight markers are, from top to bottom, phosphoryl-ase B (94,000), bovine serum albumin (68,000), andcarbonic anhydrase (30,000).

these antibodies (IVD2) was elicited from acommon antigenic determinant which was ex-tracted from all 19 species tested. Many genus-specific and broadly cross-reactive antigens ofthe mycobacteria are high-molecular-weightpolysaccharides such as arabinogalactan (6). Itis likely that similar polysaccharides were pres-ent in the crude extracts of M. leprae used forimmunizations and therefore may have elicitedantibody IVD2. Alternatively, antibody IVD2may be directed at a highly conserved region of apolymorphic protein molecule found in all spe-cies of mycobacteria tested.

In contrast to the broadly reactive antibodyIVD2, monoclonal antibodies IIIE9 and IVD8demonstrated exquisite specificity for M. lepraeby ELISA. Since the panel of antigens screeneddid not include all species of mycobacteria (14)or all serotypes of certain species (5), the defini-tion of specificity for these antibodies is takenwithin the limits of the 19 species of mycobacte-ria. Among the 18 nonreactive species, 14 are

saprophytic or nonpathogenic to humans, 3 are

important human pathogens (M. tuberculosis,M. intracellulare, and M. marinum), and 1 (M.bovis BCG) is an attenuated bovine and humanpathogen often used for human vaccination.Because of their demonstrated specificity,

antibodies IIIE9 and IVD8 are potentially im-portant. By utilizing one or more M. leprae-specific antibodies in a competition antibody-

binding experiment, it may be possible to detectan M. leprae-specific antibody in the serum ofan individual with subclinical leprosy. Thiswould allow the detection of the specific anti-body in the presence of cross-reactive mycobac-terial antibodies which may be present as aresult of infection with, or environmental expo-sure to, mycobacteria other than M. leprae. Inaddition, antibodies to BCG elicited by BCGvaccination should not interfere with a serologi-cal test of this type because of the demonstratedspecificity of the monoclonal antibody for M.leprae. Such an approach would facilitate theearly diagnosis and treatment of individuals whomight otherwise take years to develop recogniz-able clinical symptoms. A possible drawback tothe competition experiment with monoclonalantibodies from experimental animals may bethat M. leprae antigenic determinants that areimmunogenic in the animal used for the produc-tion of the monoclonal antibodies may be lessimmunogenic or non-immunogenic in humans.Accordingly, certain monoclonal antibodies maybe of limited value in a competition assay asdescribed above. However, if other determi-nants on a given antigenic molecule are immuno-genic in the experimental animal, the monoclo-nal antibodies elicited would serve as effectivereagents for the purification and subsequentcharacterization of that antigen.

Further characterization of the antigenic mol-ecule associated with antibody IIIE9 has beenslow since immunoblotting and immunofluores-cence reactivity were negative. Antibody IVD8was also nonreactive by immunoblot, but dem-onstrated immunoreactivity by indirect immuno-fluorescence on sonicated M. leprae. The treat-ment of the antigen substrate with trypsin aftersonication greatly reduced antibody IVD8 bind-ing, suggesting that the molecule which elicitedantibody IVD8 may be proteinaceous and proba-bly not exposed on the surface of the organism.The fact that other monoclonal antibodies

(IIC8, IVD2, etc.) were positive for M. leprae byELISA but negative by immunofluorescenceemphasizes the need for confirmation of mono-clonal antibody immunoreactivity by more thanone assay system. It is possible that differencesin antigen exposure concentration or labilityvary significantly between assay systems, whichmay result in conflicting data.A comparison of immunoblotting, immunoflu-

orescence, and ELISA data for monoclonal anti-body IIH9 showed good correlation. From theELISA data it was predicted that M. smegmatis,M. gastri, M. gordonae, M. flavescens, and M.leprae should all blot similarly. Of the speciestested thus far, M. leprae, M. gastri, M. smeg-matis, and M. tuberculosis all contained a68,000-dalton protein which bound antibody

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IIH9 after transfer to NCP. Immunofluores-cence of trypsin-treated M. leprae antigen sub-strate with antibody IIH9 further suggested theprotein or protein-associated nature of this epi-tope.With regard to antigenic similarities among

the 19 species tested, it is apparent that 8 of 11monoclonal antibodies reacted with M. Iepraeand primarily 3 other species (M. gastri, M.gordonae, and M. flavescens). This suggestedthat these three organisms may be closely relat-ed to M. leprae. Previous studies (2, 8, 19) withpolyclonal immune sera have focused on theantigenic similarities between M. leprae andother species, including M. avium, M. smegma-tis, M. vaccae, M. nonchromogenicum, and M.bovis BCG.

This discrepancy is largely the result of theselection process invoked in our monoclonalantibody preparation and screening procedure.Since our initial objective was to screen forclones which were potentially specific to M.leprae, we did not keep any clones which react-ed with M. smegmatis. This step excluded fromour analysis clones that produce antibodies tocommon antigens shared between M. smegmatisand M. leprae and therefore revealed the nextlevel of cross-reactivity among these 19 species.Since many of the shared antigens of the myco-bacteria are high-molecular-weight polymericand sometimes structural polysaccharides foundin the cell envelope, it is possible that theantigen(s) which elicited the cross-reactivemonoclonal antibodies may represent a highlyconserved functional enzyme(s) important incellular metabolism and physiology. Alterna-tively, the monoclonal antibodies may havebeen elicited from different epitopes on a singleimmunodominant polymorphic protein antigenofM. leprae. Immunoblotting and radioimmuno-precipitation experiments with each monoclonalantibody should clarify this point.As more information is accumulated with re-

spect to the immunochemistry of M. leprae, itshould be possible to construct an antigenic andstructural map of the surface of the organism.Some surface antigens may be virulence factorsas well as physiologically important envelopeproteins that are encoded in the genome. Genet-ic engineering experiments designed to transferspecific envelope proteins to a new host capableof rapid growth and synthesis of that protein willrequire specific probes to detect the successfultransfer of the gene and subsequent gene prod-uct. Monoclonal antibodies would serve as spe-cific probes capable of monitoring these kinds ofexperiments. These experiments could lead tothe production of large amounts of well-charac-terized antigens suitable for skin test reagentsand materials for vaccine development.

ACKNOWLEDGMENTSThis work was supported in part by Public Health Service

grant A116290 and contract A192624 from the National Insti-tute of Infectious Diseases, by Federal Health Services Proj-ect SEA 78-17, by the Immunology of Leprosy (IMMLEP)component of the UNDP/World Bank/WHO Special Pro-gramme for Research and Training in Tropical Diseases, bythe Rockefeller Foundation Program for Research on GreatNeglected Diseases, and by the Victor Heiser FoundationFellowship Program.We thank Susan Dinning for her excellent technical assist-

ance.

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