INTERNATIONAL JOURNAL OF LEPROSY^

Volume 64, Number IPrinted In the 1

Development of a SOD ELISA to Determine theImmunological Relatedness Among Mycobacterial

Channappa T. Shivannavar, Vishwa M. Katoch, Vishnu D. Sharma,Madhusudan A. Patil, Kiran Katoch, Ved P. Bharadwaj, and

Bhakt M. Agrawal 2

Serological techniques can be used toidentify and to characterize pathogenic or-ganisms, including mycobacteria, directlyfrom clinical specimens even without in-vitro isolation of the organisms ("). Suchtests mainly depend on the reaction of singleor multiple antigenic components of bac-teria with antibodies against them. De-pending upon the amino-acid sequence andstructural configuration, the antigenicity ofproteins (bacterial components) differs fromspecies to species. These changes in the ami-no-acid sequences of enzymes (protein) aremeasurable by immunological crossreactiv-ity and have been observed to reflect evo-lutionary relatedness or divergence (2 3.26').

A number of indirect immunologicaltechniques/assays have been developed tomeasure the difference in the amino-acidsequences of the polypeptide chain of a sin-gle protein/enzyme. Among these methods,complement fixation, precipitation, and im-munodiffusion techniques have been usedto study the phylogenetic and evolutionarydivergences among various species of dif-ferent genera ( 3 7' 9, 21, 30‘ .) In mycobacteria,catalases have been analyzed for the mea-surement of the immunological distances(ImDs) by using techniques such as macro-or micro-immunoprecipitation ( 14 . ' 5 . 31 ' 32 ).

While these techniques have various tech-

' Received for publication on 23 May 1995; acceptedfor publication in revised form on 8 November 1995.

= C. T. Shivannavar, Ph.D., Research Assistant; V.M. Katoch, M.D., Deputy Director; V. D. Sharma,Ph.D., Research Officer; M. A. Patil, Ph.D., formerResearch Assistant; K. Katoch, M.D., Assistant Di-rector; V. P. Bharadwaj, Ph.D., Officer-in-Charge (re-tired), Central JALMA Institute for Leprosy (ICMR),Taj Ganj, Agra 282001, India. B. M. Agrawal, M.D.,Professor and Head, Department of Microbiology, S.N. Medical College, Agra 282001, India.

Reprint requests to Dr. V. M. Katoch.

nical limitations on the stability of enzy-matic activity and the quantities required,the major problem for Mycobacterium lep-rae was the absence of any demonstrableimmunoreactive catalase in the cell-free ex-tracts of Al. leprac derived from armadillos( 15 ). To overcome these difficulties, anotherwidely distributed molecule, superoxidedismutase (SOD) (

I I. 17, IS. 33) , was chosen and

preliminary results showed its potential( 13.28 ).

This communication describes the de-velopment of an ELISA-based system to de-termine the immunological relatednessamong different mycobacterial SODs andalso to compare the trends discerned by thistechnique and conventional immunopre-cipitation methods.

MATERIALS AND METHODSMycobacterial strains. Mycobacteria in-

cluded in this study were Al. vaccae (Stan-ford 877R), M. phlei (NCTC 8156), M.smegmatis (TMC 1546), M. tuberculosisH37Ra (NCTC strain), Al. tuberculosisH37Rv (TMC 102), AI. boris BCG (Glaxo),M. avium (TMC 706), M. scronilaceum(TMC 1302) and Al. leprae (armadillo-de-rived). The cultivable mycobacterial strainsincluded in this study were initially ob-tained from Dr. J. Stanford, London; Na-tional Institutes of Health, Bethesda, Mary-land, U.S.A., and Central Drug ResearchInstitute, Lucknow, India, and are beingmaintained in the Microbiology Laboratoryat our Institute. M. leprae was obtained fromthe livers and spleens of armadillos infectedat this Institute with human leproma andstored at —70°C.

Growth and medium. Initially, all thecultivable mycobacterial strains were grownon Lowenstein-Jensen (LJ) medium (22 ),then subcultured into the liquid Sauton's

58

64, 1^Shivannavar, et al.: Mycobacterial Relatedness by SOD ELISA^59

medium ( 27 ), and incubated at 37°C untilluxurient growth was obtained. The log-phase growths were collected by centrifu-gation and washed with 0.01 M phosphatebuffer (pH 7.2) and stored at —20°C untilused. M. leprac were purified from the in-fected armadillo liver and spleen by the per-colt density gradient centri fugation-biphas-ic separation method of Draper ( 6 ) and werechecked for any cultivable mycobacteria.

Preparation of cell-free extract (CFE) andpurification of SOD enzyme. The CFEsfrom cultivable mycobacterial species wereprepared by passing the growth suspendedin phosphate buffer (1:5) through the pre-chilled French pressure cell press at 28,000psi (AMINCO, SLM Instruments, Inc., Ur-bana, Illinois, U.S.A.) or by sonication (Lu-cas Dawe Ultrasonics, U.K.) ( 10 ). Clear CFEswere obtained by centrifugation at 28,000x g for 30 min at 4°C. A part (10 ml) of theCFE from each strain was stored at —70°Cuntil used; the rest of the extract was usedfor purification of SOD enzyme by the mod-ified methods ( 16 ' 17 ). Briefly, KC1 was addedto the CFE to a final concentration of 0.1M, heated for 5 min at 65°C, and cooled tobelow 10°C immediately in an ice bath. Thenucleic acids and ribosomes were removedfrom the KC1-treated supernatant by treat-ing with streptomycin sulfate (2.5% w/v fi-nal concentration). The clear supernatantfrom the above step was made to 65% sat-uration with solid ammonium sulfate. Afterremoving the precipitate, the supernatantwas again made to 85% saturation with am-monium sulfate and incubated at 4°C over-night. The precipitate was saved and dis-solved in a minimum volume of phosphatebuffer. This was dialyzed overnight againstthe 0.1 M phosphate buffer of pH 7.0 at4°C. The dialyzed solution was used for thefinal purification of SOD by 7.5% polyacryl-amide gel electrophoresis (PAGE). The CFEfrom M. leprae was prepared by the samemethod using a mini French pressure cellpress.

Purification of SOD enzyme by polyacryl-amide gel electrophoresis (PAGE). Thepartially purified SOD enzyme preparationsfractionated with 85% saturation of am-monium sulfate were subjected to 7.5%PAGE (4 ), and the purification of the SODenzyme was repeated until a single proteinband coinciding with the achromatic en-

zyme activity band was obtained on 10%PAGE. The purified SOD enzyme was fur-ther checked for purity by SDS-PAGE.

PAGE staining procedures. The poly-acrylamide gels were stained after electro-phoresis for SOD enzyme activity by themethod of Beauchamp and Fridovich ('),and proteins were stained by the method ofHolbrook and Leaver (8 ).

Measurement of SOD activity. SOD en-zyme activity in CFE preparations obtainedin different steps of enzyme purification wasestimated spectrophotometrically by thetechnique of Nishikimi, et al. ( 25) and Kak-kar, et a!. ( 12 ).

Raising of antibodies against purifiedSOD. Each purified mycobacterial SODenzyme was mixed and emulsified with 1mg of immuno-adjuvant peptide (N-ace-tylmuramyl-L-alanyl-D-isoglutamine-M DP,Sigma) and incomplete Freund's adjuvant(IFA; DIFCO/Sigma) as per the method ofWayne and Diaz ( 31 ). One ml of this emul-sified mixture was injected intradermally/subcutaneously into the New Zealand whiterabbit at 5-8 sites. These same injectionswere repeated on day 21 and day 40. Finally,the booster (SOD enzyme intravenously)was given on day 54, and the rabbits werebled 7 days later. The serum was separatedand stored at —20°C in aliquots until used.

Purification of immunoglobulins. Thehyperim mune serum was precipitated withan equal volume of saturated ammoniumsulfate solution ( 31 ), and the precipitationwas repeated twice. The precipitate mixturewas allowed to stand for 2 hr and was thencentrifuged. The pellet was dissolved in aminimal volume of phosphate buffer of pH7.2 and was then dialyzed against 0.1 Mphosphate buffer of pH 7.2 at 4°C. Finally,the volume was made equal to the originalvolume of serum taken with phosphate buff-er.

Serological assays. 1m munopreci pita-tion of SOD with homologous and heter-ologous antibodies was carried out with aslight modification of the method originallydeveloped and used for estimation of 1mDsbetween two mycobacterial species usingcatalase enzyme as a marker by Wayne andDiaz ( 31 ). The assay procedure used in thisstudy was as follows: one unit of SOD so-lution was added to equal volumes of se-rially diluted homologous and heterologous

60^ International Journal of Leprosy^ 1996

antibodies in 1.5-m1 centrifugation tubes,mixed well, incubated at 37°C for 1 hr, fol-lowed by incubation at 4°C overnight. Thesupernatant was separated and used to mea-sure the unbound SOD present by the spec-trophotometeric method described above.Rabbit sera collected before injection wereused as controls.

ELISA. Based on the principles of anal-ogous systems (24, 34‘ ,) an ELISA for mea-suring the binding of SOD and anti-SODantibodies was developed. This was used forestimating the binding of mycobacterialSOD with homologous and heterologousanti-SOD antibodies. The essential steps ofthis procedure are as follows: 1) The purifiedSOD enzyme, 25 pg/mI (antigen) was dis-solved in carbonate bicarbonate buGr, pH9.6 (coating buffer). 2) Each well, except thelast two wells, of a microtiter plate (CookeMicrotiter, U.S.A.) received 100 pl of acoating buffer containing SOD (25 pg/mI).The last two wells received only the coatingbuffer, and the plates were incubated over-night at 4°C. 3) Wells were washed threetimes with phosphate buffer saline withTween-20 (PBS-T), 100 pl of bovine serumalbumin fraction-V (BSA-V; 2 mg/fill) inPBS-T was added to each well, and the plateswere incubated at 37°C for 1 hr. 4) Afterwashing the wells, 100 of serially dilutedtest and control rabbit sera (immunoglob-ulins) was added to respective rows and col-umns except for the number 1 well (antigencontrol) and the last well (substrate control).The second to last well of the microtiterplate received undiluted homologous serum(immunoglobulins). The plates were incu-bated at 37°C for 3 hr in a humid chamber.5) The plates were washed with PBS-T threetimes, and the traces of buffer in the wellswere removed. Then, 100 pl of diluted (1:5000) anti-rabbit immunoglobulins conju-gated with horseradish peroxidase in thePBS-T (Dakopatts) was added. The plateswere shaken carefully and incubated over-night at 4°C in a humid chamber. 6) Theunbound enzyme-labeled antibodies werewashed with PBS-T four times. 7) 100 pl of0.1% o-phenylene diamine (OPD) in citricacetate buffer (pH 5.0) containing 0.03%H 2 0,, (to be prepared just before use) wasadded and incubated at room temperaturefor 30 min in the dark. 8) The reaction wasstopped by adding 25 pl of 8 N 1-I,SO 4 to

each well. 9) The color developed was mea-sured at 490 nm using an ELISA reader (Mi-crotiter reader; Model 700: Dynatech Labs,Inc., Chantilly, Virginia, U.S.A.) and thetiter of each test serum was calculated. Thefirst well without test antibody was taken asblank, the antigen control (second to lastwell) was an antibody control, and the lastwell was a substrate control.

Calculation of immunological distances(ImDs). The binding capacity of serumagainst a given SOD preparation was thereciprocal of that dilution of serum whichprecipitated (in immunoprecipitation tech-nique) or bound (in ELISA) 1 unit or a de-fined amount of SOD. These were calculat-ed from the points on the titration curveclosest to the 50% endpoint on the proba-bility graph paper, as done in the catalasesystem earlier ( 3 ').

RESULTSThe ELISA system standardized in this

study elicited consistent and reproducibleimmunological distances between the ho-mologous and the heterologous mycobac-terial SODs. Antigen-coated plates were sta-ble and shared consistent results up to 25pg/ml (SOD enzyme).

Calculation of linDs using OD serologicaltiters by ELISA. The binding titers of se-rially diluted anti-sera (1:16, 1:32, 1:64,1:128, 1:256, 1:512, . . . 1:8192) raisedagainst SODs of cultivable mycobacter -iawere determined with homologous SODs(25 pg/m1) by the ELISA technique. TheImD units calculated between the homol-ogous and heterologous antigen-antibodytitrations of eight cultivable mycobacteriaare shown in Table 1. The reciprocal ImDsof M. tuberculosis H37Ra anti-SOD anti-bodies versus M. boris BCG SOD and M.boris BCG (serum) versus Al. tuberculosisH37Ra (SOD) were found to be 11 and 12ImD units, respectively, by the two meth-ods. The ImDs calculated for other specieswere found to be similar, consistent, andreproducible by both immunoprecipitationand ELISA methods. Table 2 comparessome of the ImDs estimated between theSODs of different cultivable mycobacteriaby immunoprecipitation and ELISA tech-niques. It should be noted that very similarImDs were discerned by these two tech-niques.

64, 1^Shivannavar, et al.: Mvcobacterial Relatedness by SOD ELISA^61

TABLE 1. Reciprocal immunological distances (I InDs) between dillerent mycobacterialSODs in cultivable mycobacteria by our ELIS.-1 technique.

Strainno. Antibodies

Antigens

3^4^5^6^7^8

I.^.1/. phfri^ 0^522. AL velem('^ 51^03. M. smej.,unatis^21^434. M. tuberculosis H37Ra^91^935. M. tuberculosis H37Rv^91^926. .1/. boris BCG^81^857. M. cilium^46^538.^M. scrolidaceum^29^41

22^91^92^81^46^29__44^93^92^84^52^43

0^82^83^76^42^2981^0^2^II^50^8383^3^0^14^52^8577^12^13^0^48^7141^51^54^47^0^1429^83^85^71^13^0

" 1, 2, 3, 4, etc., are same mycobacterial strains as in the vertical column.

The ImD values between M. leprae andother cultivable mycobacteria included inthis study, as calculated by the titration ofanti-SOD antibodies raised against SODsfrom cultivable mycobacteria versus SODantigen from A/. leprae by ELISA and im-munoprecipitation techniques, are shown inTable 3. ImD units calculated from titra-tions by the immunoprecipitation (P) andELISA (E) techniques were similar for Al.leprae versus all of these eight species. TheImDs of SOD from A/. leprac to variousspecies were: Al. phlei (69P vs 71E), A/. vac-cae (74P vs 75E), M. smegmatis (61 P vs59E), Al. tuberculosis H37Ra (30P vs 31 E),Al. tuberculosis H37Rv (31P vs 29E), M.boris BCG (23P vs 25E), Al. (11111131 (20P vs22E) and Al. scrojidaceum (31P vs 38E),respectively.

DISCUSSIONAccording to Cocks and Wilson ( 2 ), the

immunological distance (ImD) is a loga-rithmic function of the index of dissimilar-ity between two biological systems, and isdirectly related to the differences in the ami-no-acid sequence of a particular protein.These ImDs between various biological sys-tems have been estimated and have beenreported to correlate with the structural di-vergence of protein, up to substitution ofabout 40% amino-acid sequences (315.26 ).

The immunoprecipitation, immunodiffu-sion, sero-agglutination and microcompli-ment fixation tests and the ELISA are someof the techniques used for this purpose invarious biological systems, including my-cobacteria (2, 14, IS, 26, 31 ) Except for theELISA, most of these techniques require

larger quantities of antigen and antibodies,and the sensitivity of the assays are gener-ally low.

To determine the immunological dis-tances of mycobacterial catalases, Wayneand Diaz ( 31 ' 32 ) used the immunoprecipi-tation technique. Because of a lack of thepresence of an adequate quantity of im-munoreactive catalases in the cell-free ex-tracts (CFEs) in A/. leprae ('s), another wide-ly present enzyme, superoxide dismutase(SOD), has been chosen as a target moleculefor the measurement of ImDs and has shownpromising results ( 13 ). This enzyme has beenshown to be present in various mycobac-teria (' 1 . 13 ' 17 ' 18 ' 33 ), and has been demon-strated to possess a qualitative difference inmycobacteria (23 ) and other organisms, suchas those belonging to Acholeplasmataceae(20). We have earlier standardized an im-munoprecipitation technique ( 13 ) to mea-sure these divergences in the SOD molecule,and observed that there could be a problemin estimating ImD values by the immuno-precipitation technique at different inter-vals, mainly due to the loss of SOD activityon long-term storage in the laboratory. De-graded or denatured enzyme antigen re-sulted in using variable concentrations ofthe antigen in the assay which led to dis-cordance since this assay is based on theestimation of unbound enzyme in the su-pernatant. A similar problem was reportedby Wayne and Diaz ( 31 ) in their study onimmunological distances of mycobacterialcatalases. To overcome this problem, andalso with an aim to improve the sensitivity,stability of reagents used in the assay, andto handle more samples at the same time

62^ International Journal of Leprosy^ 1996

TABLE 2. Comparison of values of immunological distances (IinDs) between mycobac-terial SODs by immititoprecipilation and ELISA techniques.

Strain110.

Antibodies(antigens)

Antigens(antibodies)

ImD units"

immunopre-cipitation ELISA

I. M. tuberculosis I137Ra M. tuberculosis H37Rv 04 03bends BCG M. tuberculosis H37Rv 12 14

3. tl. phlei Al. raccae 54 514. A1. smcgmatis M. tuberculosis H 37R v 88 835. A1. raccae M. tuberculosis H37Ra 94 936. A1.^al'ill111 Al. tuberculosis H37Rv 50 517. Al. ariunz M. phlei 47 468. M. avium raccae 51 52

scrqfulaccum Al. tuberculosis H37Ra 80 8310. Al. scrolulaccum M. raccae 40 4111. A1. serglitlacmur M. avium 12 1312. Al. avium M. boris BCG 42 4713. M. smegmatis M. raccae 44 4314. Al. boris BCG M. phlei 78 81

Rounded up to the nearest whole number.

without difficulty, we have developed in thisstudy an ELISA technique to measure theimmunological crossreactivity using puri-fied SOD enzyme as antigen. It should beemphasized that purity of the SOD will beimportant for such an analysis. The purifi-cation technique described in this study ap-pears to be appropriate for this purpose, asevidenced by the final single band of pro-teins staining (PAGE and SDS-PAGE) andmatching achromatic bands of SOD activityon PAGE ( 28 ). Also, Ouchterlony's doublediffusion method showed only a single pre-

TABLE 3. Immunological distances(ImDs) of SOD of M. lepraefrom eight cul-tivable mycobacteria by both the immuno-precipitation technique and ELISA.

Antigen ofAI. leprue

Strain Antibodies (1mD units)'no. of Immuno-

precipi-tation

ELISA

1. M. phlei 69 71Al. raccae 74 75

3. M. smeginatis 61 594. Al. tuberculosis H37Ra 30 315. Al. tuberculosis H37Rv 31 296. Al. boris BCG 23 257. M. avitun 20 228. Ai. scnifulaceunt 31 38

Rounded up to the nearest whole number.

cipitation line when the antibodies were al-lowed to react with the purified SOD en-zyme or with crude extracts of different my-cobacteria. Further, there was no precipi-tation line between antibodies againstmycobacterial SOD and SODs from Esch-erichia coli and armadillo liver ( 25 ). This isalso substantiated by observing similartrends of relatedness by the immunoprecip-itation method based on measurement ofthe enzyme activity. However, it may alsobe of interest to check the purity of theseantigen preparations by other techniques,such as Western blot, or any other analyticalmethod.

In this ELISA method, the protein con-centration (25 pg/m1) of purified enzyme(SOD) was standardized and used as a con-stant for all mycobacterial SOD versus anti-SOD antibody titrations. The titers ob-tained by ELISA in the present study werehigher than those obtained by the immu-noprecipitation technique. The high sero-logical titers by this method may be becauseof the stability of the SOD as antigen ratherthan as enzyme and, also because of theinherent sensitivity of the ELISA technique.Even though high titers were obtained withthis ELISA, the ImD estimation betweenany two different mycobacterial SODs werefound to be very similar and comparable byboth the immunoprecipitation and ELISAtechniques. The ImDs calculated betweenany two mycobacterial SODs titrating either

64, 1^Shivannavar, et al.: Mycobacterial Relatedness by SOD ELISzl^63

SOD enzyme versus anti-SOD antibodiesor vice versa had reciprocally similar valuesand were reproducible. Although indica-tions of qualitative immunological differ-ences in SOD of Acholeplasmataceae ( 20 )and sonic mycobacterial species (I 7-1 ') weresuggested, the present study, for the firsttime, provides an ELISA for the quantita-tive measurement of such divergences onSOD molecules among mycobacteria. Sincethis study has shown the application of ourELISA system to tissue-derived M. /eprae,this approach can be used for taxonomicidentification of any new alleged in-vitro orin-vivo grown isolate. It is interesting to notethat, by this analysis, Al. leprae is closer toM. avium, M. bolls (BCG), M. tuberculosis;more distant from AI. scrobtlaceum and stillmore distant from rapid growers like M.smegmatis, M. vacate and Al. phlei. Similartrends have been observed by the measure-ment of the evolutionary distance by rRNAalso ( 2 '). Although Al. leprae preparationsdid not have sufficient catalase for suchanalysis, other mycobacteria have exhibitedsimilar trends of relatedness by determi-nation of ImDs of their catalases (I 5 ). Thiscorrelation between SOD ImDs and i 6SrRNA, as well as catalase ImDs, is impor-tant from the point of view of evolutionarychanges. While no relatedness between M.leprae and armadillo SOD was observed ( 25 ),it would be of interest to determine the re-latedness to various strains of armadillo-derived mycobactcria. The results of thisstudy, as well as the earlier reported findingsof Wayne and Diaz ( 32 ), further strengthenthe confidence in the use of serological mea-surements for determining the evolutionaryrelatedness/divergences of mycobacteria.SOD could be that alternate molecule forthese investigations. ELISAs for B-galacto-sidases of lactobacilli have been describedearlier ( 24 ). The results of our study furtherconfirm the validity of this approach whichcan be used for various biological systems.

The stability of the reagents and the re-producible results obtained in this studyshow that the ELISA technique standard-ized in this study should be a promisingmethod for wider application for taxonom-ical studies of mycobacteria (especially forthe noncultivable/difficult-to-grow species/strains), several members of which continueto be important pathogens of medical and

veterinary importance. Although this tech-nique may not be directly applicable to lep-rosy specimens with low bacillary load, thisinformation shows the scope to further de-velop such methods based on the SOD mol-ecule or any similar molecules. The tech-nique can, however, be directly applied fortaxonomic characterization of in vivo- (suchas armadillo/nude mouse) grown strains of

leprae as was successfully achieved ear-lier for M. lepraemurium using a catalase-based system ("). By identification of spe-cific epitopes, this strategy has the potentialof developing techniques for the diagnosisand monitoring of the treatment of leprosy,other mycobacterial and, perhaps, other in-fectious diseases.

SUMMARYThis study reports on the standardization

of an enzyme-linked immunosorbent assay(ELISA) system for the measurement of im-munological distances (ImDs) of the super-oxide dismutase (SOD) molecule among thecultivable mycobacteria, namely, Mycobac-terium vaccae, M. phlei, M. smegmatis, M.avium, M. scrofulaceum, Al. tuberculosisH37Ra, Al. tuberculosis H37Rv, and M.bolls BCG, and M. leprae. SODs from cul-tivable mycobacteria were purified, anti-bodies were raised against these molecules,and ImDs between these anti-SOD anti-bodies and antigen (SODs) were determinedby an immunoprecipitation technique stan-dardized earlier and by the ELISA tech-nique developed in this study. The ELISAsystem developed in this study showedhigher sensitivity and consistent and repro-ducible ImDs among various mycobacteria,including pathogens such as Al. tuberculosis,Al. leprae and M. (Ilium. These values werecomparable with the values derived by theimmunoprecipitation technique. Our ELISAtechnique appears to be a sensitive and rap-idly reproducible method with the addi-tional advantage of the stability of reagents,and holds promise in the taxonomy as wellas in the development of diagnostics for lep-rosy and other mycobacterial infections.

RESUMENEste estudio se refiere a la estandarizaciOn de un

cnsayo inmunoenzimntico (ELISA) par a la mediciOnde las distancias inmunolOgicas (ImDs) de la molOcula

64^ International Journal of Leprosy^ 1996

de superOxido dismutasa (SOD) entre Mycobacteriumleprae y las micobacterias cultivables Al. vaccae, M.phlei, Al. smegmcnis, Al. avium, Al. scrolidaceum, Al.tuberculosis H37Rv, y Al. boric, BCG. Las SODs delas micobacterias cultivables fucron puriticadas, contracllas se prepararon anticuerpos, y las I m Ds entre estosanticuerpos anti-SOD y los antigenos (SODs) se de-terminaron por una tecnica de inmunoprecipitaciOnestandarizada antes y por la tecnica de ELISA desa-rrollada en este estudio. El sistema de ELISA aquidesarrollado mostrO mayor sensibilidad y di6 I mDsconsistentes y reproducibles entre varias micobacte-rias, incluyendo a patOgenos tales como Al. tubercu-losis, Al. leprae y Al. avium. Estos resultados fueroncomparables con los valores dcrivados de Ia tecnica deinmunoprecipitaciOn. Nuestra tecnica de ELISA pa-rece ser un metodo sensible, rapido y reproducible, quetiene la ventaja adicional de la estabilidad de los reac-tivos. El metoclo promete ser de utilidad en estudiostaxonemicos y en el desarrollo de pruebas de diagnOs-tico de la lepra y de otras enferrnedades micobacteria-nas.

RESUME

Cette etude concerne la standardisation d'un systemeenzymatique (ELISA) pour la mesure des distances im-munologiques de la molecule de dismutase superoxide(DOS) parmi les mycobacteries cultivables (Mycobac-terium vaccae, Al. phlei„11. smeginatis, Al. avium, M.scrofulaceum, Al. tuberculosis H37Ra„l1. tuberculosisH37Rv, et le BCG a Al. bovis) et Al. leprae. Les DOSdes mycobacteries cultivables ont ête purifiees, on aprovoque Ia production d'anticorps contre ces mole-cules, et les distances immunologiques entre ces anti-corps anti-DOS et l'antigene ont ate determinees paruna technique d'immunoprecipitation standardiseeprecedemment, et par la technique ELISA developpeedans cette etude. Le systeme ELISA developpe danscette etude a montre une sensibilite plus elevee et desdistances immunologiques coherentes et reproduc-tibles parmi les difiCrentes mycobacteries, y comprisles pathogenes tels que Al. tuberculosis, .11. leprae etAl. avium. Ces valeurs etaient comparables aux valeursderivees par la technique d'immunoprecipitation. No-tre technique ELISA apparait etre une methode sen-sible et rapidement reproductible, avec l'avantage sup-plêmentaire de Ia stabilite des reactifs, et elle est pleinede promesses pour Ia taxonomic aussi bien que pourle developpement de methodes diagnostiques pour lalepre et d'autres infections mycobacteriennes.

Acknonledgment. The authors are thankful to Dr.L. G. Wayne, VA Medical Center, Long Beach, Cali-fornia, U.S.A., for valuable discussions on this studyduring his visit to this Institute; to Mr. S. K. Bhan,Mr. N. S. Singh, Mr. Sri Ram, Mrs. A. Robi and thestaff of the Animal Experimental Laboratory for tech-nical help, and to Mr. J. D. Kushwah for secretarialassistance. The gift of some reagents by LEPRA, U.K.is gratefully acknowledged.

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