INTERNATIONAL JOURNAL OF LEPROSY^ Volume 53. Number I

Printed in the U.S.A.

Serological Survey of Leprosy and Control Subjects by aMonoclonal Antibody-based Immunoassay'Sudhir Sinha, Utpal Sengupta, Gopal Ramu, and Juraj Ivanyi2

Leprosy continues to remain a seriouspublic health problem in the developingcountries and about 1.4 billion people areexposed to the risk ofcontracting the disease( 2"). The incidence of the disease could bereduced effectively by early diagnosis ofsubjects incubating the infection, who couldthen be put under close surveillance or givenprophylactic treatment ( 24). Various work-ers have developed serological assays forleprosy. The major technical approacheshave been: a) the fluorescent leprosy anti-body absorption test (FLA-ABS) for the de-tection of Mycobacterium teprac-specificantibodies in sera preabsorbed with otherspecies of mycobacteria for the eliminationof crossreactive antibodies ( 42); b) radioim-munoassay (RIA) of cross-absorbed sera forantibodies against the cell wall antigen 7 ofM. leprae ("); and c) enzyme-linked im-munoassay (ELISA) for antibodies againstthe phenolic glycolipid derived from cellwalls of M. leprae ( 4 9 ' 30).

Recently, we have reported the produc-tion of species-specific monoclonal anti-bodies against M. leprae ( 2 ') and subse-quently developed, using the ML04antibody, a serum competition RIA for theserology of leprosy ( 26). The reported pre-liminary data indicated that the competi-tion test achieved the desired degrees ofspecificity and sensitivity. In this paper, wefurther report on serological data obtainedfrom an extended collection of sera from

' Received for publication 20 January 1984; accept-ed for publication in revised form on 5 September1984.

2 S. Sinha, M.Sc.; U. Sengupta, Ph.D.; G. Ramu,M.D., Central JALMA Institute for Leprosy, Taj Ganj,Agra 282001, India. J. Ivanyi, M.D., Ph.D., Depart-ment of Experimental Immunobiology, The WellcomeResearch Laboratories, Beckenham, Kent BR3 3BS,U.K.

Reprint requests to Dr. Ivanyi, Director, MRC Tu-berculosis and Related Infections Unit, ClyclotronBuilding, 4th Floor, Royal Postgraduate Medical School,Hammersmith Hospital, Ducane Road, London W12OHS, U.K.

groups of patients with various forms oflep-rosy and from nonleprosy control subjects.

MATERIALS AND METHODSSera. The test group consisted of 96 sera

from untreated leprosy patients registeredfor treatment at the Central JALMA Insti-tute for Leprosy (CJIL), Agra, India. Pa-tients were classified by the Ridley-Joplingscale ( 23) and, accordingly, split into groupsof LL/BL (58 cases), BB (8 cases), and TT/BT (30 cases) leprosy.

Sera from 43 apparently healthy house-hold family members of proven leprosy cases(source cases) were also studied. These con-tacts were related (husband, wife, or chil-dren) either to multibacillary LL/BL/BB (28sera) or to paucibacillary TT/BT (15 sera)source cases.

Sera representing the control groups wereobtained from: a) healthy subjects, of whom18 were staff members of CJIL and fourwere U.K. citizens on a visit to India; b)proven cases of active pulmonary tuber-culosis attending the clinics of the TB Dem-onstration and Training Center, Agra; c) pa-tients suffering from autoimmune disorders(sera showing antithyroid or antinuclear an-tibodies) attending the clinics of SafdarjangHospital, New Delhi; and d) patients suf-fering from either breast or cervical cancerwhose sera were provided by the Instituteof Pathology, Safdarjang Hospital, NewDelhi.

All control subjects were clinically ex-amined and failed to manifest any signs ofleprosy.

Antigen. M. leprae were separated fromheavily infected armadillo livers by parti-tion in an aqueous two-phase polymer sys-tem ( 12). The soluble antigen was preparedby sonication of bacilli at 20 kHz, 80 Wusing a Dawe Soniprobe type 7532A, im-mersed in iced water for 20 min, followedby centrifugation at 105,000 X g for 30 min,and filtration through a 0.22 pm membranefilter. The preparation (batch V6) at 1 mg

33

34^

International Journal of Leprosy^ 1985

DIAGNOSISNUMBERPOSIT./TOTAL

PERCENT OF SACT—POSITIVE SUBJECTS

0^20^40^60^80I^I^I^I

1001

LL/BL 58/58 g23ENZ I:^40 \

BB 7/8 ^4 ^k^2AVW1..‘\.

TT/BT 14/30 7^4 ' -EL"Contacts of LL/BL/BB 13/28 8^14_L\M — 1

Contacts of TT/BT 0/15ANTIBODY

Pulmonary TB 0/31 I^ TITRE:

ITTI^=^5-25Autoimmune disorders 0/20 E =^25-125

Cancer 0/12 I^Fil^= 125-625...

Healthy subjects 0/22 I^M = > 625

I-71(i. 1. The incidence and titer of M. /eprae-specific (MY2a) antibodies in groups of leprosy patients andnonleprosy controls. Sera from a total of 224 subjects were tested in duplicate at four log s serial dilutions bythe SACT assay. Sera whic 1 failed to give 50% inhibition of 125 1-ML04 binding at the initial 1:5 serum dilutionwere scored as negative. Numbers of patients giving the corresponding ID,,, antibody titers are encircled.

per ml concentration was kindly suppliedby Dr. R. J. W. Rees, National Institute forMedical Research, London, U.K.

Monoclonal antibody. M. /eprae-specificM LO4 antibody ( 2 ') was produced as asciticfluid in BALB/c mice. Immunoglobulinswere precipitated from ascitic fluid by 18%sodium sulfate, dialyzed against phosphatebuffered saline (PBS), pH 7.4, and storedlyophilized in vials. Batches of 50 pg of thisantibody were labeled with Na ' 25 I (ob-tained from Isotope group, BARC, Bom-bay, India) using the "lodogen" technique( 15). After purification on a Sephadex G-50column, more than 90% of the radioactivitywas precipitated by trichloroacetic acid.

Serum antibody competition test (SACT).A modification of the already describedtechnique (20, 2

`') was used. Polyvinyl chloride (PVC) microtiter plates (Dynatech, Inc.)were coated with 50 Al per well of the dilutedsoluble Al. /eprae antigen (50 ag/m1 in PBS)overnight at 4°C. The plates were washedonce with PBS; subsequently the wells werefilled with a solution containing 2% bovine

serum albumin (BSA), and 0.1% sodiumazide in PBS (BSA-PBS) and stored coveredwith cellotape at 4°C until used.

Before the assay, the blocking solutionwas removed from the wells followed bytwo washings with PBS, and drying by pat-ting on a tissue paper pad. Log, serum di-lutions in BSA-PBS (four dilutions of eachserum sample) were put into duplicate wells(25 pl/well) and incubated at 37°C in a hu-midified container for 1 hr. Afterward, 125 1-ML04 was added into each well (40,000 cpmin 25 kt1 BSA-PBS) and plates were furtherincubated for 2 hr at 37°C. After washing 5times with PBS and drying the plates, in-dividual wells were cut and radioactivitiescounted in a gamma counter (model NE1600, Nuclear Enterprises, U.K.).

Expression of results. Mean binding of125 I-ML04 to wells coated with BSA alonewas taken as 0% and subtracted from eachof the test binding values. Relative bindingvalues for each serum dilution were calcu-lated whereby binding of ' 25 1-ML04 in BSA-PBS alone to M. /eprae-antigen-coated wells

53, 1^Sinha, et al.: Serology with Monoclonal Antibody^35

was taken as 100% (1000 to 3000 cpm). Theresults were expressed in terms of 1D,„ val-ues which represent the serum dilutioncausing 50% inhibition of ' 2 1-ML04 bind-ing to the antigen-coated wells.

RESULTSThe prevalence and level of serum anti-

bodies as determined by the SACT assayare expressed in Figure 1. All 58 LL/BL seratested contained high titers of antibodies.However, the antibody response was con-siderably weaker in nonlepromatous forms.In the relatively small group (8 cases) of 1313patients, antibody levels varied widelyamong the individuals. In the TT/13T formof the disease (30 cases), only 14 patients(47%) had demonstrable antibodies and ofthese, in 7 (23%) there were only low (11)„,-5-25) antibody levels.

Sera from clinically healthy family con-tacts of patients contained low antibody ti-ters in 13 (46%) subjects, who were all re-lated to multibacillary (LL/BL/BB) sourcecases. However, none of the 15 tested familycontacts of TT/I3T cases had demonstrableantibodies. As many as 85 sera were testedfrom nonlepros), control subjects, 81 ofwhom were residents of India. Although 31of these were patients with pulmonary tu-berculosis and 20 were positive for auto-antibodies, all of the subjects were negativein the ML04-based SACT assay.

The SACT assay has been standardizedfor the titration of human sera, using fourlog, dilutions. Representative curves dem-onstrating the various degrees of '"I-M LO4binding inhibition by test sera from patientsare demonstrated in Figure 2. Some controlsera showed a low degree (<50%) of ap-parently nonspecific inhibition of ' 25 1-ML04binding at 1:5 or lower serum dilutions. Thiswas observed more frequently with sera fromendemic areas, particularly from patientswith tuberculosis. The effect can probablybe attributed to steric hindrance caused byantibodies binding to sites in close prox-imity to the MY2a determinant.

DISCUSSIONThe Al. leprae bacillus is composed of

several antigens (i.e., protein, glycolipid, andpolysaccharide) which in turn are built frommultiple distinct antigenic determinants(epitopes) (3 , 5,10, 17, 19

). It seems conceivable

100-

80-

0

3cTs

60-

20 -

1/5^1/25^1/125^

1/625

TEST SERUM DILUTION

Fu 2. Demonstration of representative titrationcurves of '"1-ML04 inhibitory serum antibodies. Max-imally inhibitory nonendemic (0) or endemic (A) con-trol sera; antibody positive contact of a LL patient (^);antibody positive sera from 17/13T (A), Ill_ (•) or LL(■) patients.

that the antibody response which ensues asa result of infection is directed toward sev-eral determinants, of which the majority isshared with other species of mycobacteriaand only the minority is specific for Al. lep-rae. Using various antigenic preparations,several authors have clearly demonstrateda striking increase in antibody level in pa-tients at the lepromatous pole and a lower,marginal. or no increase at the tuberculoidpole of the disease ('' 4 ' is ' 31. Thus, the en-hanced antibody response in LL/BL pa-tients is well demonstrable even by semi-quantitative assays based on estimationswith a single dilution rather than serial di-lutions of tested sera. However, problemsmay arise in a) the definition of the marginwhich defines the border between the"background" and the "increased" anti-bod■, , level: and b) the elimination of "falsepositive" values which may result from ex-cessive stimulation with environmentalmycobacteria or with Al. tuberculosis.

The SACT assay resolves both problemslisted above on the grounds ofits specificity,

36^ International Journal of Leprosy^ 1985

which is restricted to a single antigenic de-terminant (MY2a) of M. leprae. This de-terminant is apparently immunodominantin LL/BL patients, since sera from all 58tested patients contained very high anti-body levels. Moreover, the reliable specific-ity of the SACT assay was demonstratedhere by the absence of "false positive" val-ues in any of the tested sera from variouscontrol groups comprised of patients withactive tuberculosis, autoimmune disorders,and cancer. It has been discussed previouslythat inhibition of monoclonal antibodybinding by sera from patients with schis-tosomiasis could be attributed to either an-tibodies or to circulating antigen ( 22 ). Thepossible contribution of M. leprae antigenin either free or in immune complex ( 6) formto the SACT results will need to be ad-dressed in future experiments.

The results of this study showed a con-trast between the high titers (>625 in 69%)of LL/BL and the lack of response (53.3%)or low titer (23.3%) of antibody in the TT/BT group. The latter result may be attrib-uted to the restriction of the assay to anti-MY2a-specific antibodies. However, itwould seem unlikely that TT/BT patientsproduce antibodies of different specificity,since other authors have found only low orabsent antibody responses in most patientsat the tuberculoid pole of the disease spec-trum ( 27 ' 28). The failure to detect specificantibodies in a large number ofTT/TB casesmay be due to the low antigenic load. Sincestudies in India have indicated that 74-90%of the early cases of tuberculoid leprosy maybe of a self-healing nature ( 7 ' 3 ), an increasein the antibody level could be interpretedas a prognostic signal for the shifting of thedisease toward the lepromatous pole of thespectrum.

In the group of household contacts ofmultibacillary leprosy cases, 46%) showedantibody positivity, much higher than thereported incidence rate of leprosy. Abe'sstudies with school children of Okinawa,Japan, have also shown that the FLA-ABStest was positive at least 200 times the actualincidence rate of leprosy ('). Similar obser-vations have been made in the study of lep-rosy contacts in Ethiopia (' 6). The positivelepromin skin test has also been found tobe much more frequent in a leprosy endem-ic area than the actual incidence rate of lep-

rosy ("). Presumably, the infection in con-tact subjects results in adequate protectiveimmunity and a self-healing outcome (". ' 1 ).

The much needed epidemiological test forleprosy should he highly specific and sen-sitive enough to detect early cases of theinfectious form of leprosy ( 21 ' The de-tection of such cases is mandatory for anepidemiological test if it is aimed at reduc-ing the leprosy incidence rate. Therefore,the apparent restriction of the reported anti-MY2a SACT assay as compared to othertests with a selective detection of lepro-matous cases is an apparent advantage.However, its prognostic capability to pre-dict the development of lepromatous lep-rosy at a subclinical stage is yet to be ex-plored in future long-term clinical trials.

Apart from its use in epidemiologicalstudies of family contacts, the SACT assaycould also be beneficial for monitoring theemergence of drug-resistant bacilli in re-sponse to therapy. Initial unpublished datasuggest a steady decline of ML04-specificantibody levels with continuing chemo-therapy in dapsone-responsive patients.

SUMMARYA serological antibody competition test

(SACT) using a murinc anti-Mycobacteriumleprae-specific monoclonal antibody (M L04)has been evaluated in 96 untreated leprosypatients and 128 control subjects. The testis based on the competitive inhibition byantibodies from human test sera of thebinding of the specific ' 25 I-ML04 murinemonoclonal antibody to M. leprae solubleantigen coated microtiter plates. Along thespectrum of the disease, SACT positivitywas observed in 100% of LL-BL, 87.5% ofBB, and 46.7% of the TT-BT groups of pa-tients. Moreover, 46.4% of apparentlyhealthy household family contacts of mul-tibacillary leprosy cases were also antibodypositive. Sera from 15 contacts of pauci-bacillary leprosy cases as well as from 85various nonleprosy subjects (tuberculosis,autoimmune disease, cancer, and healthy)were all found to be negative.

These results satisfy the requirements ofa leprosy-specific serodiagnostic test. Thestriking increase of antibody titer in all ofthe LL/BL patients indicates a potentialprognostic utility of this test for the lepro-matous shift of the disease. The observed

53, I^Sinha, et al.: Serology with Monoclonal Antibody^37

positivity in a fraction of healthy leprosycontacts still requires prospective evalua-tion of disease incidence in a larger test sam-ple.

RESUMENSe evaluO una "prueba serolOgica de competencia

por anticuerpo" usando un anticuerpo monoclonalmurino especifico contra Mycohaderium leprae (ML04)en 96 pacientes con lepra no tratada y en 128 sujetoscontrol. En la prueba, los anticuerpos presentee en lossueros problema inhiben competitivamente la uniondel anticuerpo monoclonal marcado ML04-1'" con elantigeno soluble derivado del M. leprae. El ensayo sehace en microplacas de pfilstico recubiertas con anti-geno. Se encontr6 que el 100% de los pacientes conlepra LL-BL, el 85% de los BB, y el 46.7% de los TT-BT, dicron un resultado positivo en esta prueba. Ade-mas, el 46.4% de los contactos familiares (aparente-mentc sanos) de los pacientes leprosos multibacilaresdicron tambien una prueba positiva. Los sueros de 15contactos de pacientes paucibacilares y los sueros de85 individuos sin lepra (con tuberculosis, con enfer-medades autoinmunes, con cancer o sanos) resultaron,todos, negativos.

Estos resultados satisfacen los requerimientos de unaprueba serodiagnOstica especifica para Ia lepra. El sor-prendente incremento en el titulo de anticuerpos entodos los pacientes LL/BL, sugiere una utilidad pro-n6stica potencial de esta prueba.

La positividad observada en una fracciOn de los con-tactos aparentemente sanos, a6n requiere de una eva-luaciOn prospectiva de la incidencia de la enfermedaden una muestra poblacional mils grande.

RESUMEChez 96 malades de la lepre non traites, et chez 128

sujets temoins, on a pratique des epreuves de compe-tition des anticorps du serum (SACT), en utilisant unanticorps monoclonal murin specifique contre Myco-bacterium leprae. Cette epreuve est bases sur une in-hibition competitive de la liaison de ]'anticorps mono-clonal murin specifique '"I-ML04 A l'antigene solublede Al. leprae dont on revel les plaques de microtitrage,par les anticorps provenant des echantillons de serumhumain que l'on etudie. Tout au long du spectre de lamaladie, on a relevê des epreuves SACT positives chez100% des malades LL-BL, 87,5% des malades BB, et46,7% des malades appartenant aux groupes TT-BT.De plus, parmi des contacts familiaux apparemmenten bonne sante partageant le foyer de malades atteintsde lepre multibacillaires, 46,7% présentait egalementune reaction d'anticorps positive. Par contre, les echan-tillons de serum obtenus chez 15 contacts de maladespaucibacillaires et chez 45 individus ne souffrant pasde lepre, mais d'autres maladies Idles que Ia tuber-culose, des maladies auto-immunitaires, des cancers,ainsi que des sujets sains, se sont tous reveles negatifs.

Ces resultats satisfont les exigences d'une epreuve

serodiagnostique specifique pour Ia lepre. L'augmen-tation frappante du titre des anticorps chez tous lesmalades LL/BL indique que cette epreuve pourrait etrefort utile pour predire ('evolution de Ia maladie versIa forme lápromateuse. Le fait que certains des contactssains de malade présentent une reaction positive merited'être etudie plus avant, en menant une etude longi-tudinale de ('incidence de la maladie dans un echan-tillon plus considerable d'individus.

Acknowledgments. We are thankful to Dr. R. J. W.Rees, National Institute for Medical Research, Lon-don, for the supply of Al. leprae antigen and for hiskeen interest in this work. We also thank Dr. M. L.Mehrotra, Director, TB Demonstration and TrainingCenter, Agra, for allowing the investigators (SS andUS) to collect sera from patients suffering from tuber-culosis, and to Dr. (Mrs.) Mithilesh Chandra of theInstitute of Pathology, New Delhi, for providing uswith sera from patients suffering from cancer and au-toimmune diseases.

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