Ten hybridoma cell lines prodacing monoclonaI antibodies (Mabs) against M. avium/intracellulare

(Mai) serotype 8 were raised by the fusion of BALB/c mouse myeloma cells

(SPZ) to spleen cells from immunized BALB/c mice. The specificity of the monoclonal

antibodies was defined using their differing abilities to bind to sonicates from a range of

mycobacterial species and strains. Tbe Mabs showed strain and species specificity. Three

Mabs bound only to Mai serotype 8 and 1 Mab bound only to Mai serotypes 8 and 16, the

only serotypes tested. The results indicate that Mabs specific for Mai species and serotypes

can be produced. These could be useful for serodiagnostic and for epidemiological purposes

Coates, Anthony R M

Jackett, Paul S

Lowrie, Douglas B

Mirchison, D A

Paramasivan, C N

NIRT Institutional Repository

Indian J Med Res 88, July 1988, pp 13-17Monoclonal antibodies against Mycobacterium avium/intracellulareC.N. Paramasivan, Paul S. Jackett, Anthony R.M. Coates, Douglas B. Lowrie& Denis A. MitchisonDepartment of Bacteriology, Royal Postgraduate Medical School, London, UK &Tuberculosis Research Centre, MadrasAccepted March 16, 1988Ten hybridoma cell lines prodacing monoclonaI antibodies (Mabs) against M. avium/intrace-llulare (Mai) serotype 8 were raised by the fusion of BALB/c mouse myeloma cells(SPZ) to spleen cells from immunized BALB/c mice.The specificity of the monoclonalantibodies was defined using their differing abilities to bind to sonicates from a range ofmycobacterial species and strains.Tbe Mabs showed strain and species specificity.ThreeMabs bound only to Mai serotype 8 and 1 Mab bound only to Mai serotypes 8 and 16, theonly serotypes tested.The results indicate that Mabs specific for Mai species and sero-types can be produced. These could be useful for serodiagnostic and for epidemiologicalOrganisms of the Mycobacterium avium/intracellulare complex (Mai) are of fairlyfrequent occurrence in man, especially incountries such as India1, where either theycolonise the tracheal mucosa or more rarelyare responsible for disease. Monoclonalantibodies (Mabs) specific for Mai couldtherefore be of value in the study of myco-bacterial disease. Firstly, their high speci-ficity might be used to subdivide species forepidemiological purposes.Secondly, theMabs could form the basis of serodiagnostictests specific for Mai infection, which isposing an increasing problem in immunocom-promised persons, particularly those withAIDS. Thirdly, the Mabs might be used toisolate the specific epitopes from Mai anti-gen. These molecules could then be in-valuable in testing the hypothesis that priorexposure to environmental Mai impairs thefficacy of BCG vaccination2,3. Mai islikely to be more important as an environ-mental immunogen than other mycobacteria,because it was the most frequent non-tuber-culosis mycobacterium isolated from sputumin the south Indian region where BCG vacci-nation was found to give poor protection1,4.Material & MethodsBacteria : M. avium/intracellulare serotype 8(NCTC 10610) and serotype 16 (NCTC10425) were used M. tuberculosis strainswere the laboratory strain H37Rv (phage13type B), strain S1 (isoniazid-sensitive, phagetype A) obtained before chemotherapy froma British patient and strain 7219 (isoniazid-sensitive; low virulence in the guineapig, witha rootindex of virulence of 0.57; phage type I)from a south Indian patient.Strains of M.bovis were Vallee (NCTC 5693) and BCG-Glaxo (both phage type A). Other bacteriawere M. scrofulaceum (NCTC 10803), M.kansasii (NCTC 10268), M. gordonae(NCTC 10267), M. flavescens (NCTC 10271),M. terrae (NCTC 10856), M. fortuitum(NCTC 10394),M. chelonei (abscessus)(NCTC 10882) and Escherichia coli strainK12.Antigen preparation : Antigens used forimmunisation and screening were ultrasoniclysates of mycobacteria. The mycobacterialcultures were grown on the surface of liquidSauton’s medium until confluent colonies werejust apparent. The cultures were then har-vested and sonicated in phosphate bufferedsaline (Dulbecco ‘A’) in an ice bath for 15min (in 5 sec bursts) using a Rinco ModelMP ultrasonicator (Rinco Ultrasonics UKLtd., London) at 70 per cent maximumintensity. After ultracentrifugation of thelysates at 100,000 g for 1 h (Kontron Instru-ments; Switzerland) the supernatants weresterilised by filtration (0.22 pm Millipore,SLGV membrane) and adjusted to a standardprotein content as determined by a modifiedLowry’s method with tartrate replaced bycitrate5.Immunisation and fusion : BALB/c mice wereimmunised ip with Mai serotype 8 antigencontaining 100 µg protein with incompleteFreund's adjuvant on days 0 and 15. Tendays after the second inoculation, sera fromthe mice were tested for antibody activityagainst Mai serotype 8 using solid phaseradioimmunoassay (RIA) as described pre-viously6. Mice which showed high levels of14 Monoclonal antibodies against M. avium/intracellulareantibody were selected and a third injection,with ut adjuvant, was given ip three daysbefore harvesting the spleen cells for fusion.Polyethylene glycol mediated fusions weredone with the mouse myeloma cell line SP27.The culture supernatants from numeroushyb ds were initially tested by RIA againstsonicates of Mai serotype 8 and Esch. coli,to determine the broad specificity of theclones. After two limiting dilutions, 14clones were selected and fuller specificitieswere determined by testing their culturesupernatants against sonicates of the 14mycobacterial species and Esch. coli. Ofthese clones, 10 which showed diverse speci-ficities were injected ip into pristane-primed mice to induce antibody-rich ascitesand each ascitic fluid sample was also testedfor specificity.Characterisation studies : The monoclonalantibodies (Mabs) were further characterisedby determining their immunoglobulin iso-types by immunofluorescent staining of theclones. Three clone supernatants wereselected forimmunoblotting to determinethe approximate molecular weight of theantigenic proteins to which the Mabs boundand to confirm the specificities shown by solidphase RIA8. Blotting was against the myco-bacterial sonicates that had been run onsodium dodecylsulphate-polyacrylamide slabgel electrophoresis (SDS-PAGE) under redu-cing conditions9.ResultsTh  binding of ascitic fluid rich in MabAI/1 to the antigen sonicate of Mai serotype8 as well as the control binding to the plasticwell is show in the Fig., and is representativeof all of the curves obtained with the asciticfluids, Based on such titrations, each asciticParamasivan et al 15Fig. Titration curves of binding of monoclonal anti-body AI/10 (ascitic fluid) to (a) sonicate of Mai sero-type 8 (O–O), (b) to empty plastic wells ( l – l )and net binding ( n – n , a–b). Binding is expressedas a percentage in relation to a control well coatedwith normal mouse serum (100% reference).fluid was diluted to an extent (to 10-4; inFig.) consistent with retaining net binding ofantibody to Mai serotype 8 antigen that wasat least 20 per cent of the values for normalmouse serum-coated wells (i.e.,percentagebinding >20%). The ascitic fluid was thenassayed at this dilution for cross-reactivitywith other mycobacterial sonicates.The binding patterns and antibody isotypesfor ascitic fluids from 10 hybridomas areshown in the Table. The binding of theseMabs suggested 6 distinct patterns. Thefirst is shown by a group, containing AI/4,AI/6 and AI/7, binding only to Mai serotype8 and not to any of the other mycobacterialantigens including Mai serotype 16; AI/2shows species specificity in that it boundonly to the Mai strains tested and not evento the antigenically closely related M.scrofulaceum; the third pattern was shownby AI/5 and AI/8, which reacted only withMai serotype 8 and, to a low degree, M.tuberculosis H37Rv; in contrast, AI/l boundslightly to M. bovis BCG in addition to Maiserotype 8 ; the fifth pattern is shown by AI/3,which bound to Mai serotype 8, M. bovisVallee and M. fortuitum. AI/9 bound toabout half to the mycobacterial sonicatestested and AI/10 reacted positively with allof them.The binding patterns for all 10 Mabs werealso assessed using undiluted supernatantfrom tissue culture wells and there was goodagreement with results from ascitic fluids.Further, assays of subclones of AI/9 (toconfirm purity of AI/9) were in close agree-ment with the results shown.In immunoblotting, AI/10, which boundto all of the mycobacterial antigens tested byRIA, bound uniformly to a common antigenof approximately 45 kdaltons. AI/8,chosen for its RIA specificity for Mai serotype8 and to M. tuberculosis H37Rv, boundselectively to an antigen of molecular weightof approximately 110 kdaltons that appearedto be present only in blots prepared fromextracts of these two organisms.AI/7 boundonly to an antigen of approximately 90kdaltons in Mai serotype 8 blots.DiscussionDiscrimination between mycobacterialtrains and species was achieved with 9 ofthe 10 Mabs (Table), though more strainsshould be tested before firm conclusions onspecificity of these Mabs can be reached.Mabs that distinguish between Mai serotypes4, 8 and 9 have recently been reported10.Of particular interest was the ability of mostof our Mabs to differentiate between Maiserotype 8 and serotype 16. Our evidenceis insufficient to conclude whether or notAI/4 and AI/6 bind to the Schaefer antigenof serotype 8 which is a small peptidogly-colipid11. In contrast, it seems unlikelythat AI/7 reacts with the Schaefer antigen16 Monoclonal antibodies against M. avium/intracellulareTable. Specificity of monoclonal antibodies†AntigenMonoclonal antibodyAI/4 AI/6 AI/7 AI/2 AI/5 AI/8 AI/1 AI/3 AI/9 AI/10M. avium/intracellulare (s rotype 8) 41 78* ** *31***3112****************2a21**16**24**41*******10**M. intracellulare (serotype 16)M. scrofulaceumM. tuberculosisH37Rv * *SI * *7219 * *M. bovisVallee * *BCG * *M. kansasii * *M. flavescens * *M. gordonae * *M. terrae * *M. fortuitum ** *M. chelonei * *Esch. coli * *Isotype 2a 2a† Percentage binding, defined as described in the text, is‘ shown only for positive reactions (binding of > 10 %).** 5–9.9 % binding;* < 5% binding**********2b 2b10*************3********11*******13******16**2a 2a54 100** 2514 32** 26** 2019 2.541 2719 24** 3852 35** 39* 3052 3076 61* *1 1because immunoblotting revealed that thisMab binds to a 90,000 dalton molecule.Further, this antigen was detected as a singleband with protein stains such as Coomassieblue and Amido black. It is neverthelesspossible that it contains the antigeniccarbohydrate residues present in the Schaeferantigen and that the Mab binds to thiscarbohydrate epitope.Isolation of antigens with Mabs such asthese would open two important avenuesof research into the reasons for the relativefailure of the BCG trial in south India.For example, a Mab such as AI/2 could beused to purify an antigen with an epitopeshared between several serotypes of M.avium/intracellularebut absent in othersp cies of mycobacteria. If this antigenwas more specific for Mai than PPD B itcould be useful as a skin test reagent or as astimulant of circulating T lymphocytes toestablish the incidence of exposure of indi-viduals in south India to Mai. However,if thi  antigen was a large protein moleculeit would probably bear many differentepitop s, some of which could be presentin other mycobacteria and thus compromiseParamasivan et althe specificity of the AI/2 epitope. In thissituation fractionation of the molecule mightyield the specific epitope. Secondly, anti-gens isolated by Mabs AI/9 or AI/10 wouldbear epitopes which were shared betweenMai, M. bovis BCG and M. tuberculosis. Oneof the hypotheses to explain the low protec-tive value of BCG vaccination in southIndia, is that exposure to environmentalmycobacteria, likely to be Mai, provides adegree of immunity which cannot be in-creased by subsequent BCG vaccination.An exploration of any interaction betweenshared epitopes and the specific epitope inprotective cellular immunity might help toclarify the basis of success and failure inBCG vaccination.AcknowledgmentAuthors are grateful to Dr P.A. Jenkins, for adviceand provision of M. avium/intracellulare strains.The first author was supported by a British CouncilTCTP fellowship.References1. Paramasivan, C.N., Govindan, D., Prabhakar,R., Somasundaram, P.R., Subbamal, S. andTripathy, S.P. Species level identification ofnon-tuberculous mycobacteria from South IndianBCG field trial area during 1981. Tubercle 66(1985) 9.4.5.6.7.8.9.10.2.3.Vaccination against tuberculosis. Report of an   11.ICMR/WHO Scientific Group. WHO TechRep Ser 651 (1980) 1.Edwards, M.L., Goodrich, J.M., Muller, D.,Pollack, A., Ziegler, J.E. and Smith, D.W.fection with M. avium-interacellulare andprotective effects of BCG. J Infect Dis(1982) 733.17In-the145Tuberculosis Prevention Trial, Madras. Trialof BCG Vaccines in South India for tuberculosisprevention.Indian J Med Res 72 suppl (1980) 1.Lowry, O. In :Techniques in protein chemistry,J. Leggett Bailey, Ed. (Elsevier, Amsterdam)1967 p 340.Coates, A.R.M., Hewitt, J., Allen, B.W., Ivenyi,J. and Mitchison, D.A. Antigenic diversity ofM. tuberculosis and M. bovis detected by meansof monoclonal antibodies. Lancet i (1981) 167.Kohler, G. and Milstein, C. Continuous cul-tures of fused cells secreting antibody of prede-fined specificity. Nature 256 (1975) 495.Towbin, H., Staehelin, T. and Gordon, J. Elec-trophoretic transfer of proteins from polyacry-lamide gels to nitrocellulose sheets : proceduresand some applications. Proc Natl Acad SciUSA 76 (1979) 4530.Van Raamsdonk, W., Pool, C.W. and Heyting,C. Detection of antigens and antibodies by animmunoperoxidasemethod applied on thinlongitudinal sections of SDS-polyacrylamidegels. J Immunol Methods 17 (1977) 337.Nlishimori, K., Yugi, H., Naiki, M., Sugimura,T., Tanaka, Y., Nonomura, I., Yokomizo, Y.and Kubo, S. Production and characterizationof serovar-specific monoclonal antibodies toserovars 4, 8 and 9 of Mycobacterium intracel-luclare. Infect Immun 55 (1987) 711.Brennan, P.J. Antigenic peptidoglycolipids,phospholipids and glycolipids. In : The Myco-bacteria, Part A, G.P. Kubica and L.G. Wayne,Eds (Marcel Dekker, New York) 1984 p 467.Reprint requests :Dr C.N. Paramasivan, Deputy Director (Bacteriology), Tuberculosis Research Centre,Spurtank Road, Chetput, Madras 600031

Monoclonal antibodies against mycobacterium avium/intracellulare

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