Seand. J. Immunol. 36, 697-702, 1992

Isoelectrophoretic Characterization of Protein Antigens Present in Mycobacterial Culture Filtrates and Recognized by Monoclonal Antibodies Directed Against the Mycobacterium bovis BCG Antigen 85 Complex A. D R O W A R T . J. DE BRUYN*, K. H U Y G E N * . G. D A M I A N I ' . H. P. GODFREY*, M. S T E L A N D R E * . J.-C. Y E R N A U L T & J.-P. VAN VOOREN Universilc Libre de Bruxelles, Belgium. *Instituut Pasteur van Brabant, Belgium. 'Institute of Biological Chemistry, University of Genoa, Genoa. Italy, and ^New York Medical College, Valhalla, New York, USA Drowart A, De Bruyn J. Huygen K. Damiani G. Godfrey HP. Stclandre M. Ycrnault J-C, Van Vooren J-P, Isoelectrophoretic Characterization of Protein Antigens Present in Mycobacterial Culturc Filtrates and Recognized by Monoclonal Aniibodies Directed Against ihc Mvcohaclerium bovis BCG Antigen 85 Complex. Scand J Immunol 1992:36:697-702 Nine monoclonal antibodies (MoAbs) were produced againsi Mycchaclerium hmi.s BCG aniigen 85 complex. Using isoelectric focusing combined with Western (immtinoblot) blot analysis, anligenicaliy related proteins could be identified incuUurefiltratesfrom M. luhercuio.fi.s. M. hovis. M. kamasii. M. aviurti. M. .\enopi. M. gunlimeu'. M. fornimmi., M. phlciand M. .^mcgrtmtis. Most ofthe MoAbs were found to he broadly cross-reactive between the various mycobacterial species, albeit some minor ditfcrcnccs were observed. These MoAbs reacted generally, in each species. with different components. One MoAb (VIDl-14) was found to be specifically direeted only against antigen 85B from M. hons. M. tiiherciilosi.'i and M. kiimasii. J.-P. Van Vooren. Clie.st Department. Hopiiul Era.mu: Rtc pi-dni^A\n M. kansasii and M. fortuitum. To our surprise, the majority of the MoAbs were found to be hroadly cross-reactive, recognizing antigen S5 hotnologucs in virtually all species. The one exception was MoAh VlDl-14 whieh reacted strongly and exelusiveiy against the componenl 85B of M. hovis. M. tuherculc.sis and M. kan.susii. Reeognition of M. avium and M. xenopi was very weak; the other strains were not reeognized al all with this particular MoAb. Some of the monoclonal antibodies (TD 32/4, VinH2-6, IIG8-4 and 233.27) reacted with a fourth band in M. hovis and M. tuberculosis culture filtrate, with an IP close to antigen 85A. This could possihly correspond to a post-lranslational processing of the moleeule. as only three antigen 85 genes have been identified [1, 2).

MoAhs Agaitisi the Comple.x 85 ofM. bovis BCG

69

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Fi(i. I. SDS PAGE (Icfl) and IEF (riglil) elect ropliorcsis of BCG culture ftltriitc (A and D) and of molecular wciglil (MW)(irisoclci:lricpoint.s(IP) m;irkers stained bvCoomas.sie blue, SDS-PAGE (lefl) and IFK(right) electrophoresis or BCG culture liltratc analysed in Wcslern blot with MoAb TD 17/4 (Band E) and MoAb VlDl-i4 (C and F). S5A = 32 kDa: »5B = .^0 kDa; 85C = .13 kDa.

DISCUSSION We have applied one-dimensional IEF combined with immunoblot analysis for the characicrizalion of antigen S5 related proteins in various myeobaeterial speeies using len monoclonal antibodies directed against the aniigen 85 eomplex of M. bovis BCG. In M. bovis culture filtrate, we identified the components A, B and C of the 85 eomplex by comparison of their location on a gel with that of the purified proteins. The IPs subsequently determined in a pH 2.5 6.5 gradient, i.e. 4.55, 4.15 and 4.35 respectively for the eomponents A, B and C of the 85 complex [S, 9], are close lothe IPs4.7.4.2 and4.6, reported by Nagai f/ii/. [16] who used a pH 3-10 gradient. The IPs of the antigen 85 components of M. lubvrculosis H37Rv were found to be identical to those of M. bovis BCG. This is not surprising since the protein sequences of both species have been determined and were found to be almost identical [1. 2]. All MoAbs except VIDI-t4 reacted against the 85A. B and C component in both M. bovis BCG

and M. tuberculosis culture filtrates. In BCG culture filtrate, however, reaction was generally strongest against the 85A component whereas in M. lubcrculosis culture filtrate, the antigen 85B was the most intensely stained. One exception was MoAb 233.27, that reacted less strongly lo antigen 85B than to antigen 85A in M. tuberculosis culture filtrate, in this respect, it is interesting to note that antigen 85A was found to be ihe major antigen 85 component in M. bovis BCG culture filtrate but not in M. tuberculosis filtrate in which the 85B component is the mosl abundant, irrespective of culture period (unpublished data). The ratio of antigen 85A/antigen 85B was respectively 3/1 in M. bovis BCG culture filtrale and 1/1.5 in M. tuberculosis filtrate as estimated by get scanning after Coomassie blue staining. This observation could explain why aniigen 85B ( = aniigen 6^alpha-antigen = MPB59) [6, 10] was more easy to purify frorn the H37Rv culture filtrate [17] than from the BCG filtrate, whereas the opposite was observed for antigen 85A ( =^ P32^ MPB44) [4]. Interestingly, antigen 85B was also found

700

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MoAbs Againsi the Comple.x 85 ofM. bovis BCG more abundantly in culture filtrate from virulent M. bovis (strain An5) (data not shown). It is possible that preponderance of antigen 85B is related to virulence but this point remains hypothetieal and needs to be elucidated in more detail. MoAb VIDI-14 reacted strongly and specifically with antigen 85B from M. hovis and M. tuberculosis. This MoAb also selectively recognized one component wilh an IP of 4.025 which probably corresponds to the so-called alphaantigen described in M. kansasii [18], It did not react with the proteins present in the M. gordonae. M. smegtnatis. M. fortuitutv and M. phlei culture filtrates and only faintly with one antigen of M. .xenopi and one antigen of M. avium. When testing ihe other MoAbs, broad erossreactivity was ohserved for all. However, some minordifFerences were observed. The MoAbs TD 32/4 and XDlO-3 were reactive with all myeobacterial species whereas MoAbs TD 17/4 and 233.27 reacted with all species except M. smegtnaiis. Antigen 85 homologues could noi be stained in M. gordonae or in M. avium by MoAbs V1I1H2-6 and VIIHl 1-4. MoAb XFI1-7 did not react with M. avium nor MoAb I1G8-4 with M. phlei. Finally, the previously described MoAb HYT 27 recognized all mycobacterial species except M. fortuilum. In eonclusion, IEF combined with Western blot analysis of culture filtrates of mycobacteria allows a better separation than SDS PAGE of molecules antigenieally related to the antigen 85 complex [12]. Most of the MoAbs direeted against the cotnplex 85 of M. hovis BCG were found to be eross-reactivc in various mycobacterial species and furthermore against several components in each species. One MoAb (VIDI-14), however, did recognize only one of the components of the complex 85 (antigen 85B) in M. lubereulosis., M. bovis BCG and M. kansasii. This could indicate that a smalt number of dominant B-cell epitopes are shared by all mycobacterial speeies and that only few specics-specifie epitopes exist. IEF followed by Western biol could be of use to test the reactivities of new monoclonal antibodies directed against this important mycobacterial antigen.

ACKNOWLEDGMENTS This work was supported by the National Fund for Scientific Research (Belgium), by the Fonda-

701

lion Erasme (Universite Libre de Bruxelles). We ihank F, Portaels for providing the myeobacterial strains, M, Weckxand J.Nyabenda for myeobacterial culture preparalions and L. Ljungqvist for providing MoAb HYT 27. Exeellent technical assistance was given by M. Decock, A. Alexander, K. Palfliet, F. Jurion and C. Lenoir. REFERENCES 1 Content J, de la Cuvcllcric A, De Wit A, VincentLevy-Frebault V, Ooms J, I)c Bruyn J, The genes coding for ihe antigen 85 complexes of A/, luherculosis and M. hnri.s BCG are members ofa gene family: Cloning sequence determination and genomic organization of the gene coding !or antigen 85C of M. luherculosis. Infect Immun iy9l;59:.12O5 12, 2 WikerHG, Sletten K. Nagai S. Harboe M, Evidence for three separate genes eneoding the proteins ofthc mycobacterial aniigen 85 complex. Infect Immun 1990:58:272-4, 3 Abou-Zeid C, Ratliff TL, Wiker HG. Harboe M, Bennedscn J, Rook, GAW, Characlerisalion of fibroneetin binding antigens released by MycohacIcritim tiihcrtuliisis and Mycohaelerium hovis BCG, Infect Immun 1988;56:3O46-5I, 4 De Bruyn J. Huygen K, Bosmans R ei al. Purification, partial characteri7ation and idenlilication of the 32 kDa-proteIn antigen of Myeohacterium hovis BCG, MicrobPathog 1987:2:351-60, 5 Drowarl A, Huygen K. De Bruyn J. Yernault JC, Farber CM, Van Vooren JP. Anlibody levels io whole culture liltrate and to purified P32 during treatment of smear positive tubereulosis. Chest I991;IOO(3):6S5 7, 6 Sada ED. Ferguson LE, Daniel TM, An ELISA for ihc serodiagnosis of tuberculosis using a 30,000 Da native antigen of Mycohaelerium tuherculosis. J Infeet Dis 1990:162:928 31, 7 Turneer M. Van Vooren JP, Dc Bruyn J. Serruys E, Dirckx P. Yernault JC, Humoral immune response in human tuberculosis : immunoglobulins G, A and M directed against ihe purified P32 protein antigen of M hovis bacillus Calmette et Gucrin, J Clin Microbiol 1988:26:1714 19, 8 Drowart A. Launois P, De Cock M ei al. An isoelectrie focusing method for the study of the humoral response against the antigen 85 complex of Mycobticterium hovis BCG in the different forms of leprosy, J Immunol Methods l991;145:223-8, 9 Van Vooren JP, Drowart A. De Cock M et al. Humoral immune response of tuherculosis patients against the three eomponenls of the Mycohaaeriutn hovis BCG eomplex separated by isoelectric focusing. J Clin Microbiol 1991:29:2348-50. 10 Yoneda M, Fukui Y, Yamanouchi T. Extracellular proteins of tubercle bacilli, V, Distribution of alpha and beta antigens in various Mvcohacteria. Biken J 1965:8:201 223. 11 Harboe M. Mshana RN, Closs O, Kronvall G, Axelsen NH, Cross reactions between .Mycohacteria. 11. Crossed immunoelectrophoretie analysis of

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antigens in differeni mouse strains infeeted with Mycohacterium horis BCG, Infecl Immun 1990;5S(7)2192 7. 16 Nagai S. Wiker HG. Harboe M. Kinomoto M, Isolation and partial characterisation of major protein antigens in the culture fluid of Mycohacterium luhi'fciito.'iis. [tifect Immun I99I;59;372 9, 17 Salata RA. Sarison AJ. Malhotra IJ cf ul. Purification and characlcrization of the .'^0,000 Dalton native antigen of Mycohucterium luherculo.sis and characterization of six monoclonal antibodies reactive with a major epitope of this antigen, J Lab Clin Med 1991:11S:589 598, 18 Matsuo K. Yamaguchi R. Yamazaki A, Tasaka H, Yamada T, Cloning and expression of gene for the cross reactive alpha-antigen, lnfeet Immun 1990: 58:550 6,

Respir DIS I99O;14O:83O 8,

15 Huygen K. Ljungqvist L, Ten Berg R. Van Vooren JP, Repertoires of antibodies to culture filtrate

Received 16 March 1992 Accepted in revised form 23 June 1992