We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay

Folds, J

Saunders, J

PubMed

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INFECTION AND IMMUNITY, Aug. 1983, p.844-847 Vol. 41, No. 20019-9567/83/080844-04$02.00/0Copyright C 1983, American Society for MicrobiologyDevelopment of Monoclonal Antibodies that RecognizeTreponema pallidumJAMES M. SAUNDERS AND JAMES D. FOLDS*Department of Microbiology and Immunology, School of Medicine, University of North Carolina, ChapelHill, North Carolina 27514Received 14 February 1983/Accepted 13 May 1983We developed a panel of monoclonal antibodies to Treponema pallidum(Nichols) antigens, some of which recognize treponemal antigens on T. pallidum(Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. Theantibodies were detected by either an enzyme-linked immunosorbent assay or aradioimmunoassay.Until recently, little specific informationabout the mechanisms of resistance in syphilisor the antigenic structure of Treponema palli-dum have been available. Both the humoral andcell-mediated immune responses have been im-plicated in acquired or protective immunity (orboth) to syphilis; however, their roles have notbeen fully delineated. Detailed knowledge aboutthe antigenic structure of the organism and itspathogenicity have been limited, partly due totechnical problems such as the inability to growT. pallidum in vitro for sustained periods oftime. Recently, several new techniques havebeen utilized to characterize T. pallidum proteinantigens. Alderete and Baseman (2) utilized ra-dioimmunoprecipitation to assess T. pallidumsurface proteins, whereas Lukehart et al. (5) andHanff et al. (4) used a Western blotting tech-nique (11) to study T. pallidum antigens. Thesereports indicate that T. pallidum generates acomplex humoral response to a number of trepo-nemal antigens during syphilitic infection. Rob-ertson et al. (10) reported the development ofhybridomas producing antibodies to T. pallidum.In this report, we describe additional monoclo-nal antibodies, detected by an enzyme-linkedimmunosorbent assay (ELISA), which recog-nize treponemal surface components.T. pallidum (Nichols) (obtained from J. Base-man) was maintained by intratesticular passagein rabbits treated with cortisone as previouslydescribed (3, 6). A street strain of T. pallidum(strain 14; a more recent human isolate, obtainedfrom Sandra Larsen of the Centers for DiseaseControl, Atlanta, Ga.) was grown in rabbits thatdid not receive cortisone.T. pallidum organisms were extracted in phos-phate-buffered saline-10% heat-inactivated nor-mal rabbit serum as previously described (9).Treponema phagedenis biotype Reiter wasgrown in spirolate broth supplemented with 10%heat inactivated normal rabbit serum (5). T.pallidum antigens used for the ELISA wereeither phosphate-buffered saline-washed organ-isms or organisms purified by Percoll densitygradients (4, P. A. Hanff, S. J. Norris, M. A.Lovett, and J. N. Miller, Abstr. Annu. Meet.Am. Soc. Microbiol. 1983, 130, p. 176).The T. pallidum, T. phagedenis biotype Rei-ter, and normal testicular extracts were sonicat-ed as previously described (J. D. Folds, A. S.Rauchbach, E. Shores, and J. M. Saunders,submitted for publication). The ELISA proce-dure was performed as described by Folds et al.(submitted for publication). The radio-immunoassay (RIA) procedure was similar tothe method described by Zeltzer et al. (12)except that 125I-labeled goat anti-mouseimmunoglobulin (New England Nuclear) at 2.5x 105 cpm per well was used to detect antibody.Antigens used to prepare RIA plates were eithersonicated organisms as used for the ELISA orwhole organisms (106 cells per well).BALB/c mouse spleen cells were sensitized toT. pallidum (Nichols) by using a 6-week immuni-zation schedule. Mice were initially injectedintradermally with virulent T. pallidum (107 or-ganisms) in complete Freund adjuvant. Addi-tional injections made intraperitoneally with 107T. pallidum, and a final intraperitoneal boosterinjection of T. pallidum antigen (100 ,ug of pro-tein) was given 3 days before cell fusion. Thecell fusion method was similar to previouslydescribed procedures (8). Briefly, washed, sen-sitized mouse spleen cells and myeloma cells(either P3x63-Ag 8.653 or Sp2/0-Ag 14) werecombined and then copelleted and exposed to30% polyethylene glycol (PEG 1500) to effectcell fusion. The cells were washed and suspend-ed to approximately 2.5 x 106 cells per ml andportioned to microtiter wells. The cultures werefed Dulbecco modified Eagle medium-hypoxan-844TABLE 1. ELISA for eight representative hybridoma culture supernatants and controlsAntigen attached (optical density at 492 nm)°Prepn T. pallidum T. pallidum NormalT.pallidum (Nichols) strain 14 T. phagedenis testicular(Nichols) (Percoll (Percoll biotype Reiter extractpurified) purified)ClonesIV 0.2 0.3 0.5 bVI 0.3 0.6 0.9X 0.5 0.7 1.0 -XVI 0.3 0.7 1.2 -III 0.4 0.6 1.0 0.2 0.3XIV 0.3 0.5 0.7 0.2 0.2II 0.3 0.5 0.8 0.2VII 0.4 0.8 0.8 - 0.2ControlsSyphilitic mouse 0.65 0.9 0.8 0.6 0.45sera (1:50)cNormal mouse 0.04 0.06 0.05 0.07 0.08serum (1:50)P3X-Ag8.653 culture 0.02 0.01 0.02 0.01 0.03supernatanta Measured in a Titertek ELISA reader.b , Not reactive, optical density of less than 0.20.Serum taken from BALB/c mice at least 45 days after inoculation with T. pallidum (Nichols).thine-aminopterin-thymidine as the selectionmedium. When the hybrids exhibited goodgrowth, the culture supernatants were screenedfor anti-T. pallidum activity by using an ELISAtechnique. Cultures which were identified aspositive were expanded, and those which con-tinued to produce relevant antibody were clonedby using a soft agar method. Hybrid cell cloneswere selected and again tested for antibodydirected against T. pallidum, and those cellcultures positive for antibody activity were fur-ther expanded.The hybridoma cell lines were selected byusing the ELISA and sonicated T. pallidum(Nichols) antigen. To evaluate the specificity ofthe monoclonal antibodies, ELISAs were per-formed with different antigens. T. pallidumstrain 14, T. phagedenis biotype Reiter, and anextract of normal rabbit testicular tissue wereused as antigens. Table 1 illustrates severalrepresentative hybridoma culture supernatantswhich react with both T. pallidum (Nichols)and T. pallidum street strain 14; however, thePercoll-purified organisms displayed greater re-activity than did the non-Percoll-purified organ-isms. Some culture supernatants showed cross-reactions with T. phagedenis biotype Reiter ornormal testicular extract (or both).The monoclonal antibodies were also charac-terized by using the RIA procedures to evaluatethe specificity of the hybridomas. Table 2 indi-cates that the hybridoma culture supernatantswere reactive in the RIA with either sonicated orintact T. pallidum; this correlated well with theELISA results. When sonicated or whole T.phagedenis biotype Reiter was used as the anti-gen in the RIA, several culture supernatants thatwere not reactive by the ELISA were either notreactive or weakly reactive by the RIA, whereassome monoclonal antibodies displayed signifi-cant reactivity toward T. phagedenis biotypeReiter.The isotypes of the monoclonal antibodieswere determined via immunodiffusion and mostwere immunogobulin Gl (IgGl); however, onewas IgG2B and four were not typable by stan-dard sera against IgG subclasses, IgM, or IgA.By using an ELISA and RIA method, themonoclonal antibodies strongly reacted withboth T. pallidum strains, suggesting that theypossess common antigens. This is an importantobservation since T. pallidum (Nichols) hasbeen maintained in cortisone-treated rabbits andmay have altered antigens. It is also interestingto note that Percoll purification of T. pallidumresulted in greater reactivity with the monoclo-nal antibodies. This may be due to the concen-tration of T. pallidum-specific components in theantigenic suspension used for testing or perhapsto the decreased rabbit testicular tissue contami-nation, which may interfere with the immunoas-says. For the most part, there was good correla-tion between the ELISA and RIA results;however, there were several clones whose anti-body showed greater reactivity by RIA than inthe ELISA.Some monoclonal antibodies showed reactiv-ity against T. phagedenis biotype Reiter by RIA;VOL. 41, 1983 NOTES 845TABLE 2. RIA for eight representative hybridoma culture supernatants and controlsAntigen attached (cpm x IO)'Prepn Sonicated Whole Sonicated WholeT. pallidum T. pallidum T. pallidum T. pallidum(Nichols) (Nichols) strain 14 strain 14ClonesIV 10.7 9.3 11.0 8.6VI 31.1 28.8 31.4 24.4X 23.4 20.2 24.3 15.3XVI 27.2 24.2 29.8 23.7III 30.1 24.9 28.7 21.9XIV 17.3 15.5 19.4 14.7II 15.6 12.2 17.6 14.1VII 28.0 24.4 29.0 16.1ControlsSyphilitic mouse 43.6 42.2 46.0 37.7sera (l:50)bNormal mouse 4.4 4.4 4.5 4.3serum (1:50)P3X-Ag8.653 culture 3.6 3.7 3.9 3.3supernatanta All antigens were Percoll purified.b Serum taken from BALB/c mice at least 45 days after inoculation with T. pallidum (Nichols).however, in the ELISA there was minimal activ-ity. This is not unusual since T. pallidum and T.phagedenis biotype Reiter are known to sharesome antigenic determinants. This is also consis-tent with the results of Robertson et al. (10) whoused slightly different methods to develop mono-clonal antibodies to T. pallidum and found thatsome monoclonal antibodies cross-reacted withT. phagedenis biotype Reiter. Several of ourmonoclonal antibodies showed reactivity withrabbit normal testicular extract. Once again thismight be expected since the treponemes used forsensitization of the mice were grown in rabbittestes, and the treponemes were not highlypurified. Furthermore, Miller et al. (7) showedthat there is cross-reactivity with treponemesand rabbit testicular tissue, and Alderete andBaseman (1) have demonstrated that host pro-teins avidly bind to T. pallidum.We have generated a number of hybridomalines which produce monoclonal antibodies di-rected against T. pallidum (Nichols) as well as amore recently established strain, T. pallidumstrain 14. This indicated that the antibodiesdetect antigens on more than one strain of T.pallidum and not antigens that are unique to theNichols strain. These monoclonal antibodieswill be further characterized via Western blot-ting, radioimmunoprecipitation, or other proce-dures to identify the specific antigens recog-nized. It may then be possible to utilize themonoclonal antibodies to purify and identifyspecific T. pallidum surface components.This investigation was supported by Public Health Servicegrant AI 10536 from the National Institute of Allergy andInfectious Diseases.We thank Janne Cannon and Pat Stewart for technicaladvice and Phil Hanff for reviewing the manuscript. We alsothank Renee Woodlief for excellent secretarial assistance.LITERATURE CITED1. AMderete, J. F., and J. B. Baseman. 1979. Surface-associat-ed host proteins on virulent Treponema pallidum. Infect.Immun. 26:1048-1056.2. Alderete, J., and J. Baseman. 1980. Surface characteriza-tion of virulent Treponema pallidum Infect. Immun.30:814-823.3. Baseman, J. B., J. C. Nichols, J. W. Rumpp, and N. S.Hayes. 1974. Purification of Treponema pallidum frominfected rabbit tissue: resolution into two treponemalpopulations. Infect. Immun. 10:1062-1066.4. Hanff, P., T. Fehniger, J. Miller, and M. Lovett. 1982.Humoral immune response in human syphilis to polypep-tides of Treponema pallidum. J. Immunol. 129:1287-1291..5. Lukehart, S., S. Baker-Zander, and E. Gubish. 1982.Identification of Treponema pallidum antigens: compari-son with a nonpathogenic treponeme. J. Immunol.129:833-838.6. Maret, S. M., J. B. Baseman, and J. D. Folds. 1980. Cell-mediated immunity in Treponema pallidum infected rab-bits: in vitro response of splenic and lymph node lympho-cytes to mitogens and specific antigens. Clin. Exp.Immunol. 39:38-43.7. Miller, J. N., J. H. Bekker, J. H. DeBruUn, and P. C.Onvlee. 1966. The immunologic response of goats tonormal and syphilitic rabbit testicular tissue. J. Immunol.97:184-188.8. O, V., and L. Herzenberg. 1980. Immunoglobulin-produc-ing hybrid cell lines. p. 351. In B. Mishell and Shiigi (ed.),Selected methods in cellular immunology. W.H. Freemanand Co., San Francisco.9. Pavia, C. S., J. D. Folds, and J. B. Baseman. 1977.RI" NOTES INFECT. IMMUN.VOL. 41, 1983 NOTES 847Selective in vitro response of thymus-derived lympho-cytes from Treponema pallidum. Infect. Immun. 18:603-611.10. Robertson, S., J. Keltia, J. MUfler, and M. Norgprd.1982. Murine monoclonal antibodies specific for virulentTreponema pallidum (Nichols). Infect. Immun. 36:1076-1085.11. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electro-phoretic transfer of proteins from polyacrylamide gels tonitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. U.S.A. 76:4350-4354.12. Zeltzer, P. M., J. S. Pepose, N. H. Bishop, and J. N.Miller. 1978. Microassay for IgG antibodies to Treponemapallidum with radioiodinated protein A from Staphylococ-cus aureus: immunoglobulin G response in experimentalsyphilis in rabbits. Infect. Immun. 21:163-170.

Development of monoclonal antibodies that recognize Treponema pallidum.

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Development of monoclonal antibodies that recognize Treponema pallidum.

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