Characterization of syngeneic anti-idiotypic antibody against the idiotype fo BALB/c myeloma protein J558.

BALB/c mice were immunized against the idiotype of BALB/c myeloma J55,. Syngeneic, specific anti-idiotypic antibodies against this germ line idiotype were shown by the isoelectric focusing technique to be markedly heterogeneous. In fact, the heterogeneity of isogeneic anti-idiotypic sera appeared to be comparable to those of allogeneic anti-J558 sera, raised in A/J and CB20 mice. As a rule, individual mice exhibit different clonal spectra. By spleen cell transfer experiments, each individual spectrum of clones could be expanded in order to arrive at an estimate of the size of anti-idiotypic repertoires. These were found to be of the order of 7-16 different anti-idiotypic clones for an individual BALB/c mouse. From the infrequency of clonal repetition we conclude that the repertoire of the BALB/c strain must be well in excess of one hundred anti-idiotypic molecular species.     

Regulatory effects of isologous anti-idiotypic antibodies on the formation of different immunoglobulin classes in the immune response to phosphorylcholine in BALB/c mice

BALB/c mice were immunized with purified phosphorylcholine (PC)-specific myeloma protein of the TEPC-15 tumor, which bears the major idiotype of the anti-PC response (T15 Id) in this mouse strain. Four months after establishing the anti-idiotypic response, animals were immunized with PC-keyhole limpet hemocyanin under conditions which normally lead to IgE antibody formation. The expression of anti-PC antibodies of each immunoglobulin class was compared to a group of matched control mice not immunized with T15. In BALB/c mice producing anti-idiotypic antibodies the formation of anti-PC IgM, IgG1, IgG2, IgG3 and IgE was suppressed to various extents and for different lengths of time. An exception to this observation was the formation of anti-PC IgA antibodies, in that no significant difference was measured between the two groups of mice. BALB/c mice immunized in the same way with the PC-specific myeloma proteins of the MOPC-167, MOPC-511 and MOPC-603 tumors produced normal levels of anti-PC IgE and IgG antibodies. These results suggest that T15 idiotype-positive antibodies are probably formed within all classes of specific antibodies expressed during an immune response to PC. The possibility of extending the phenomena of anti-idiotype-induced suppression to the level of various classes of specific antibodies, including IgE, might be of interest in the treatment of some allergic diseases.     

Molecular heterogeneity and fine specificity of BALB/c antibodies elicited by isologous myeloma protein.

BALB/c mice immunized with the isologous myeloma protein MOPC-315 (M315; α, λ₂) develop antibodies most of which are directed to idiotypic antigens (Id315) located in or near the CNP binding sites of M315, In order to more precisely establish the fine specificity of anti-Id315 antibodies, and to obtain an estimate of the size of the BALB/c anti-Id315 repertoire, we examined anti-Id315 antibodies from individual mice by analytical isoelectric focusing followed by autoradiography. ¹²⁵I-labeled probes were prepared from purified mildly reduced and alkylated M315 protein (RA315) (7 S monomer), Fab'315, Fv315, L315, H315, DNP affinity-labeled RA315, mildly reduced and alkylated MOPC-460 (RA460; 7 S; α, k, Fab'460, H460, and DNP affinity-labeled RA460. We observed that (a) the majority of BALB/c antibodies elicited by immunization with RA315 are specific for idiotypic antigens in the variable region which are rendered inaccessible in the presence of DNP-hapten; (b) distinctly separate antibodies are specific for idiotypic antigens also located in the variable regions but which are not influenced by DNP hapten; (c) about 30 % of BALB/c mice develop antibodies which appear to be specific for a determinant located in the second or third, homology regions of the constant region of the α chain which is not influenced by DNP hapten, and which is also present in the same region of H460; (d) none of the antibodies elicited by M315 recognize free L315; (e) the total antibody response is quite restricted; (f) shared spectrotypes are common between individual mice; and (g) unique or infrequently shared spectrotypes do occur. These studies demonstrate, by a direct technique, the fine specificity and the clonal and molecular heterogeneity of the B cell response induced by immunization with RA315.     

Monoclonal vs. heterogeneous anti-H-8 antibodies in the analysis of the anti-phosphorylcholine response in BALB/c mice

Biological activities of monoclonal A/J antibodies to the T15 idiotype in BALB/c mice were compared to heterogeneous antibodies raised by conventional immunization procedures. Two monoclonal antibodies, AB1-2 and GB4-10, which are of the γ₁,? class, appeared to have identical specificities by binding criteria and reacted similarly to conventional antibodies in their abilities to induce neonatal suppression, inhibit plaque-forming cell induction by phosphorylcholine (PC) antigens and to inhibit specifically, anti-PC plaque-forming cells. However, in functional analyses of anti-PC responses in various strains of mice, discrepancies were noted in the T15 responses as defined by monoclonal antibodies and conventional antisera. This heterogeneity was also observed in adult mice suppressed with the GB4-10 monoclonal antibody. These animals eventually produced an anti-PC response of AB1-2 idiotype but lacking the GB4-10 marker. These results show that the T15 IgM anti-PC response in BALB/c and other strains of mice is heterogeneous and probably consists of a family of clones. Particular clones can be precisely eliminated by the use of appropriate monoclonal antibodies, and the anti-PC response that eventually recovers is still T15⁺ but lacking the suppressed clones.     

Anti-T-cell humoral and cellular responses in healthy BALB/c mice following immunization with ovalbumin or ovalbumin-specific T cells

It is known that T-cell vaccination (TCV) elicits cellular immune responses against the immunizing T cells in vivo. However, it is still unclear whether similar anti-T-cell responses are also induced in individuals responding to foreign antigen (Ag) challenge. This study was undertaken to characterize and compare anti-T-cell cellular and humoral responses of BALB/c mice after ovalbumin (OVA) immunization or TCV. Splenocytes from OVA-primed BALB/c mice proliferated in response to stimulation with a syngeneic OVA-specific T-cell clone, OVA-T3, and secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4). Cytotoxic T-lymphocyte (CTL) activity against the T-cell clone was also observed. Serum samples from these animals discriminated between activated and resting murine T cells, as determined by indirect immunofluorescence staining. Vaccination of BALB/c mice with OVA-T3 cells induced similar, but stronger, cellular and humoral responses. TCV-induced antibodies were not only able to distinguish between activated and resting syngeneic T cells but also positively stained allogeneic T cells from BXSB mice. Furthermore, TCV resulted in hyporesponsiveness of BALB/c mice to subsequent Ag challenge, and antisera from the immunized animals inhibited T-cell proliferation in vitro. Our data suggest that anti-T-cell antibodies, and CD4+ and CD8+ regulatory T lymphocytes may form a complex and co-ordinated regulatory network that plays an important role in regulating the adaptive immune responses in vivo. Implications of this observation for our understanding of the immunoregulation and peripheral tolerance are discussed.     

Anti-idiotypic immunization provides protection against lethal endotoxaemia in BALB/c mice.

Against lipid A (the conserved moiety of lipopolysaccharides from Gram-negative bacteria) neutralizing IgM monoclonal antibodies (mAb) 8-2 and 26-20 anti-idiotypic (Ab2) mAb were produced: Ab2 mAb KM-04 (IgG1) against mAb 8-2, and Ab2 mAb PW-1 (IgG2a) and PW-2 (IgG1) against mAb 26-20. The binding of Ab2 mAb KM-04 to 8-2 (Ab1) was strongly inhibited by a lipopolysaccharide (LPS) extract from either Salmonella minnesota R595 (Re LPS) or Escherichia coli J5 (Rc LPS), whereas the binding of Ab2 mAb PW-1 and PW-2 to 26-20 (Ab1) was only marginally inhibited by both Re LPS and Rc LPS. The results indicated that Ab2 mAb KM-04 recognizes a lipid A-binding site related idiotope on mAb 8-2 and therefore KM-04 might bear the internal image of a neutralization determining epitope of lipid A. Consequently Ab2 KM-04 might induce antibodies to lipid A. Indeed anti-idiotypic immunization of syngeneic BALB/c mice with Ab2 mAb KM-04 resulted in development of lipid A-binding anti-anti-idiotypic (Ab3) antibodies in serum. Similar immunizations with Ab2 mAb PW-1 and PW-2 were unsuccessful. However, induction of lipid A-binding Ab3 by mAb KM-04 proved to be genetically restricted to BALB/c mice. DBA/2 mice, Swiss mice and rabbits did not develop lipid A-binding antibodies upon immunization with mAb KM-04. In protection experiments, it was shown that BALB/c mice vaccinated with mAb KM-04 showed significantly enhanced survival from challenge with either rough (Re) LPS from Salmonella minnesota or smooth LPS from E. coli 0111:B4 when compared to BALB/c mice immunized with a non-relevant Ab2 mAb. The results suggest that mAb KM-04 constitutes a non-internal image vaccine to the lethal effect of lipid A in BALB/c mice. Furthermore an Ab3 mAb was prepared against Ab2 mAb KM-04 that showed reactivity with Re LPS. This Ab3 mAb, designated LE-21 (IgG2a) protected mice against an otherwise lethal challenge of Re LPS.     

Idiotypic cross-reaction between MRL autoantibodies and a BALB/c myeloma

Y2, a monoclonal anti-Sm antibody of MRL origin, demonstrates an idiotype commonly expressed in autoimmune MRL mice, although not necessarily associated with anti-Sm activity. To identify non-anti-Sm antibodies with this common idiotype, a rabbit anti-Y2 anti-idiotypic antiserum was tested for activity with other MRL hybridoma products (HP) as well as BALB/c myelomas. Idiotypic cross-reactivity was thus demonstrated for Y2 and the product of the BALB/c fusing cell line 45.6TG1.7 (M45), an antibody without known antigen binding activity. This determinant was denoted the Y2-M45 idiotype and its presence in serum and other antibodies tested by an inhibition ELISA. The idiotype was demonstrated on some, but not all, MRL HP's tested and was not confined to antibodies of a unique specificity. The idiotype was also present in sera of some normal as well as autoimmune strains of mice with highest levels in two strains bearing the lpr gene. These results indicate that idiotypes of anti-Sm antibodies are not exclusive to the pathologic setting but may be found commonly expressed in mice with antibodies of a variety of binding activity.     

Biological and serological comparison of syngeneic and allogeneic anti-idiotypic antibodies

The majority of BALB/c antibodies with specificity for the (1→3)-glucosidic linkage of dextran B1355, fraction S (-dextran), share an idiotype crossreactive with the -dextran-binding BALB/c myeloma proteins J558 and MOPC104E. This crossreactive idiotype (IdX558) is denned by an allogeneic antiidiotypic antiserum raised in mice of strain A/HeJ against J558 myeloma protein (Carson & Weigert, 1973). Allogeneic anti-IdX558 antisera induce a long lasting complete suppression of the total anti--dextran immune response when given neonatally to BALB/c mice. Mice which recover from suppression fail to express the IdX558. In contrast, when BALB/c mice are immunized by use of J558 myeloma protein, no measurable effect on the subsequent total anti--dextran immune response or on the expression of IdX558 is found in mice making anti-J558 themselves nor in those mice which received the syngeneic anti-J558 idiotype antibody neonatally. In addition, no significant alteration of the anti--dextran response can be detected in BALB/c mice which were immunized against MOPC104E protein prior to -dextran immunization. This different suppressive capacity may be explained by different specificities of the allogeneic and syngeneic antiidiotype antisera. Hemagglutination and isoelectric focusing studies of the antisera show that BALB/c mice fail to produce antibodies against the crossreactive idiotype whereas in the allogeneic situation the anti-IdX558 is the overwhelming one. Syngeneic anti-idiotypic antibodies react only with the corresponding myeloma protein used for immunization, thus defining individual idiotypes (IdI558 and IdI104). IdI558 and IdI104 are expressed by every BALB/c mouse in response to -dextran but as a minor component of their idiotypic spectrum.     

Specificity of T and B lymphocytes for myeloma protein 315.

The antibody response of BALB/c mice directed against the idiotype of the BALB/c myeloma protein MOPC 315 (M315) is T cell-dependent. The specificity of helper T cells for M31 5 was investigated. Helper cells were primed with M31 5, M315 affinity-labeled with N^α-bromoacetyl-N^ε-2,4-dinitrophenyl-L-lysine, M315 L-chain and H-chain the M315 Fv fragment, myeloma protein MOPC 460 and monoclonal human kappa and lambda chains. The primed cells were transferred together with (4-hydroxy-5-iodo-3-nitrophenyl(acetyl)) (NIP)-primed B cells to 500 R-irradiated recipient syngeneic BALB/c mice which were challenged with NIP_sM315. The anti-NIP antibody responses were augmented by helpers primed with all derivatives and subunits of M315. This effect disappeared almost completely when the helper cells were treated with anti-Thy-1,2 and C prior to transfer. Priming with M460 induced weak and inconsistent helper effects, which opens the possibility that some of the helper cells may recognize nonidiotypic determinants on M315. The strong and consistent helper effect induced by free M315 L-chain (L-31 5)-primed cells was not due to dissociation of NIP-L-315 from intact NIP₅ M315 used for challenge, because L-315-primed T cells also augmented the antibody response against the idiotype of M315 which is only expressed on associated (H+L) chain pairs. In contrast, BALB/c antibodies elicited by free L-315 were specific for a determinant which was present on free L-chains but which was not or only minimally accessible on the complete M315. The data indicate that the helper cells recognize determinants which are separate from the ligand binding site of M315 and which are accessible on both the complete M315 7 S subunit and its isolated chains. In contrast, B cells stimulated by free L-315 recognize determinants which are hidden on associated (H+L) chains. Moreover, the associated (V_L+V_H) domains of M31 5 Fv fragment, which lacks C domains are immunogenic for T helper cells.     

Functional relationship between T15 and J558 idiotypes in BALB/c mice.

In inbred strains of mice, antiphosphorylcholine (PC) and anti-alpha 1,3 dextran (DEX) antibodies are structurally distinct from each other and have been shown to exhibit noncross-reactive antigen binding and idiotypic specificities. However, the prototype anti-PC and anti-DEX antibodies, TEPC15 and J558, respectively, were shown to be connected via a common autoantiidiotypic monoclonal antibody isolated from newborn BALB/c mice. The capacity of various monoclonal anti-PC and anti-DEX antibodies as well as the antigens PC and DEX to modulate T15 and J558 idiotypes in BALB/c mice was tested by their administration to newborn mice. Anti-PC antibodies of the T15 idiotype injected into 2-4-day-old mice, at a time when T15+ anti-PC precursors develop in BALB/c mice, suppressed the anti-PC response of these mice at 6 weeks of age. Similarly, J558 antibodies injected into 8-12-day-old mice, at a time when J558 precursors normally develop, suppressed the response to DEX. As a further demonstration of this connectivity, the injection of J558 into 4-day-old mice led to a down modulation of T15 idiotype, whereas both T15 and a minor idiotype-expressing antibody M167 when injected into 8-12-day-old mice caused a reduction in expression of the J558 idiotype. As predicted from in vitro analysis, injection of anti-PC antibodies of the M167 idiotype 2 to 4 days after birth enhanced the subsequent response to PC. However, anti-PC antibodies expressing another minor M603 idiotype did not affect the PC response. The results parallel the in vitro enhancement of M167 antibodies but not M603 on T15 binding to antiidiotype in vitro. Similarly, anti-DEX antibodies expressing the M104E idiotype had no detectable effects on the capacity to respond to PC or DEX or on the expression of T15 and J558 idiotypes as adults. Exposure of newborn mice to PC led to a dramatic reduction in the response to DEX as adults, whereas exposure to DEX at this stage of development had no effect on response to PC as adults. Collectively, these observations provide evidence for a complex functional connectivity between T15 and J558 idiotype-bearing B cells during ontogeny and extend our previous observations that development of these idiotypes is regulated by idiotype-directed interactions between B cells or their immunoglobulin products.     

Functional Relationship Between T15 and J558 Idiotypes in BALB/c Mice

In inbred strains of mice, antiphosphorylcholine (PC) and anti-α1,3 dextran (DEX). antibodies are structurally distinct from each other and have been shown to exhibit noncrossreactive antigen binding and idiotypic specificities. However, the prototype anti-PC and anti-DEX antibodies, TEPC15 and J558, respectively, were shown to be connected via a common autoantiidiotypic monoclonal antibody isolated from newborn BALB/c mice. The capacity of various monoclonal anti-PC and anti-DEX antibodies as well as the antigens PC and DEX to modulate T15 and J558 idiotypes in BALB/c mice was tested by their administration to newborn mice. Anti-PC antibodies of the .T15 idiotype injected into 2-4-day-old mice, at a time when T15 anti-PC precursors develop in BALB/c mice, suppressed the anti- PC response of these mice at 6 weeks of age. Similarly, J558 antibodies injected into 8-12-day-old mice, at a time when J558 precursors normally develop, suppressed the response to DEX. As a further demonstration of this connectivity, the injection of J558 into 4-day-old mice led to a down modulation of T15 idiotype, whereas both T15 and a minor idiotypeexpressing antibody M167 when injected into 8-12-day-old mice caused a reduction in expression of the J558 idiotype. As predicted from in vitro analysis, injection of anti-PC antibodies of the M167 idiotype 2 to 4 days after birth enhanced the subsequent response to PC. However, anti-PC antibodies expressing another minor M603 idiotype did not affect the PC. response. The results parallel the in vitro enhancement of M167 antibodies but not M603 on T15 binding to antiidiotype in vitro. Similarly, anti-DEX antibodies expressing the M104E idiotype had no detectable effects on the capacity to respond to PC or DEX or on the expression of T15 and J558 idiotypes as adults. Exposure of newborn mice to PC led to a dramatic reduction in the response to DEX as adults, whereas exposure to DEX at this stage of development had no effect on response to PC as adults. Collectively, these observations provide evidence for a complex functional connectivity between T15 and J558 idiotype-bearing B cells during ontogeny and extend our previous observations that development of these idiotypes is regulated by idiotype-directed interactions between B cells or their immunoglobulin products.     

Incomplete expression of the MOPC 460 idiotype in the SERA of BaLB/c mice immunized either with DNP antigen or with anti-idiotypic

The MOPC 460 idiotype, as defined by polyclonal probes, has been described as a recurrent marker among the anti-DNP antibodies synthetized by IgHC^a mice. In this paper, we demonstrate, using syngeneic monoclonal anti-idiotypic probes, that only a part of the idiotopes of this idiotype are indeed ercurrently expressed in BALB/c mice (IgHC^a) after immunization with DNP antigen. We will also show that the immunization of BALB/c mice with monoclonal anti-idiotypic antibody specific for the recurrent determinants results firstly in the synthesis of anti-DNP antibodies and secondly in the expression of the same recurrent M460 idiotope present on a part of induced anti-DNP molecules. Contrary to this, the immunization with the monoclonal anti-idiotypic antibody specific for the private idiotope never resulted in the synthesis of anti-DNP antibodies. These results clearly suggest that, after DNP or anti-idiotypic immunization, the M460 idiotype is not expressed in its entirety.     

Anti-idiotype to monoclonal anti-swine SLA antibody detects a common idiotype shared by mouse anti-SLA sera and elicits an anti-SLA activity

Heterologous anti-idiotypic reagents have been prepared against a BALB/c anti-swine MHC (SLAd) monoclonal antibody (74-11-10) in order to test for possible interspecies idiotypic cross-reactions of anti-MHC antibodies. Using these reagents to examine xenoantisera produced in BALB/c mice immunized with swine SLAdd peripheral blood lymphocytes, all animals tested were found to express detectable levels of the 74-11-10 idiotype (Id). The Id could also be detected in one out of five BALB/c mice immunized with swine SLAcc PBL. Swine anti-SLAdd alloantibodies were also tested, but failed to show detectable levels of the 74-11-10 Id. The in vivo administration of anti-idiotypic reagents to BALB/c mice induced detectable levels of 74-11-10 Id positive antibodies that bound specifically to SLAdd PBL. Similar treatment of SLAgg swine (recombinant swine expressing the class I MHC molecules of c) with anti-idiotypic antibodies failed to induce anti-SLAd antibody activity. These results thus indicate that 74-11-10 represents a shared idiotype of BALB/c anti-SLAdd antibodies. The presence of 74-11-10 Id in one mouse immunized with SLAcc PBL suggests that the failure of pigs to express the 74-11-10 Id following treatment with anti-idiotypic antibodies may be the result of tolerance rather than absence of the relevant variable region gene(s).     

Idiotype-Anti-Idiotype Interactions of VHIX-Coded Anti-Progesterone and Anti-Arsonate Antibodies

The reactivity and specificity of potyctonal and monoclonal anti-idiotypic antibodies raised against monoclonal anti-progesterone and anti-arsonate antibodies have been studied by solid phase radioimmunoassay (RIA) with immobilized idiotype and by passive haemagglutination with idiotype-coupled red cells. The sensitivity of the two methods was comparable, though some cross-reactions were only detected by RIA. Passive haemagglutination was found to be especially suitable in screening for monoclonal anti-idiotypes in hybridoma supernatants and ascites. and had advantages over RIA in detection of syngeneic anti-idiotypes. Demonstration of binding site-associated idiotopes was possible by haemagglutination inhibition. RIA and haemagglutination were used to investigate the idiotypic relationships between BALB/c antiprogesterone and anti-arsonate monoclonal antibodies which share heavy chains encoded by V_HIX variable region genes.     

A method of generating antibodies against exogenously administered self-antigen by manipulating CD4+CD25+ regulatory T cells

The generation of antibodies against self-antigens or antigens having a high degree of structural homology with self-antigens is a difficult task because of immunological tolerance. CD4(+)CD25(+) regulatory T cells play an important role in maintaining peripheral tolerance. Sakaguchi et al. previously reported that the transfusion of CD25(+) cell-depleted mouse splenocytes into syngeneic nude mice results in a breaking of peripheral tolerance that leads to the development of autoimmunity. In this study, we attempted to apply this mouse model to the generation of antibodies against self-antigens. We depleted CD25(+) cells from BALB/c mouse splenocytes and transferred the rest of the cells into syngeneic nude mice. The animals were immunized with mouse thyroglobulin. We observed a significant increase of the anti-mouse thyroglobulin antibody titer in the group of mice immunized twice within 10 days after the cell transfer (P<0.05). From these mice, we established hybridoma cell lines producing anti-mouse thyroglobulin monoclonal antibodies, including a clone with a dissociation constant of 10(-8)M. Control nude mice which received CD25(+) cell-containing BALB/c splenocytes did not produce anti-mouse thyroglobulin antibodies. When the CD25(-)cell-transferred nude mice were immunized with mouse Gα12, another self-antigen, anti-Gα12 antibodies were produced in the sera. This method should prove highly useful in the generation of antibodies against self-antigens or antigens for which the structure is highly conserved across species.     

Thymus-independent induction of idiotype suppression in newborn mice by syngeneic anti-idiotype antisera

BALB/c and BALB/c nu/nu mice were shown to express to a variable extent in their response against dextran B1355S (Dex), an idiotype which is present on the Dex-reactive BALB/c myeloma protein MOPC 104E. Injection of minute amounts of syngeneic anti-MOPC 104E idiotype antisera into neonatal euthymic or athymic BALB/c mice suppressed this idiotype in the Dex-specific response of the adult animals. When spleen cells from suppressed BALB/c mice were transferred into irradiated BALB Ighb mice the state of suppression persisted. Data are discussed with respect to possible mechanisms regulating expression of this idiotype.     

Suppression of phosphorylcholine-specific IgE antibody formation in BALB/c mice by isologous anti-T 15 antiserum

In order to study the regulation of IgE antibody formation, isologous anti-idiotypic antisera against the phosphoryl choline (PC)-specific BALB/c myeloma proteins T 15 and M 167 were passively administered to BALB/c in the course of an anti-PC IgE response. Isologous anti-T 15 antiserum had a long-lasting suppressive effect on the formation of IgE antibodies with PC specificity, whereas administration of anti-M 167 antiserum had no or only little effect, similar to that of normal BALB/c serum. This indicates that anti-PC IgE antibodies consist mainly of the T 15 idiotype or of cross-reacting idiotypes, and that IgE response is accessible to regulation with anti-idiotypic antibodies. This murine model may permit the study of regulation of an IgE response largely restricted to few defined idiotypes characterized as tumor proteins.     

The use of immunodeficient male (CBA/N x BALB/c) F1 mice to produce monoclonal antibodies directed to proteins of Leptospira interrogans rather than to immunodominant lipopolysaccharides.

A method, using an immunodeficient mouse strain, for the production of monoclonal antibodies directed exclusively against the proteins in an antigen mixture also containing immunodominant LPS, is described. Male (CBA/N x BALB/c) F1 mice were immunized with an outer envelope antigen mixture from Leptospira interrogans strain Wijnberg containing both lipopolysaccharides and proteins. The immune response in these mice was shown to be predominantly directed against protein antigens. Hybridoma cell lines were generated by fusing spleen cells from a (CBA/N x BALB/c) F1 mouse with BALB/c Sp2/0 plasmacytoma cells. Hybridoma cell lines producing monoclonal antibodies reacting with the outer envelope preparation were identified by ELISA. All epitopes recognized by the monoclonal antibodies are sensitive to proteinase K degradation and resistant to oxidation by periodate indicating that they are located on proteins. All epitopes are located on a 35 kDa protein and specific for the pathogenic L. interrogans species.