Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Functional and molecular analysis of three distinct HLA-DR4 beta-chains responsible for the MLR between HLA-Dw4, Dw15, and DKT2

Differences in structure and function of HLA-class II molecules between HLA-DR4-related HLA-D specificities HLA-Dw4, Dw15, and DKT2 were investigated. Anti-HLA-DR framework monoclonal antibody HU-4 completely inhibited the one-way mixed lymphocyte reaction (MLR) between these specificities. HU-4 precipitated a monomorphic alpha-chain and a polymorphic beta-chain of human class II molecules from each B lymphoblastoid cell line (BLCL) homozygous for these three HLA-D specificities. We established IL 2-dependent T cell lines specific for streptococcal cell wall (SCW) antigen from peripheral blood lymphocytes (PBL) from four DR4-positive donors: an HLA-Dw4/DKT2 heterozygote, an HLA-Dw4/Dw12 heterozygote, an HLA-DKT2/D-blank heterozygote, and an HLA-Dw15/D-blank heterozygote. These T cell lines showed a proliferative response to SCW antigen in the presence of autologous or allogeneic antigen-presenting cells (APC) when T cell donors and APC donors shared at least one of the HLA-D specificities. This cooperation between the T cell line and APC was completely blocked by HU-4. We conclude from these data that the T cells could distinguish three distinct DR4 molecules expressed on APC of HLA-Dw4, Dw15, and DKT2 as restriction molecules at the presentation of SCW antigen.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

Three cytotoxic monoclonal antibodies, HU-11, HU-32, and HU-33, specific for human Ia-like antigens were used to analyze the two HLA-DR2-associated HLA-D specificities, HLA-Dw2 and HLA-Dw12. In the HLA-Dw2, DR2, MB1 homozygous B-cell line EB-CMG, the binding of radiolabeled HU-32 and HU-33 was strongly inhibited by the addition of nonlabeled HU-11, whereas no inhibition occurred in the HLA-Dw12, DR2, MB1 homozygous B-cell line EB-KT. To confirm this differential inhibition pattern further, F(ab')2 fragments were prepared from HU-11, and their ability to inhibit complement-dependent lysis mediated by HU-32 and HU-33 was assessed against a total of five homozygous typing cell lines homozygous for HLA-Dw2, DR2, MB1 or HLA-Dw12, DR2, MB1, including EB-CMG and EB-KT. Here again, the same differential inhibition pattern as that observed in the radiobinding inhibition assays was obtained. Thus, the data suggest that the two kinds of HLA-DR2-positive homozygous typing cell lines with distinct HLA-D specificity can be distinguished from each other by using solely serologic methods. This is the first clear-cut serologic distinction made between homozygous typing cells defining HLA-Dw2 and those defining HLA-Dw12, since no serologic means that enables one to distinguish one from the other has been available.     

Two-dimensional gel analysis of a second family of class II molecules by polymorphic HLA-DR4,5, and w9 monoclonal antibodies

Human Ia-like, class II molecules were isolated by immunoprecipitation with monoclonal antibodies from various HLA-D/DR homozygous cell lines and were analyzed by two-dimensional gel electrophoresis. The monoclonal antibody PLM12 reacted with B cells carrying DR4, DR5, DRw6.2, and DRw9 phenotypes, and its reactivity perfectly correlated with the previously defined TB21 (MB3-like) specificity. Class II molecules detected by PLM12 were structurally distinct from those precipitated by the anti-DR monoclonal antibody NC1 on all HLA-DR4, DR5, DRw6.2, and DRw9 homozygous cell lines and showed polymorphism in heavy and light chains among these cell lines. The monoclonal antibodies PLM2 and PLM9 only reacted with B cells carrying DR5 and DRw6.2 and also detected a distinct set of class II molecules from those precipitated by NC1 but identical to those of PLM12. Thus, PLM2 and PLM9 serologically detected a new subtypic antigen of the PLM12-reactive class II molecules. Furthermore, the antibody NC1 precipitated two light chains and one heavy chain from HLA-DRw6.2 homozygous cell line EBV-Sh. The result indicated the presence of three sets of class II molecules: two in a DR family and another carrying the polymorphic determinants detected by PLM2, PLM9, and PLM12 in a second family.     

Two distinct class II molecules encoded by the genes within HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating and restriction molecules

By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we investigated the difference in the HLA class II molecule between HLA-Dw2 and Dw12, both of which are typed as HLA-DR2 serologically. The anti-HLA-DR framework monoclonal antibody (MoAb) HU-4 precipitated an alpha-chain and two beta-chains of human class II molecules from both Dw2 and Dw12 homozygous B lymphoblastoid cell lines. It was demonstrated clearly that an alpha-chain (alpha 1) and one of the beta-chains (beta 1) showed no difference in mobility in the 2D-PAGE between Dw2 and Dw12, but that another beta chain (beta 2) of Dw2 was distinct from that of Dw12 in the 2D-PAGE profile. Thus, MoAb HU-4 precipitated alpha 1 beta 1 and alpha 1 beta 2 molecules from Dw2 and Dw12, and the alpha 1 beta 1 molecule appears to be an HLA-DR2 molecule. The alpha 1 beta 2 molecule, on the other hand, is a class II molecule distinct from those precipitated with anti-DR2, anti-DQw1 (DC1, MB1, MT1), or anti-FA MoAbs. MoAb HU-4 completely inhibited the mixed lymphocyte culture reaction (MLR) between Dw2 and Dw12, but anti-DR2 MoAb HU-30, which reacts only with the alpha 1 beta 1 molecule, did not show an inhibitory effect on the MLR between Dw2 and Dw12. The alpha 1 beta 2 molecule is therefore the molecule which elicits MLR between Dw2 and Dw12. An IL 2-dependent T cell line established from an HLA-Dw12/D blank heterozygous high responder to the streptococcal cell wall antigen (SCW) clearly distinguished the Dw2 specificity from Dw12 specificity expressed on the antigen-presenting cell (APC). Moreover, MoAb HU-4 markedly inhibited the cooperation between the T cell line and APC to respond to SCW. These observations indicate that the alpha 1 beta 2 molecule is recognized as a restriction molecule by the T cell line at the antigen presentation of SCW through APC MoAb HU-30 on the other hand partially inhibited the MLR between Dw2 or Dw12 homozygous cell as a stimulator cell and non DR2 cell as a responder cell. It markedly inhibited the proliferative response of the Dw12/D- heterozygous T cell line to SCW, presented by Dw2+ but Dw12- allogeneic APC, and the peripheral response of Dw2 or Dw12 homozygous peripheral blood lymphocytes to SCW. Thus, two distinct class II molecules encoded by the genes within the HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating molecules in the MLR and as restriction molecules in the antigen presentation by APC.     

Complexity of the supertypic HLA-DRw53 specificity: two distinct epitopes differentially expressed on one or all of the DR beta-chains depending on the HLA-DR allotype

The supertypic HLA-DRw53 specificity is associated with three allelic class II specificities defined by alloantisera: HLA-DR4, -DR7, and DRw9. The present study demonstrates the complexity of this supertypic DR specificity by comparing two DRw53-related determinants defined by the monoclonal antibodies PL3 and 109d6. For every HLA-DR4 cell line tested, both monoclonal antibodies were found to bind to the same subpopulation of DR molecules. This PL3+, 109d6+ DR subpopulation is also found on most, but not all, DR7+ cell lines with a beta-chain pattern that is identical to the beta-chain pattern of the PL3+, 109d6+ subpopulation on DR4 cell lines. However, some DR7+ cells which carry the HLA haplotype Bw57, DR7, DRw53, DQw3 were also found which completely lack the expression of the 109d6 determinant, but continue to express the PL3 determinant and some of the DRw53 determinants recognized by alloantisera. This results from the fact that the PL3 determinant is expressed on all of the DR molecules found on DR7 cells, including the distinct subpopulation of molecules that carry the HLA-DR7 determinant recognized by the monoclonal antibody SFR16-DR7. This PL3+, SFR16-DR7+ subpopulation does not carry the 109d6 determinant, demonstrating that the PL3 and 109d6 DRw53-related determinants are distinct and can be expressed on a different number of DR molecules, depending on the allotype of the cells. Blocking studies were also performed by using these monoclonal antibodies with alloreactive HLA-DR7-specific cytotoxic T cell clones. In these studies, the T cell-defined HLA-DR7 determinants were found to be carried by the same subpopulation of DR molecules recognized by the HLA-DR7-specific monoclonal antibody and not carried by the DR molecules recognized by 109d6. The DR7+ cell lines which do not express the 109d6 determinant also fail to express another supertypic determinant recognized by the monoclonal antibody IIIE3 carried on this molecule. Furthermore, no additional allelic forms of this unique DR beta-chain were found associated with the nonpolymorphic DR alpha-chain on these cells, suggesting that this DR beta-chain gene is not expressed. These cells also behave as homozygous typing cells for the Dw11 subtype of DR7 in HLA-D typing in the mixed lymphocyte culture assay. This suggests that the lack of expression of a specific class II gene may contribute additional genetic polymorphism within the known HLA-DR allotypes.     

Expression of HLA-DR, MB, MT and SB antigens on human mononuclear cells: identification of two phenotypically distinct monocyte populations

The expression of HLA-DR, SB, MB, and MT antigens in different populations of human mononuclear cells was investigated with the use of monoclonal antibodies that recognize distinct human Ia-like antigens. Our results indicate that in man, as previously reported in other species, two phenotypically distinct populations of monocytes or macrophages can be identified on the basis of expression of Class II MHC antigens. Virtually all circulating monocytes displayed determinants associated with HLA-DR, SB, and MT. In addition, a subpopulation of human monocytes expressed MB/DS-associated antigens, as detected with monoclonal antibodies specific for MB1, MB3, and DS-framework determinants. Most B lymphocytes expressed antigens associated with HLA-DR, and the specificities SB2, SB3, MB1, MB3, MT2, and MT3 were also present. Resting T lymphocytes were unreactive with antibodies that recognize all of the Class II MHC antigens tested. T lymphocytes activated by soluble antigen or alloantigens, and expanded in culture, expressed DR, SB, MB, and MT. The majority of the MB/DS+ cells present in the adherent population were monocytes, because they were phagocytic and had the monocyte-specific marker 63D3. The rest of the cells were not identified. They are likely to include mostly B lymphocytes. The presence of other cells, such as dendritic cells, in this subset needs to be determined.     

Association of PLT specificity of alloreactive lymphocyte clones with HLA-DR,MB and MT determinants

The HLA-D region encodes for several serologically defined systems, including DR, MB, and MT. The antigens of MB and MT are strongly associated with two or more DR specificities. The purpose of this study was to determine the role of MB and MT antigens in lymphocyte alloactivation. A soft agar colony assay was used to generate alloreactive lymphocyte clones primed in mixed leukocyte culture against a stimulator who typed as HLA-DR4,-;MB3,-; MT3,-. In secondary primed lymphocyte typing (PLT) assays, several clones were identified with PLT specificities strongly associated with DR4, MB3, or MT3. The data suggest that HLA-D controls different lymphocyte-activating determinants associated with the serologically defined DR, MB, or MT antigens.     

Molecular relationships of the human B cell alloantigens, MT2, MB3, MT4, and DR5

The human class II, HLA-linked, B cell alloantigens include the HLA-DR, MB, MT, and Te determinants. Interest in the molecular relationships of these antigens has recently intensified because of their homology to the murine Ia antigens and their possible importance in disease predisposition and transplantation. We have used alloantisera with carefully defined immunochemical as well as serologic specificity, and two immunochemical techniques, sequential immunoprecipitation with analysis by SDS-PAGE and two-dimensional gel electrophoresis, to explore the molecular relationships of the MT2, MB3, MT4, and HLA-DR5 antigenic determinants. The data presented here indicate that 1) all class II molecules that bear the DR5 antigenic determinant also bear the MT2 antigenic determinant; (2) the homozygous DR5 cell line, Swei, expresses at least two structurally distinct class II molecules, both of which bear MT2: one bears the MT2, MB3, and MT4 antigenic determinants, and the second bears the MT2, but not the MB3 or MT4 antigenic determinant; and (3) the DR5 determinant is located on at least one and possibly both of these distinct class II molecules.     

Serologic heterogeneity in I-J determinants associated with functionally distinct T cell regulatory factors

Information transfer among regulatory T cell subsets is mediated by biologically active T cell factors. Many of these factors are comprised of two molecules: one that binds antigen, and another that is I-J+ and determines the self recognition capability of the factor (I-J molecule). In the in vitro response to sheep red blood cells, we used three functionally distinct I-J+ factors to study the relationship between polymorphic I-J determinants and the biological activity of these factors. Our study shows that several monoclonal I-J antibodies react with I-J molecules associated with T suppressor-inducer factor (TsiF) and T suppressor-effector factor (TseF), but not with T contrasuppressor inducer factor (TcsiF). In contrast, a different set of monoclonal I-J reagents reacts with TcsiF but not TsiF or TseF. Finally, some monoclonal I-J antibodies distinguish between I-J molecules associated with TsiF and TseF. Thus anti-I-J reagents differentially react with I-J determinants on regulatory factors, and this differential pattern of reactivity correlates with the functional activity of the factors. The possible relationship between I-J heterogeneity and the biological function of I-J molecules in regulation is discussed.     

Antigen-specific HLA-restricted human T-cell lines

Human T-cell lines responsive to the polypeptide antigens GAT and (T, G)-A--L were developed. They were specific for the priming antigens and required the participation of accessory cells matched for HLA-linked determinants, as shown in family studies. In panel studies, the ability of accessory cells to present antigen was shown to be associated with HLA-D-region antigens. However, the specificity of these determinants did not fully correspond to any HLA antigens as currently defined. One GAT-specific subline, derived by limiting dilution, utilized a restriction determinant associated with, but distinct from, the DQw3 (MB3) allospecificity. Blocking studies with mouse monoclonal antibodies indicated that this restriction determinant was carried by HLA-DQ molecules. The epitopes recognized in these molecules appear to be distinct from the alloantigenic determinants currently defined by serology.     

Polymorphisms within the HLA-DR3 haplotypes

HLA-DR molecules were isolated from eight different HLA-DR3 homozygous B-cell lines by immunoprecipitation with monoclonal antibodies, and they were subsequently analyzed by two-dimensional gel electrophoresis. We found that HLA-DR3 homozygous B-cell lines of consanguineous origin express two types of HLA-DR molecules. One type of HLA-DR molecule was present in all the cell lines tested, whereas the second DR molecule appears to be polymorphic. DNA isolated from the different HLA-DR3 homozygous cell lines was studied by Southern blot analysis to determine whether any DR restriction fragment length polymorphism could be observed. Polymorphisms detected at both the product and genomic level have been compared to each other, and their relations to the serological (HLA-DR) and cellular (HLA-D and LB-Q1) typing data will be discussed.No reprints available     

The role of three distinct Ia-like antigen molecules in human T cell proliferative responses: effect of monoclonal anti-Ia-like antibodies

Murine monoclonal antibodies (MoAb) to three distinct Ia-like molecules were studied for their inhibitory effects on antigen- and alloantigen-induced T cell proliferations. The MoAb were classified into three groups according to the molecules they recognized. Both the group I MoAb reacting with DR molecules and the group III MoAb were capable of inhibiting T cell proliferative responses to PPD- and HSV-Ag-pulsed APC, autologous B-LCL, and alloantigens. On th other hand, the group II MoAb, which reacted with a determinant on the molecule carrying MB1 determinants, was only capable of inhibiting T cell responses to alloantigens. These results suggest that the structure of the molecules correlates with the functional repertoire of the human Ia-like antigens.     

Serologic and immunochemical characterization of the specificity of four monoclonal antibodies to distinct antigenic determinants expressed on subpopulations of human Ia-like antigens

Serologic and immunochemical assays showed that the monoclonal antibodies Q2/70, Q2/80, Q5/6, and Q5/13 react with human Ia-like antigens. Each monoclonal antibody recognizes distinct antigenic determinants that are different from those defining the serologic polymorphism of Ia-like antigens defined by conventional alloantisera and are expressed on subpopulations of Ia-like antigens. The determinants recognized by the MoAb Q2/70 and Q5/13 are expressed on all HLA-DR allospecificities tested, whereas those reacting with the MoAb Q2/80 and Q5/6 are not detectable on HLA-DR5 and HLA-DR7 allospecificities, respectively.     

Production and characterization of species-specific monoclonal antibodies against Leishmania donovani for immunodiagnosis

Sixteen species-specific monoclonal antibodies were produced against membranes of Leishmania donovani. These antibodies only reacted with determinants present on L. donovani. No cross-reactions were found with any other species of Leishmania or with membranes of Trypanosoma cruzi. An extensive analysis of the binding specificities of selected antibodies was carried out by using whole promastigote homogenates as antigen. Monoclonal antibodies D-1, D-2, D-3, and D-4 correctly identified all 44 L. donovani stocks from a cross-panel of 84 New and Old World Leishmania stocks. Antibodies D-1 and D-2 were also useful for species classification by immunofluorescence. No cross-reactions were observed with any other Leishmania species examined. Based on either Western blot and/or radioimmunoprecipitation analyses, five distinct groups of molecules associated with L. donovani-specific antigenic determinants were identified. These molecules range in m.w. from 18 to 84 kilodaltons. The antigenic molecules recognized by antibodies D-2, D-10, and D-13 are also recognized by antibodies present in sera from patients with visceral leishmaniasis (kala-azar). Kala-azar sera obtained from cases in both the Old and New World specifically compete with these monoclonal antibodies for the appropriate antigenic determinants in Western blot analysis. These monoclonal antibodies and/or the purified protein antigens may be useful in the development of a serologic assay for the clinical diagnosis of visceral leishmaniasis caused by L. donovani and in epidemiologic studies of leishmaniasis.     

Highly polymorphic products of both HLA-DR and HLA-DQ genes contribute to the polymorphism of the HLA-DR w13 haplotype

We studied the polymorphisms of HLA-DR and HLA-DQ products from HLA-DRw13 haplotypes by analyzing the restriction of influenza A-specific cloned T cells from an HLA-DRw13,DQw1,Dw19 homozygous individual. The results show that (1) some functional epitopes, which can be borne by either HLA-DR or HLA-DQ molecules, are strictly correlated with the HLA-Dw19 subtype of HLA-DRw13. This clearly indicates that both HLA-DR and HLA-DQ products contribute to the HLA-Dw19 subdivision of HLA-DRw13. (2) At least two different restricting epitopes are borne by DR products: one is correlated with the HLA-DRwl3 serologically defined specificity, which includes Dw19 and Dw18 haplotypes; the other is correlated with the only HLA-Dw19 subtype of HLA-DRwl3. (3) Restricting epitopes borne by DQ molecules have been found on Dw19 cells only. (4) DQ-restricted clones were unable to react with DQwl APC of any other haplotypes tested, including DR1, DR2-long, DR2-short, and DRw14, demonstrating a high degree of functional polymorphism among the serologically defined DQw1 specificities.Abbreviations used in this paper: APC antigen-presenting cells - cpm count per minute - HAU hemagglutinin units - IL-2 interleukin 2 - MHC major histocompatibility complex - mAb monoclonal antibody - PBM peripheral blood mononuclear cells - PHA phytohemagglutinin - pl isoelectric point - PMA phorbol myristic acetate - SD standard deviation     

Restimulation properties of human alloreactive cloned t-cell lines. Dissection ofHLA-D-Region alleles in population studies and in family segregation analysis

Alloactivated human lymphocytes were cloned by limiting dilution. After 1 month in culture with T-cell growth factor several clones incorporated tritiated thymidine when stimulated with the appropriate allogeneic cells. Specificity of restimulation of two primed lymphocyte clones, designated 12-2 and 12-8, was studied in detail after varying periods of culture (up to 50 days). Clone 12-2 cells were stimulated only by cells expressing the HLA-Dw antigens of the original priming cells (Dw3); furthermore, this primed lymphocyte reagent specifically recognized antigens associated with only one of the three distinct Dw3-bearing haplotypes from an informative family (KOH). Clone 12-8 cells, on the other hand, failed to recognize Dw3 antigens in the random panel or on homozygous typing cells (including the original priming cell), but were strongly restimulated by certain cells expressing Dw4 antigens. In addition, within family KOH, these restimulating products segregated with another one of the three Dw3-bearing haplotypes but with none of the three Dw4-bearing haplotypes. These two clones exemplify a hitherto unknown precision in cellular typing of theHLA-D region. Clone 12-2 allows the discrimination of a probably rare and as yet undetected HLA-Dw3 subtypic specificity. Clone 12-8, on the other hand, apparently identifies an allelic system segregating withHLA but distinct from the HLA-D determinants definable by HTC-typing.Abbreviations used in this paper MHC major histocompatibility complex - HLA human leukocyte antigens - PBL peripheral blood leukocytes - HTC homozygous typing cells - MLC mixed leukocyte culture - PLT primed lymphocyte testing - TCGF T-cell growth factor - CTC cultured T cells - Tdr tritiated thymidine     

Studies on species cross-reactivity of hemopexin by use of monoclonal and polyclonal antibodies

The extent of immunological cross-reactivity between hemopexins of four species (rat, human, rabbit and chicken) was assessed with four affinity purified polyclonal antibodies and three monoclonal antibodies using RIA, Western blotting and rocket immunoelectrophoresis. Neither the two monoclonal antibodies to rabbit hemopexin (Rb3D11 and Rb3H9), the monoclonal antibody (R4B3) to rat hemopexin nor any of the polyclonal antibodies showed shared antigenic determinants between avian and mammalian hemopexins as judged by RIA or rocket immunoelectrophoresis. Western blotting with polyclonal antibodies revealed some reactivity raising the possibility of a few shared, though distantly related, epitopes. Polyclonal antibodies, raised to the mammalian hemopexins cross-reacted to variable extents with the respective antigens by RIA, results paralleled by data obtained by Western blotting. Anti-rat monoclonal antibodies reacted only with rat hemopexin in Western blots and minimally with rabbit hemopexin in RIA. The anti-rabbit monoclonal antibodies recognized two distinct epitopes one of which is shared with human hemopexin and presumably highly conserved.     

Carbohydrate determinants associated with carcinoembryonic antigen (CEA)

The reactivities of eight purified preparations of carcinoembryonic antigen with monoclonal antibodies directed to tumor-associated carbohydrate determinants have been studied. All eight preparations showed strong reactivities with AH6, which defines Y structure (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3] GlcNAc beta 1----R), whereas only a few preparations showed reactivity with FH4-defining dimeric X determinants, (Gal beta 1----4 [Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4 [Fuc alpha 1----3]GlcNA beta 1----3Gal beta 1----R). No other antibodies tested showed any reactivity with these preparations. These carbohydrate markers associated with carcinoembryonic antigen will be useful to enhance the diagnostic value of the antigen.     

Monoclonal antibodies identify individual determinants on mouse mammary tumor virus glycoprotein gp52 with group, class, or type specificity

Hybrid cell lines producing monoclonal antibodies against the C3H strain of mouse mammary tumor virus (C3H MMTV) were prepared by the fusion of mouse myeloma cells with the lymphocytes of BALB/c mice that were immunized with C3H MMTV. Approximately 10% of the hybrid cells initially plated after cell fusion produced immunoglobulins that reacted in antibody-binding assays with C3H MMTV; 40 of these cells were cloned, and 6 eventually yielded stable cell lines. High concentrations of monoclonal antibodies (5 to 20 mg/ml) were obtained from serum and ascites fluid of syngeneic mice inoculated with the hybrid cells. All of the monoclonal antibodies were directed against the envelope glycoprotein gp52. Three of the hybrid cell lines produced immunoglobulins of the immunoglobulin M subclass and three produced immunoglobulin G2a. The monoclonal antibodies showed limited charge heterogeneity in light and heavy chains when analyzed by high-resolution, two-dimensional gel electrophoresis. Three serologically distinct specificities were observed when these ascites fluids were tested against different strains of MMTV. The antigenic determinants detected were the following: (i) a type-specific determinant unique to the C3H strain of MMTV; (ii) class-specific determinants shared between C3H and GR MMTVs; and (iii) a group-specific determinant found on C3H, GR, RIII, and the endogenous C3H (C3Hf) MMTVs. Because monoclonal antibodies recognize single antigenic determinants, these results demonstrate for the first time that the three patterns of antigenic reactivity for MMTV are related to individual determinants on the gp52 molecule and also clearly show that one strain of MMTV can be distinguished from other strains.