Syngeneic anti-idiotypic antisera to murine monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens

BALB/c mice were immunized with syngeneic anti-HLA class I monoclonal antibodies. The latter included the anti-HLA-A2, A28 monoclonal antibody (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to HLA-B antigens and the MoAb CR10-215 and CR11-115 to the same (or spatially close) monomorphic determinant. Anti-idiotypic antibodies could be detected in bleedings obtained 3 days after the first booster, increased in titer in bleedings obtained after the second booster, and persisted at high levels in subsequent bleedings. The four anti-HLA class I MoAb did not differ in their ability to elicit syngeneic anti-idiotypic antibodies. Cross-blocking studies with a panel of anti-HLA class I, anti-HLA class II, and anti-human melanoma-associated antigen (MAA) MoAb showed that the anti-MoAb CR10-215 and anti-MoAb CR11-115 antisera contain only antibodies to private idiotopes, whereas the anti-HLA MoAb CR11-351 and anti-MoAb Q6/64 antisera also contain antibodies to public idiotopes. The latter are expressed by the anti-HLA class I MoAb CR11-351, Q1/28, Q6/64, and 6/31, and by the anti-HLA class II MoAb Q5/6, Q5/13, 127, and 441. Public idiotopes were not detected on the nine anti-MAA MoAb tested. Public idiotopes do not interfere with the binding of anti-HLA MoAb with the corresponding antigenic determinants. On the other hand private idiotopes are located within the antigen-combining site, because anti-idiotypic antisera specifically inhibit the binding of the corresponding immunizing anti-HLA class I MoAb to cultured human lymphoid cells in a dose-dependent manner. Analysis by isoelectric focusing of the anti-HLA class I MoAb antisera showed that the spectrotype of the anti-MoAb CR11-351 antiserum comprises four components that focus in the pH 6.9 to 6.2 range, the spectrotype of anti-MoAb Q6/64 antiserum comprises three components that focus in the pH 6.5 to 6.1 range, the spectrotype of the anti-MoAb CR10-215 antiserum comprises three components that focus in the pH 6.4 to 6.1 range, and the spectrotype of the anti-MoAb CR11-115 antiserum comprises three components that focus in the pH 6.6 to 6.4 range.     

Structural relatedness of distinct determinants recognized by monoclonal antibody TP25.99 on beta 2-microglobulin-associated and beta 2-microglobulin-free HLA class I heavy chains

The association of HLA class I heavy chains with beta2-microglobulin (beta2m) changes their antigenic profile. As a result, Abs react with either beta2m-free or beta2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both beta2m-associated and beta2m-free HLA class I heavy chains. The reactivity with beta2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194-198 in the alpha3 domain. The reactivity with beta2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239-242, 245, and 246 in the alpha3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs.     

Interaction of alpha 1 with alpha 2 region in class I MHC proteins contributes determinants recognized by antibodies and cytotoxic T cells

The structure-function relationship of individual coding regions of class I mouse major histocompatibility complex proteins was studied by a combination of recombinant DNA, gene transfer techniques, and serologic and functional characterization. To examine the role of alpha 1 and alpha 2 regions in antibody and CTL recognition, the third exon of H-2Dd, Kd, and Ld transplantation antigen genes was replaced by the homologous coding region of the Qa-2-coded class I gene, Q6. We have chosen to carry out the exon shuffling experiments between these two different types of class I genes, because they are structurally similar and did not evolve to carry out identical functions. Therefore, it is less likely that the hybrid proteins will fortuitously recreate alpha 1-alpha 2 controlled functionally important determinants. The replacement of H-2 alpha 2 coding region with its Q6 counterpart had different effects on the expression of the three genes. The mutant H-2Dd gene transfected into L cells was expressed at high levels and retained several of the serologic determinants found on parental H-2Dd and Q6 domains. The serologic epitopes on the mutant H-2Kd-transfected cells were detectable at very low levels, whereas the product of the mutant H-2Ld gene could not be identified at all. Analysis of cells transfected with mutant H-2Dd gene with alloreactive and minor antigen(s)-restricted cytotoxic T cells indicated that the hybrid proteins lost the ability to be recognized by T cells. Our data suggest that cytotoxic T cells recognize conformational determinants composed of amino acids from alpha 1 and alpha 2 regions. Alternatively, it could be proposed that T cell recognition sites located in a single alpha 1 or alpha 2 protein region are susceptible to distortion upon alpha 1-alpha 2 interactions. Such susceptibility to conformational changes of the amino-terminal domain of transplantation antigens could be of functional importance for H-2-restricted antigen presentation.     

Structural analysis of the epitopes recognized by monoclonal antibodies to angiotensin II

Six clones were obtained that secrete anti-angiotensin II antibodies after somatic cell fusions between splenocytes of immunized BALB/c or outbred OF1 mice and NS-1 myeloma cells. The dissociation constants for angiotensin II ranged from 0.3 to 2.9 nM. A panel of 20 structural analogs of the hormone were used as probes to analyze the specificity of binding. From the binding studies and the putative three-dimensional structures of the tested peptides, three families of antibodies could be distinguished that recognized overlapping epitopes; the conservation of the native conformation of the angiotensin II molecule in the analogs appeared essential for the preservation of a high affinity to the antibodies. With one antibody, the affinities of the angiotensin II analogs have been correlated with their intrinsic biologic activities (as measured by in vivo pressor tests), and not with their binding affinity to the membrane receptor. These results are interpreted as mimicry, by the antibody binding site, of the active conformation of the receptor site.     

Lack of a role of monocytes in the inhibition by monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens of PHA-P-induced peripheral blood mononuclear cell proliferation

This study aimed at characterizing the mechanism(s) underlying the regulatory role of distinct determinants of HLA Class I antigens in PHA-P-induced T cell proliferation and the involvement of monocytes in this phenomenon. The anti-HLA-A2,A28 monoclonal antibodies (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to the gene products of the I antigens HLA-B locus, and the MoAb CR10-215 and W6/32 to distinct monomorphic determinants of HLA Class I antigens were found to inhibit PHA-P-induced peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent fashion. The inhibition is specific and reflects neither inhibition of PHA-P binding to cells nor a toxic effect of the anti-HLA Class I MoAb. The latter differed in the concentration required to induce inhibition, in the influence of the concentration of PHA-P used as mitogen, in the differential effect on the donors used as a source of PBMC, and/or in the requirement of the Fc portion to induce inhibition. At variance with the information in the literature, the inhibitory effect of anti-HLA Class I MoAb on PHA-P-induced PBMC proliferation neither reflected their interaction with accessory cells nor was mediated by suppressor factors released by monocytes stimulated with PHA-P in the presence of anti-HLA Class I MoAb. Therefore, the regulatory role of HLA Class I antigens in T cell proliferation is not likely to be mediated by monocytes and/or factors released from them, but may reflect an involvement of these molecules in T cell activation pathways.     

Antigenic determinants on human choriogonadotropin alpha-subunit. I. Characterization of topographic sites recognized by monoclonal antibodies

Immunochemical studies were designed to localize antigenic regions recognized by two monoclonal antibodies directed against the alpha-subunit of human choriogonadotropin (hCG-alpha) and to provide information on the three-dimensional structure of hCG and its alpha-subunit. Monoclonal antibody HT13 bound to a region accessible on both hCG and the free alpha-subunit, whereas monoclonal antibody AHT20 recognized a site localized only on the free alpha-subunit. By studying the cross-reactivity of these antibodies to homologous proteins, we found that antibody HT13 did not bind to equine or ovine lutropin, whereas AHT20 was capable of binding to both subunits. This observation suggests that AHT20 recognized a structurally related antigenic determinant on alpha-subunits of different species. To delineate the portions of hCG-alpha contributing to the antigenic determinants of AHT20 and HT13, we performed competitive inhibition assays using reduced and carboxymethylated hCG-alpha, deglycosylated hCG-alpha, hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core 1), or disulfide-bridged peptides comprising residues 1-35 and 52-91 of hCG-alpha (hCG-alpha core 2). Reduced and carboxymethylated hCG-alpha did not inhibit the binding of 125I-labeled hCG-alpha to both antibodies, whereas deglycosylated hCG-alpha was as active as hCG-alpha, suggesting that antigenic determinants of both antibodies are mainly discontinuous and do not reside on the oligosacharide part of the alpha-subunit. hCG-alpha core 1 had the same capacity as intact hCG-alpha to inhibit the binding of 125I-hCG-alpha to both antibodies, indicating that the 5 COOH-terminal residues of hCG-alpha do not participate in the antigenic determinants. hCG-alpha core 1 was as potent as hCG-alpha in inhibition experiments performed with HT13, whereas, in striking contrast, hCG-alpha core 2 did not compete with 125I-hCG-alpha for binding to AHT20, suggesting that the peptides released after proteolysis of the alpha-subunit by trypsin participate in the epitope of AHT20 and are not included in the antigenic determinant of HT13. In an attempt to elucidate the amino acid residues constituting the antigenic sites of HT13 and AHT20, hapten inhibition experiments were carried out using as competitive inhibitors five different synthetic peptides spanning the primary structure of hCG-alpha. None of these peptides inhibited the binding of 125I-hCG-alpha to HT13. In contrast, two peptides analogous to regions 23-43 and 33-59 of hCG-alpha exhibited significant potency in competing with 125I-hCG-alpha for binding to AHT20.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and functional analysis of integrin alpha2I domain interaction with echovirus 1

Integrins are cell surface receptors for several microbial pathogens including echovirus 1 (EV1), a picornavirus. Cryo-electron microscopy revealed that the functional domain (alpha(2)I) of human alpha(2)beta(1) integrin binds to a surface depression on the EV1 capsid. This three-dimensional structure of EV1 bound to alpha(2)I domain provides the first structural details of an integrin interacting with a picornavirus. The model indicates that alpha(2)beta(1) integrin cannot simultaneously bind both EV1 and the physiological ligand collagen. Compared with collagen binding to the alpha(2)I domain, the virus binds with a 10-fold higher affinity but in vitro uncoating of EV1 was not observed as a result of attachment of alpha(2)I. A molecular model, constructed on the basis of the EV1-integrin complex, shows that multiple alpha(2)beta(1) heterodimers can bind at adjacent sites around the virus 5-fold symmetry axes without steric hindrance. In agreement with this, virus attachment to alpha(2)beta(1) integrin on the cell surface was found to result in integrin clustering, which can give rise to signaling and facilitate the initiation of the viral entry process that takes place via caveolae-mediated endocytosis.     

The domain of platelet glycoprotein I as recognized by various antibodies, both monoclonal and polyspecific

Platelet membrane glycoprotein Ib plays a major role in the binding of factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium. We have used polyspecific and monoclonal antibodies to glycoprotein Ib and have demonstrated that both antibodies were directed to glycoprotein Is, a soluble fragment of glycoprotein Ib. By showing an inhibition of the binding of factor VIII/von Willebrand factor to control platelets in presence of the antibodies, it can be concluded that glycoprotein Is is involved in these binding sites.     

Topographic analysis of antigenic determinants recognized by monoclonal antibodies to the photoreceptor guanyl nucleotide-binding protein, transducin

Antigenic sites for six monoclonal antibodies that bind to the alpha subunit (G alpha) of the photoreceptor guanyl nucleotide-binding protein (G-protein or transducin) have been determined. Five of these antibodies (4A, 7A, 7B, 7C, and 7D) were shown in the preceding paper (Hamm, H. E., Deretic, D., Hofmann, K. P., Schleicher, A., and Kohl, B. (1987) J. Biol. Chem. 262, 10831-10838) to block G-protein-rhodopsin interaction. We have blotted tryptic and chymotryptic peptides of G-protein to nitrocellulose paper and found that these antibodies bind to peptides that contain the COOH-terminal end of the protein assessed by 32P-ADP-ribosylation of the COOH-terminus by pertussis toxin. The antigenic site is not exactly at the COOH-terminus since the antibodies also bind two peptides which lack a 2-kDa piece from the COOH-terminus. Antigenic sites are therefore on the 7-kDa chymotryptic peptide and 5-kDa tryptic peptide more than 2 kDa away from the COOH-terminus. Further evidence for this antigenic site comes from the ability of these antibodies to block pertussis toxin-mediated ADP-ribosylation while still binding to the previously ADP-ribosylated protein both on nitrocellulose blots and in immunoprecipitations. Antibody 4H, which was shown not to interrupt any of the functions studied, binds to the 11-kDa major tryptic fragment. To aid in the mapping of these sites onto the surface of G alpha, a model of the three-dimensional structure of G alpha has been generated using the G alpha primary sequence, predicted secondary structure, hydropathy plot, and the constraints of the GDP-binding site of the GTP-binding protein elongation factor Tu solved by Jurnak (Jurnak, F. (1985) Science 230, 32-36).     

Implication of HLA class I molecule from monocyte in T-cell activation by CD2 or CD3 monoclonal antibodies

The role of monocytes in human T-cell activation by monoclonal antibodies (mAb) recognizing CD3 molecule or by 2 mAb pairs directed against different epitopes of CD2 "GT2+T11(1)" or "D66+T11(1)" has been studied. It appears that HLA-cl I molecules from monocytes are involved in the early activation phase of T-cells stimulated by CD3 or CD2 when direct contacts between T-cells and monocytes are required. Thus, pretreatment of monocytes with HLA-cl I mAb inhibited IL 2 receptor appearance and IL 2 synthesis on T-cells stimulated by CD3 mAb or CD2 "GT2+T11(1)" mAb pair.     

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.     

Differential regulatory role of monomorphic and polymorphic determinants of histocompatibility leukocyte antigen class I antigens in monoclonal antibody OKT3-induced T cell proliferation

Monoclonal antibodies (mAb) to monomorphic and polymorphic determinants on the heavy chain of histocompatibility leukocyte antigen (HLA) class I antigens inhibit mAb OKT3-induced T cell proliferation, whereas the anti-beta 2-microglobulin mAb NAMB-1 does not affect it. The inhibitory effect of anti-HLA class I mAb is specific, is not an Fc-mediated phenomenon, does not require accessory cells, and does not involve early stages of T cell activation. Distinct determinants of HLA class I antigens regulate T cell proliferation by different mechanisms, because the anti-HLA-A2, A28 mAb CR11-351, and the mAb W6/32 to a framework determinant of HLA class I antigens block interleukin 2 (IL-2) secretion and IL-2 receptor expression, whereas the mAb CR10-215 to a monomorphic determinant blocks only IL-2 receptor expression. The mAb CR10-215 and W6/32 induced a 50% of maximal inhibition of T cell proliferation, when added after 27 and 12 hr, respectively, of incubation of peripheral blood mononuclear cells with mAb OKT3. On the other hand, the mAb CR11-351 inhibited T cell proliferation even when added after 38 hr of incubation of peripheral blood mononuclear cells with mAb OKT3 and was the only one to inhibit proliferation of cycling T lymphocytes. It is suggested that HLA class I antigens regulate T cell proliferation by interacting with cell-surface molecules involved in T cell activation. The differential inhibitory activity of the anti-HLA class I monoclonal antibodies tested may reflect the different ability of the corresponding determinants to interact with activation molecules.     

Impact of peptides on the recognition of HLA class I molecules by human HLA antibodies

MHC class I molecules expressed on cell surfaces are composed of H chain, beta2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.     

Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells. As expected, elimination of class II HLA-positive AC with anti-class II HLA MoAb plus complement caused a decrease in proliferation of CTC in response to all the mitogens tested. In contrast, elimination of class II HLA-positive CTC was shown to clearly increase proliferation of CTC, perhaps because this may deplete class II HLA-positive suppressor cells.     

Regulatory role of monomorphic and polymorphic determinants of HLA class I antigens in the proliferation of T cells stimulated by autologous and allogeneic B and T lymphoid cells

Monoclonal antibodies (mAb's) to monomorphic and polymorphic determinants of HLA Class I antigens were shown to inhibit proliferation of T cells stimulated with autologous and allogeneic B and T lymphocytes. Inhibition of proliferative responses was lower when T cells were used as stimulators than when B cells were used. The inhibitory activity was similar for mAb's to monomorphic and polymorphic determinants of HLA Class I antigens, suggesting that the density of antigen-antibody complexes on the cell membrane does not play a major role in the phenomenon. The anti-HLA Class I mAb's exerted their inhibitory effect at the level of both the responding and the stimulating cells. Addition of exogenous interleukin 2 to the mixed cultures did not affect the mAb-mediated inhibition.     

Hordein variation in the genus Hordeum as recognized by monoclonal antibodies.

The composition of the major storage protein, hordein, in wild barley species has been studied by using gel electrophoresis, Coomassie staining, and immunoblot assays. We have shown earlier that it is possible to obtain cross-reaction outside the cultivated barley, with monoclonal antibodies raised against hordeins from the barley cultivar Bomi. These antibodies have now been used to investigate the hordein composition in all species of the Hordeum genus. The results showed that polypeptides similar to the two major hordein groups of cultivated barley, the B- and C-hordeins, are produced in all wild Hordeum species, and that there are both similarities and differences between the two hordein groups. The similarities indicate a common evolutionary origin, while the distinction between B- and C-hordeins in the entire genus clearly shows that the divergence of their coding genes preceded the divergence of the Hordeum species. The presence of the same antigenic site in two different species indicates that they are evolutionarily related. Among the wild species, two rarely occurring sites were exclusively found in H. vulgare ssp. spontaneum and H. bulbosum, which confirms that they are the cultivated barley's closest relatives. Some of the antibodies also gave an extensive reaction pattern with H. murinum, which suggests a fairly close relationship to H. vulgare, though not as close as between H. vulgare and H. bulbosum.     

Prolamin variation and evolution in Triticeae as recognized by monoclonal antibodies.

The isopropanol-soluble seed storage proteins, prolamins, were studied in 18 different genera of the tribe Triticeae by gel electrophoresis, Coomassie staining, and immunoblot assays. The monoclonal antibodies were originally raised against cultivated barley (Hordeum vulgare L.) hordein, and their reactions had been tested earlier on wild Hordeum species. The study showed that all the investigated Triticeae species produce prolamins and that structural similarities can be found throughout the tribe. The presence of the same antigenic sites in all the species indicates that the polypeptides contain well-conserved regions. They also indicate that the prolamins of the Triticeae species have a common evolutionary origin. In all the investigated species an antigenic site that is common to the B- and C-hordeins of barley was detected. Some of the reacting polypeptides also contained a site that is only present in the B-hordeins. The B-hordein specific site was found in all genera except Agropyron, Hordelymus, and Secale. This shows that although there are similarities between individual polypeptides, the composition of the various prolamin groups may vary between different genera. In the polyploid Elymus species different banding patterns were observed depending on what basic genomes were represented. The results suggest a direct correlation between the presence of a fast migrating polypeptide containing the B-hordein specific site and the presence of the H genome.     

Detection of cross-reactive determinants shared by human monoclonal IgM reacting with myelin-associated glycoprotein

Monoclonal IgM from patients with peripheral neuropathy frequently have anti-myelin-associated glycoprotein (MAG) activity. We investigated the idiotypes of 10 monoclonal anti-MAG by using rabbit polyclonal antisera. Three groups of anti-idiotypic antisera could be distinguished. Four sera reacted only with the immunizing protein. Two sera reacted with a single other anti-MAG IgM in addition to the immunizing one. Immunoenzymatic studies showed that these two couples of anti-MAG IgM reacted identically with 100% cross-inhibition, indicating that the whole set of idiotypes identified by the rabbit antiserum was present on both IgM antibodies. The four other anti-idiotypic sera reacted with the homologous IgM, as well as with most of the other anti-MAG IgM. In contrast to the previous antisera the binding of these four sera to the homologous IgM could not be inhibited by other cross-reacting anti-MAG IgM. However, when a heterologous IgM was used for coating, these antisera with one exception showed complete cross-inhibition. The absence of inhibition by purified MAG of the patient of the anti-idiotypic antisera sera suggests that these antibodies are most likely to be directed against framework determinants rather than against combining site epitopes.     

Analysis of class I MHC antigens in the rat by monoclonal antibodies

Monoclonal antibodies (mAb) were made against class I MHC antigens of the i (mAb 42,70,39) and u (mAb 68-D) haplotypes in the rat by using specific strain combinations in order to obtain reagents for identifying the products of the RT1.An, RT1.Au, and RT1.Eu loci. These antibodies were hemagglutinating only; were IgG except for mAb 68-D3, which had a defective heavy chain; reacted identically with MHC-congenic strains and with their inbred donor strains; and precipitated class I MHC antigens. Strain distribution, sequential immunoprecipitation, and peptide mapping studies were used to define the specificities of the mAb, and the assignments were checked by comparing the specificities of the mAb with those of haplotype-specific alloantisera. The specificities were the following: mAb 42, An; mAb 68-D, Au; mAb 70, Eu; and mAb 39, an antigen encoded by a locus different from A and E. This new locus was designated RT1.F, and the allele detected by mAb 39, as Fa. The serologic data place RT1.F between RT1.A and RT1.D. The plasma membranes of DA.1I(BI) lymphocytes contain comparable amounts of An, Eu, and Fa antigens but express them on the cell surface in the order An much greater than Eu greater than Fa.