Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive molecules on human mononuclear cells

It is known that the antibody to human myelin-associated glycoprotein (MAG) reacts with a subset of human mononuclear cells (MNC) mediating a natural killer (NK) activity. The properties of the target molecule of the anti-MAG antibody, however, have not yet been elucidated. Three (GC-J4, MC-P2, and MC-P4) of five murine monoclonal antibodies (mAb) to MAG bound to human MNC. Moreover, MC-P2 and MC-P4 inhibited the binding of 125I-labeled anti-Leu-7 to MNC in a dose-dependent fashion. Conversely, anti-Leu-7 inhibited the binding of MC-P2 and MC-P4 to MNC, but did not inhibit the binding of GC-J4. Therefore, it is possible that MC-P2 and MC-P4 bind directly to or close to the Leu-7 epitope, and that GC-J4 binds to the epitope which is distinct from the Leu-7 epitope. The electrophoretic patterns of immunoprecipitates with GC-J4, MC-P2 and anti-Leu-7 from detergent lysates of surface-labeled human MNC were very similar. The target molecules of anti-Leu-7 and anti-MAG mAb have apparent m.w. of 205, 170, 150, 135, 110, 85, 65, and 55 kDa. All of the molecules precipitated by these mAb are monomeric or noncovalently associated proteins, because the electrophoretic mobilities of the proteins remained unchanged whether the samples were reduced or not. MC-P4 may have a higher affinity for the 65 kDa molecule than the other mAb, and precipitates the 58 kDa molecule as well. Therefore, the fine antigenic specificity of MC-P4 is slightly different from those of anti-Leu-7 or MC-P2. The implication of these results is that mAb, whose specificity is directed to the carbohydrate part of human MAG, reacts with the Leu-7 reactive molecules on human MNC, and that at least two epitopes detected by anti-MAG mAb coexist on the surface molecules with various apparent m.w.     

Murine antigen H-2.7: Localization of its antigenic determinant to the Ss (C4) molecule

Murine blood group antigen H-2.7 is encoded by a locus mapping in the vicinity of theS locus which codes for the Ss antigen carried by the fourth component of the complement pathway (C4). Normal mouse serum of H-2.7-positive strains contains a substance which inhibits anti-H-2.7 hemagglutination. This substance cannot be removed by passage of the serum through an anti-Ss immunoabsorbent column indicating that the Ss and H-2.7 antigens are present on separate molecules or molecular fragments in the serum. In contrast, fresh plasma either does not contain the H-2.7-bearing substance at all or it contains it at a far lower concentration than normal serum, although it has a normal level of the Ss-antigen-bearing substance. However, the H-2.7-positive substance appears when the plasma is allowed to stand for several hours, or when it is dialyzed and treated subsequently in a manner favoring spontaneous degradation of complement components. Removal of the Ss substance from the fresh plasma prevents the appearance of the H-2.7 antigen at any time thereafter. These findings indicate that the Ss and H-2.7 antigens are carried by the same molecule or molecular complex. The intact molecule expresses only the Ss antigen; the H-2.7 antigen is either hidden or masked so that it is inaccessible or poorly accessible to H-2.7 antibodies. Degradation of these molecules results in the generation of two fragments, a large fragment carrying the Ss antigen and a smaller H-2.7-positive fragment. The data are consistent with the interpretation that the H-2.7 antigen is encoded by the S locus, and that it is carried by that portion of the C4 molecule split off during complement activation.Abbreviations used in this paper NMS normal mouse serum - DNP dinitrophenyl - EDTA ethylene-diamine-tetraacetate - PVP polyvinylpyrrolidone     

Murine antigen H-2.7: localization of its antigenic determinant to the Ss (C4) molecule

Murine blood group antigen H-2.7 is encoded by a locus mapping in the vicinity of the S locus which codes for the Ss antigen carried by the fourth component of the complement pathway (C4). Normal mouse serum of H-2.7-positive strains contains a substance which inhibits anti-H-2.7 hemagglutination. This substance cannot be removed by passage of the serum through an anti-Ss immunoabsorbent column indicating that the Ss and H-2.7 antigens are present on separate molecules or molecular fragments in the serum. In contrast, fresh plasma either does not contain the H-2.7-bearing substance at all or it contains it at a far lower concentration than normal serum, although it has a normal level of the Ss-antigen-bearing substance. However, the H-2.7-positive substance appears when the plasma is allowed to stand for several hours, or when it is dialyzed and treated subsequently in a manner favoring spontaneous degradation of complement components. Removal of the Ss substance from the fresh plasma prevents the appearance of the H-2.7 antigen at any time thereafter. These findings indicate that the Ss and H-2.7 antigens are carried by the same molecule or molecular complex. The intact molecule expresses only the Ss antigen; the H-2.7 antigen is either hidden or masked so that it is inaccessible or poorly accessible to H-2.7 antibodies. Degradation of these molecules results in the generation of two fragments, a large fragment carrying the Ss antigen and a smaller H-2.7-positive fragment. The data are consistent with the interpretation that the H-2.7 antigen is encoded by the S locus, and that it is carried by that portion of the C4 molecule split off during complement activation.     

Limited proteolysis of human leukocyte interferon-alpha2 and localization of the antigenic determinant binding the monoclonal antibodies

Large peptide fragments of human leucocyte interferon-alpha 2 (INF-alpha 2) were obtained by limited proteolysis with trypsin, pepsin, thermolysine and Bacillus amyloliquefaciens intracellular serine proteinase. The ability of the fragments to bind murine monoclonal antibodies NK2 raised against INF-alpha 2 was studied by the immunoblotting technique. The region of sequence 110-149 is the most sensitive to proteolytic attack, being probably exposed on the surface of the INF-alpha 2 molecule. INF-alpha 2 fragments 1-139, 1-147, 1-149 are capable of binding antibodies, whereas fragments 1-109 and 1-112 do not bind antibodies NK2. A comparison of the primary structure of human leucocyte and murine leucocyte INF families in the region of sequence 110-139 and an analysis of the ability of human INF differing in amino acid sequences to bind antibodies NK2 demonstrated that the antigenic determinant for antibodies NK2 is the sequence Glu114-Asp115-Ser116-Ile117 of the INF-alpha 2 molecule.     

Expression of HLA-B27 in transgenic mice is controlled by gene(s) mapping between H-2D and H-2L loci

The level of HLA-B27 transgene expression on the cell surface is dependent on the host H-2 haplotype. Mice homozygous for the H-2 ^b , H-2 ^f , H-2 ^f , H-2 ^p , H-2 ^r , and H-2 ^k haplotypes express B27 at high levels. An intermediate level of B27 expression is observed in H-2 ^v mice whereas low levels of B27 are expressed in H-2 ^q and H-2 ^d mice. The decreased expression of B27 maps to the D region of the major histocompatibility complex. Recombinant strain B10.RKDB (D^dL^b) mapped the low expression gene centromeric to H-2L. In order to determine the low expression within the H-2D region, the B27 transgene was introduced into B10.D2-H-2 ^dm1 and BALB/c-H-2 ^dm2 mice. Expression of B27 in both of these strains was high indicating that neither H-2D ^d nor H-2L ^d is responsible for the low expression. This maps the effect between the H-2D and H-2L loci. In addition, introduction of human ₂-microglobulin (₂m) into B10.D2-B27 transgenic mice caused a marked enhancement of B27 expression on the cell surface suggesting that the defect in B27 expression in certain haplotypes is due to an inability of B27 to associate with endogenous mouse ₂m. We propose that gene(s) mapping between D and L (either D2, D3, D4, or some as yet unidentified gene) may be involved in class I assembly by helping association of ₂m with class I. This putative molecule, designated Assembly Enhancer (AE) might have a negative influence in the association between human class II and mouse ₂m.     

New antigenic determinant (Sa) on human lymphocytes and phagocytes

Presence of antigenic determinants reacting with homogeneous IgM/kappa cold agglutinin (CA) of a new specificity, tentatively called Sa, was investigated by bithermic cytotoxicity assay and by immunofluorescence. CA Sa killed on average 38% allogeneic peripheral blood lymphocytes (PBL) and up to 74% of autologous PBL. There was preferential kill of B-PBL compared to T-PBL. Some preference toward B cells was also noted using tonsillary B and T lymphocytes. Cytotoxic activity of CA Sa against chronic lymphocytic leukemia cells of B-type was almost equal to that of potent anti-I CA and much stronger than anti-i CA. Presence of additional B-cytotoxic factor in the serum was excluded by the use of red blood cell eluate composed solely of homogeneous CA. Thymocytes and helper-type T cells from a patient with T cell chronic lymphocytic leukemia were very susceptible to the cytotoxic action of Sa. CA Sa killed 39% of monocytes, but there was almost no kill of polymorphonuclear leukocytes. Lymphocytotoxicity of CA Sa was abolished by sialyllactose and was not influenced by I-active glycoproteins. Comparison of CA Sa to CA of other specificities showed marked differences, supporting the view that Sa has new, previously unrecognized specificity.     

Radioimmunochemical evidence for a role of carbohydrate in antigenic determinant(s) on human liver alpha-L-fucosidase.

A competitive-binding radioimmunoassay method was employed to investigate the role of carbohydrate in antigenic determinant(s) of human liver alpha-L-fucosidase. Competition curves were used to quantify the concentrations of competitors needed to cause 30% inhibition of the precipitation of 125I-labelled alpha-L-fucosidase. The isoelectric forms of alpha-L-fucosidase, which are related by sialic acid residues, were separated preparatively and used as competitors in the radioimmunoassay. A pattern of increasing effectiveness as competitors with increasing acidity of the forms was found, suggesting that sialic acid may be involved in the antigenic determinant(s) of alpha-L-fucosidase. Specificity was exhibited when sugar and sugar derivatives were used as competitors in the radioimmunoassay: a 51-fold range of competitive ability was found, and sialic acids (N-acetylneuraminic acid and N-glycollylneuraminic acid) and colominic acid (a polymer of N-acetylneuraminic acid) were the best competitors. The results of our studies suggest that carbohydrate contributes to antigenic determinant(s) of alpha-L-fucosidase and that sialic acid is probably the major sugar involved.     

Generation of anti-peptide monoclonal antibodies which recognize mature CSF-2 alpha (IL 3) protein

The colony-stimulating factor CSF-2 alpha (IL 3) has been purified to homogeneity, the protein sequenced, and the gene encoding this lymphokine cloned. Knowledge of the protein sequence permitted the synthesis of peptides corresponding to the amino terminus of the molecule. These peptides, after conjugation to palmitic acid, were used to immunize mice. Spleen cells from mice immunized with one of these peptides (CSF-2 alpha 1-14) were fused with the myeloma cell line NS-1. The fusion resulted in the isolation of two hybridoma cell lines, designated 6A5 and 4D4, that secreted antibodies that were specific for the immunizing peptide. The antibodies did not react with a closely related peptide CSF-2 alpha 7-16. The antibodies were capable, however, of recognized CSF-2 alpha protein as judged by the ability of the antibodies to remove CSF-2 alpha activity from culture medium of PHA-stimulated LBRM-33-5A4 cells, to immunoprecipitate radiolabeled CSF-2 alpha protein, and to detect CSF-2 alpha protein bound to nitrocellulose membranes.     

An antigenic determinant on human centromere protein B (CENP-B) available for production of human-specific anticentromere antibodies in mouse.

Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromere heterochromatin throughout the cell cycles. Some components of mammalian centromeres including CENP-B are target antigens for autoimmune disease patients, often those with scleroderma. Recent isolations of CENP-B genes from human and mouse suggested that CENP-B was highly conserved among mammals. From the previous analysis of the reactivity of patient anticentromere sera, two autoepitopes have been located on the DNA binding domain at the amino-terminal region. The amino acid sequences for both the epitopes are perfectly conserved in the two species, human and mouse. In this study, to identify a human-specific antigenic determinant, the remaining two epitopes were further located in separate carboxyl-terminal regions of human CENP-B. Although the amino acid sequence of one epitope is identical to that of the corresponding region in mouse CENP-B, the other has a less homologous sequence. To confirm that the latter epitope was available for production of human-specific anticentromere antibodies, mice were immunized with the recombinant human CENP-B product. One serum that exclusively stained human centromere structure, but not that of other mammals, was identified in the immunofluorescence microscopic observation. The epitope analysis showed that the less conserved one was recognized by this serum. These results suggested that the corresponding region defines the antigenic determinants for the species specificity.     

An antigenic determinant of human beta 2-microglobulin masked by the association with HLA heavy chains at the cell surface: analysis using monoclonal antibodies

Four anti-human beta 2-microglobulin monoclonal antibodies were produced against whole lymphoid cells or against urinary beta 2-microglobulin. Their reactivity was fully inhibited by purified soluble beta 2-microglobulin. The B1.1G6, C23.24.2, and B2.62.2 antibodies bound either to free or to HLA-complexed beta 2m. They recognized the same or very close determinants, since they achieved mutual blocking. In contrast, the C21.48A1 antibody did not bind to the cell surface and did not recognize the same determinant on the purified beta 2-microglobulin molecule as the others. It was able to bind and to form a specific complex with membrane beta 2-microglobulin only, once separated from the HLA heavy chain. These data strongly suggest that the C21.48A1 antigenic determinant might be hidden when the beta 2-microglobulin is complexed with the HLA heavy chains at the cell surface.     

Evidence for shared antigenic determinants on rabbit and human interleukin 1 (IL 1)

An antiserum to human interleukin 1 (IL 1) was prepared by immunizing a goat with the isoelectric point (pI) 6.9 type of IL 1 in Freund's complete adjuvant. Serum-mediated inhibition of the biological activity of IL 1 appeared within 4 wk after the first immunization, and showed a progressive rise in titer over a 9-mo period. The inhibitory moiety was purified by sequential ammonium sulfate fractionation and DEAE-Sephacel ion exchange chromatography, and the activity was found to co-purify with the IgG fraction of the serum. The antibody neutralized the biological activity of the pI 6.9 type of human IL 1 derived from either human placental tissue or human peripheral blood adherent cells, but did not neutralize the pI 5.2 type of IL 1 derived from either source. When used as an affinity reagent, the antibody selectively absorbed the pI 6.9 human IL 1, but not the pI 5.2 human IL 1. Furthermore, the antibody neutralized the pI 7.4 type of IL 1 derived from rabbit alveolar macrophages, but had no activity against the pI 4.6 IL 1 derived from the same source. No inhibitory activity against rat spleen cell-derived IL 1 or murine P388D1 cell line-derived IL 1 was detected. These experiments support the concept that the differing pI types of IL 1 derived from the same species are both biochemically and antigenically distinct molecules, and IL 1 of similar pI type derived from different species may share antigenic determinants.     

The binding of monoclonal antibodies to cell surface molecules. Quantitative analysis of the reactions and cross-reactions of an antibody (MB40.3) with four HLA-B molecules

The MB40.3 monoclonal antibody binds to four distinct HLA-B molecules; B7, B40, B40*, and B27. With Fab' fragments only the interaction with B7 and B40 was detected and the affinity for both was the same (1-2 X 10(8) M-1) suggesting the epitope is shared by the two molecules. Unlike many antibodies for which low affinity is due to a high-dissociation constant, that of MB40.3 results from a very low-association rate constant, coupled with a low-dissociation constant. In consequence, the affinity and avidity of Fab', F(ab')2, and IgG for B7 and B40 were found to be of a similar magnitude, soluble B7 was a more efficient competitor for antibody than cell surface B7 and in practice antibody bivalency was of little importance. The forward rate constant could be increased by removing Fc from the antibody or by removing sialic acid from the cells by treatment with neuraminidase. The neuraminidase treatment also produced an increase in the number of detectable cell surface HLA-A,B molecules. The affinity of MB40.3 for B40* and B27 was estimated to be less than 4 X 10(6) as no binding with Fab' was detected due to a high-dissociation rate. For these two HLA-B molecules bivalent attachment was critical, and it increased the strength of interaction with cell surface B40* and B27 to a point where the avidities were comparable to those obtained with B7 and B40, with B40* interacting more strongly than B27. The epitopes recognized by MB40.3 on B40* and B27 were thus shown to be structurally different from each other and from those on B7 and B40. The properties of this antibody contrast with those of other anti-HLA-A,B we have studied (Ways, J.P., and Parham, P. (1983) Biochem. J. 216, 423-432).     

MCF-specific murine monoclonal antibodies made against AKR-247 MCF virus recognize a unique determinant associated with the gp70-p-15(E) complex.

Hybridomas obtained from (NFS X AKR)F mice immunized with syngeneic cells infected with AKR-247 MCF virus produced antibodies specific for only AKR-247 or closely related MCF viruses which hare a previously defined MCF antigen (MCFA-3). These monoclonal antibodies recognized a new type of viral antigenic determinant which appeared to be a conformational determinant associated with the env precursor polyprotein (pr80env) or its disulfide-linked gp70-p15(E) complex (gp80) but not with free gp70 or p15(E) or any other virion or virus-induced protein.     

Cross-reactive and serotype-specific neutralization epitopes on VP7 of human rotavirus: nucleotide sequence analysis of antigenic mutants selected with monoclonal antibodies.

The neutralization epitopes of human and simian rotavirus protein VP7 were studied by producing six neutralizing monoclonal antibodies (N-MAbs) and using these N-MAbs to select antigenic mutants that resisted neutralization by the N-MAbs used for their selection. Cross-neutralization tests between the N-MAbs and the antibody-selected antigenic mutants identified one cross-reactive and five distinct serotype-specific neutralization epitopes which operationally overlapped one another and constituted a single antigenic site. In addition, the amino acid substitutions in human rotavirus VP7 that are responsible for the antigenic alterations in the mutants selected with anti-VP7 cross-reactive or serotype-specific N-MAbs were identified. All the amino acid substitutions in the antigenic mutants occurred in one of two variable regions: amino acids 87 to 101 and 208 to 221.     

Murine monoclonal antibodies directed to the human histo-blood group A transferase (UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase) and the presence therein of N-linked histo-blood group A determinant

Mouse MAbs (WKH-1 through -3) to the human histo-blood group A glycosyltransferase (Fuc alpha 1----2Gal alpha 1----3 galactosaminyltransferase) were established by immunization with the purified native A transferase protein. Hybridomas were selected on the basis of solid-phase reactivity with the purified native A transferase, cell immunofluorescence and immunoprecipitation of transferase activity, and absence of reactivity with blood group ABH carbohydrate determinants. Three MAbs, thus selected, were found most likely to react with the protein epitopes unrelated to carbohydrate epitopes of purified A transferase. The MAbs reacted with cells having high A transferase activity and immunoprecipitated the A transferase activity as well as the 40,000 MW iodinated transferase protein. The antibodies were shown, however, to immunoprecipitate and partially inhibit not only A1 and A2 but also B transferase activity from plasma and A transferase from human lung, and to react with B cells expressing B transferase, thus indicating a cross-reactivity with B transferase. In contrast, they showed no reactivity with various cells having the O phenotype and did not immunoprecipitate the A transferase from porcine submaxillary glands or the alpha 1----2fucosyltransferase from Colo205 cells. The purified A glycosyltransferase was found to carry blood group A carbohydrate determinants by immunochemical detection with a panel of anti-carbohydrate MAbs. These determinants are believed to be N-linked, since treatment of the purified A transferase with N-glycanase removed activity. Immunohistological studies of three epithelial tissues showed that the antibodies stained the Golgi area of cells in epithelia from A and B, but not O, individuals.     

The antigenic determinants recognized by three monoclonal antibodies to keratan sulphate involve sulphated hepta- or larger oligosaccharides of the poly(N-acetyllactosamine) series

The carbohydrate determinants of keratan sulphate recognized by three monoclonal antibodies (5-D-4, 1-B-4 and MZ15) have been investigated by solid-phase radioimmunoassay using bovine corneal keratan sulphate as the immobilized reference antigen. The antibodies appeared highly specific for sulphated poly(N-acetyllactosamine) sequences, for their binding was strongly inhibited by preparations of keratan sulphate, but not by glycoproteins with non-sulphated poly(N-acetyllactosamine) sequences of I and i antigen types, a desulphated keratan sulphate hexasaccharide, an array of neutral and sulphated mono- and disaccharides and other glycosaminoglycans. Inhibition of binding assays using a series of structurally characterized sulphated di, tetra-, hexa-, octa- and decasaccharides, and partially characterized larger oligosaccharides, isolated from bovine corneal keratan sulphate after digestion with endo-beta-galactosidase (see preceding two papers in this journal) showed that the smallest oligosaccharide reactive with all three antibodies was the linear pentasulphated hexasaccharide, E-II although antibody 1-B-4 reacted with a tetrasulphated analogue. The heptasulphated octasaccharide, G-III, was more active; among the structurally characterized keratan sulphate oligosaccharides the nonasulphated decasaccharide, I-IV, was the most active. Thus, the hepta- and octasaccharide sequences, indicated by brackets below are proposed as candidate antigenic structures recognized by the three monoclonal antibodies. (Formula: see text). Antibody 5-D-4 differs from the other two antibodies in reacting relatively strongly with a minor oligosaccharide which chromatographs as a hexasulphated octasaccharide, G-I, and most strongly with a minor sulphated, linear dodecasaccharide, J-II, which has been partially characterized [Tang, P.W., Scudder, P., Mehmet, H., Hounsell, E. F. & Feizi, T., unpublished results] and may contain N-sulphated glucosamine residues.     

Identification of structural differences between different forms of interleukin-2 (IL-2) using anti-(human recombinant) IL-2 monoclonal antibodies.

We have generated a panel of murine monoclonal antibodies (mAbs) directed against recombinant human interleukin-2 (rh IL-2). All mAbs have similar affinities (Kaff approximately equal to 10(-8) M) and are of the IgG1 isotype. Specificity of the mAbs has been established using an ELISA method and immunoprecipitation of native human interleukin-2 (nh IL-2) present in supernatants from PHA-stimulated human mononuclear cells (PBMNC). Four of the nine mAbs inhibit the rh IL-2-dependent proliferation of a murine cytotoxic T-lymphocyte line (CTLL-2). The estimated ID50, in the presence of 0.75 IU rh IL-2/ml, ranges from 2.0 micrograms/ml to 30 micrograms/ml final concentrations of the antibodies. Using different forms of IL-2 we found that the mAbs give different patterns of inhibition of the IL-2-dependent proliferation. One mAb (AMCIB 27) is able to discriminate between rh and nh IL-2. Findings with another mAb (AMCIB 24) indicate that possible functional differences between human IL-2 and recombinant murine (rm) IL-2 are caused by differences in the active sites. The results of this investigation show that it is possible to obtain mAbs, after immunization with rh IL-2, that differ in their ability to inhibit the biological action of different forms of IL-2.