Human leucocyte surface glycoprotein CDw44 and lymphocyte homing receptor are identical molecules.
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Antibodies, Monoclonal↗
Biomedical subjects
V Horejsí
Publications and source records attributed to V Horejsí.
Structural relationship between the soluble and membrane-bound forms of human monocyte surface glycoprotein CD14.
The carboxy-terminal amino acid sequence of the soluble form of the 53,000 mol. wt monocyte surface antigen, CD14, was determined by carboxypeptidase Y digestion and compared with the complete amino acid sequence of this protein as predicted from the structure of cloned cDNA [Goyert et al. Science 239, 497-500 (1988)]. The soluble antigen isolated from urine appears to lack eight C-terminal amino acid residues predicted for the full-size translation product, but possesses a major part of the C-terminal hydrophobic domain originally suggested as the membrane-spanning segment. The CD14 antigen can be removed from the monocyte surface by phosphatidylinositol-specific phospholipase C treatment, indicating that this glycoprotein is anchored in the membrane by a phospholipid and is not a transmembrane protein. The soluble form occurring in serum and in supernatants of cultured monocytes thus probably arises by phospholipase-mediated cleaving off the cell surface antigen. A sensitive sandwich ELISA was developed using a monoclonal anti-CD14 antibody, MEM-18, and polyclonal rabbit anti-CD14 antiserum for quantitation of the soluble antigen concns in sera and cell culture supernatants. Using this assay, the antigen present in the supernatant of phospholipase treated peripheral blood mononuclear cells could be estimated. The assay was also used for estimation of the concns of the soluble form of the CD14 antigen in human sera.
Amino Acid Sequence↗
Characterization of a broadly expressed human leucocyte surface antigen MEM-43 anchored in membrane through phosphatidylinositol.
A monoclonal antibody MEM-43 was prepared, which recognizes an antigen expressed on all peripheral blood leucocytes, on erythrocytes and several cell lines, but is absent from U937, Nalm-6, Daudi and Raji cell lines. The antigen isolated by immunoaffinity chromatography from several cell lines is an 18,000-25,000 mol. wt glycoprotein. An apparently identical antigen isolated from erythrocytes binds to several lectins and has a 14,000 mol. wt polypeptide backbone, modified by an endoglycosidase F-sensitive carbohydrate moiety. The epitope recognized is reduction-sensitive. The sequence of N-terminal 17 amino acid residues was determined; five out of six N-terminal amino acids are identical to those found at the N-terminus of the mouse lymphocyte surface antigen Ly-6C. The antigen is completely released from the cell surface after treatment with phosphatidylinositol-specific phospholipase C.
Amino Acid Sequence↗
Monoclonal antibodies against human leucocyte antigens. III. Antibodies against CD45R, CD6, CD44 and two newly described broadly expressed glycoproteins MEM-53 and MEM-102.
Monoclonal antibodies against several human leucocyte cell surface antigens were prepared and characterized: (1) MEM-56, MEM-93, MEM-66, and MEM-104 against the CD45R antigen (220 kDa and 205 kDa mol. wt. forms of the leucocyte common antigen CD45 expressed on B-lymphocytes, myeloid cells and a subpopulation of T-lymphocytes); (2) MEM-98 and MEM-100 against the T-lymphocyte antigen CD6 (mol. wt. 100 kDa); (3) MEM-85 against the broadly expressed antigen CD44 (mol. wt. 85 kDa) which was recently shown by us to be identical with the lymphocyte homing receptor; (4) MEM-53 against a newly described broadly expressed antigen of 35-40 kDa mol. wt. and (5) MEM-102 against another newly described glycoprotein of 40 kDa mol. wt. anchored in membrane through a phosphatidylinositol moiety.
Animals↗
Surface proteins and glycoproteins of human leucocytes.
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Animals↗
Monoclonal antibodies against human alpha-fetoprotein. Exploitation of an unusual calcium-dependent interaction with the antigen for analytical and preparative purposes.
Seven murine hybridomas were established producing IgG1 monoclonal antibodies (mAbs) against three different epitopes of human alpha-fetoprotein (alpha FP). The interaction of two of these mAbs (AFP-01 and AFP-02) with the antigen was strongly inhibited by calcium-chelating agents. The mAb AFP-11 could be used for estimation of alpha FP by RIA in the concentration range of 5-100 ng/ml. Several combinations of the mAbs could be used in a two-site sandwich-ELISA for similar purposes. Immunoaffinity chromatography on a column of immobilized mAb AFP-01 permitted the purification of alpha FP.
Animals↗
Human monocyte activation induced by an anti-CD14 monoclonal antibody.
An anti-CD14 mAb RoMo-1 rapidly induces in human monocytes a transient oxidative burst activity as detected by chemiluminescence assay. Pretreatment of these cells with the mAb markedly suppresses the monocyte chemiluminescence response to opsonized zymosan. In addition, the antibody induces a significant increase of IL-1 production and secretion by mononuclear cells, comparable to a similar effect of rIFN-gamma or LPS. Electron microscopy demonstrates internalization of the CD14 molecules after interaction with the mAb in a characteristic receptor-like manner.
Antibodies, Monoclonal↗
Characterization of a new murine monoclonal antibody against human DP antigens.
Murine Hybridoma cell line BraFB6 was prepared producing monoclonal antibody reactive with an antigen identified as DP. This identification is based on the results of immunoprecipitation from radiolabeled cell lysates, competitive binding-inhibition assay with a reference anti-DP antibody B7/21, sequential immunoprecipitation and specific binding to the purified DP glycoprotein. In addition to this anti-DP monoclonal antibody, two anti-DR and two anti-DR +DP monoclonals were obtained.
Animals↗
Characterization of a 95 kDa human leucocyte sialoglycoprotein: its identity with CD43, gpL115, leukosialin and sialophorin.
A monoclonal antibody, MEM-59, was prepared, which recognizes a heavily sialylated glycoprotein antigen expressed on most human leucocytes, the apparent molecular mass of which is strongly dependent on the concentration of polyacrylamide gel used for SDS-PAGE. The antigen isolated on immobilized MEM-59 reacted in Western blotting with mAbs described previously by others as recognizing antigens of similar properties. These experiments indicate that the molecules called CD43, gpL115 (sialophorin) or leukosialin are identical and different from a 75-85-kDa glycoprotein of similarly broad tissue distribution (CDw44). Most of the anti-CD43 mAbs recognize sialic acid-dependent epitopes. None of the anti-CD43 mAbs tested stimulated proliferation of peripheral blood leucocytes in vitro.
Antibodies, Monoclonal↗
Monoclonal antibodies against human leucocyte antigens. II. Antibodies against CD45 (T200), CD3 (T3), CD43, CD10 (CALLA), transferrin receptor (T9), a novel broadly expressed 18-kDa antigen (MEM-43) and a novel antigen of restricted expression (MEM-74).
The specificities of several monoclonal antibodies described by us earlier were determined more exactly: (1) antibodies MEM-31 and MEM-32 are directed against CD8 and CD5 T cell antigens, respectively: (2) MEM-15 and MEM-18 react with the CD14 monocyte antigen; (3) MEM-28 recognizes the CD45 pan-leucocyte antigen. Several new monoclonal antibodies are described: (4) MEM-55 and MEM-58, reactive with subsets of CD45-related molecules; (5) MEM-43, recognizing a broadly expressed 18-kDa glycoprotein; (6) MEM-57, reacting with the CD3 antigen complex: (7) MEM-59 recognizing the CD43 leucocyte antigen; (8) MEM-74, which reacts with an unidentified antigen strongly expressed on several cell lines and many leukaemic cells but absent from most resting leucocytes; (9) MEM-75, directed against transferrin receptor, and (10) MEM-78, recognizing CD10 (CALLA) antigen.
Antibodies, Monoclonal↗
Purification and characterization of class II histocompatibility antigens from a homozygous human B cell line.
Human class II histocompatibility antigens were purified from the Epstein-Barr virus-transformed human B lymphoblastoid cell line LG-2 by immunoaffinity chromatography. This is the first time all three subsets have been prepared as nonradioactive materials on a milligram scale. The yields of DR, DQ, and DP from 10 g of cells were approximately 12, 2, and 0.2 mg, respectively. Cross-contamination of the subsets was found to be less than 2% when assayed by measuring the binding of antigen-specific monoclonal antibodies to antigen immobilized on fixed erythrocytes. The three purified subsets were extensively characterized. They contained no detectable invariant chain. The three proteins were distinguished by their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The denatured antigens were susceptible to partial removal of carbohydrate by endoglycosidase H and apparently complete removal of carbohydrate by endoglycosidase F. The isolated, denatured chains differed in their affinities for radiolabeled lectins, suggesting differences in carbohydrate structures. A water-soluble form of each antigen was prepared by a controlled papain digestion of the native antigen. Both native and denatured antigens were analyzed for their reactivities with a panel of class II antigen-specific monoclonal antibodies, allowing a precise definition of the specificities of the antibodies.
Alkylation↗
Murine monoclonal antibodies to human A erythrocytes: differential reactivity with N-acetyl-D-galactosamine.
Six different hybridoma cell lines were obtained producing IgM monoclonal antibodies (MAbs) against human blood group A antigen. All these MAbs agglutinate the A1, A1B and A2 erythrocytes, while only one of them agglutinates the A2B erythrocytes. The agglutination activity of 3 of these MAbs is inhibited by N-acetyl-D-galactosamine (D-GalNAc). Dissociation constants of complexes of these MAbs with D-GalNAc are approximately 2.2 X 10(-3) mol/l.
ABO Blood-Group System↗
HLA-DQ1 + DQ3-specific monoclonal antibody cross-reacts with a rat MHC-encoded polymorphic determinant.
From a panel of seven mouse monoclonal antibodies against human class II molecules only one, HL-37, directed against the beta chain of human DQ1 and DQ3 antigens cross-reacted in membrane immunofluorescence with the RT1b haplotype of the rat major histocompatibility complex. From the results obtained using an indirect immunoprecipitation method with 35S- and 125I-labelled rat samples from RT1b-carrying lymph node cells it can be supposed that the rat homologue of the human DQ beta chain was detected.
Animals↗
Simple polyacrylamide gel electrophoresis in continuous carbonate buffer system suitable for the analysis of ascitic fluids of hybridoma bearing mice.
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Animals↗
Biochemical characterization of a soluble form of the 53-kDa monocyte surface antigen.
Two monoclonal antibodies, MEM-15 and MEM-18, were prepared which recognize a monocyte 53-kDa antigen. A soluble form of this antigen was found in most normal sera and in urine of some nephrotic patients. Milligram amounts of this glycoprotein were isolated by immunoaffinity chromatography from urine and used for quantitative amino acid and carbohydrate analysis and N-terminal amino acid sequencing. The reactivity of the isolated antigen with several previously described monoclonal antibodies indicates that it is the antigen previously called MY-4 or MY-23. A significant homology exists between the sequenced N-terminal portion of the antigen and sequences found in the kinase-related transforming protein src and in the precursor of epidermal growth factor.
Amino Acid Sequence↗
Characterization of seven new monoclonal antibodies against human DR, DR + DP and DQ1 + DQ3 antigens.
Seven murine monoclonal antibodies were prepared that react with human class II antigens. Four of them (HL-39, MEM-12, MEM-24G, and MEM-32B: react with a monomorphic determinant dependent on association of heavy and light chains of DR antigens, two others (HL-38 and HL-40) recognize a monomorphic determinant localized on the light chains of DR and DP antigens. The antibody HL-37 is directed against a determinant present on DQ1 and DQ3, but not DQ2 molecules; at least in the case of DQ1, the epitope recognized is located on the light chain.
Animals↗
Monoclonal antibodies against human leucocyte antigens. I. Antibodies against beta-2-microglobulin, immunoglobulin kappa light chains, HLA-DR-like antigens, T8 antigen, T1 antigen, a monocyte antigen, and a pan-leucocyte antigen.
Monoclonal antibodies against several human leucocyte cell surface antigens were prepared and characterized: B2M-02, a non-cytotoxic antibody against beta-2-microglobulin; MEM-09 and MEM-40, against immunoglobulin kappa-type light chains; MEM-12, MEM-24G and MEM-32B, all against a monomorphic determinant in MHC class II antigens, presumably HLA-DR, dependent on the association of alpha and beta chains; MEM-15 and MEM-18, against a monocyte antigen of 55 kDa; MEM-28, against a 200 kDa antigen expressed on all leucocytes; MEM-31, against the T8 antigen of the cytotoxic/suppressor T-lymphocyte subpopulation; MEM-32, against the T1 antigen of T lymphocytes.
Animals↗
Equilibrium in the protein-immobilized-ligand-soluble-ligand system: estimation of dissociation constants of protein-soluble-ligand complexes from binding-inhibition data.
The equilibrium in the protein-immobilized-ligand-soluble-ligand system was examined theoretically and the equations found were used for determination of dissociation constants of protein-soluble-ligand complexes (K). These constants can be obtained from the s/b vs C plot [s/b = ratio of soluble and bound forms of the protein at equilibrium established in the presence of the soluble ligand (concn C)], which is linear if: (1) the concns of the complexes are much lower than the total concns of the immobilized and soluble ligands, and (2) if multiple interactions of an n-valent protein with the immobilized ligand essentially do not occur (i.e. the binding to the immobilized ligand is monovalent). The effect of violation of condition (1) is examined by computation simulation and is shown to be manifested as a non-linearity of the plot. Heterogeneity of the immobilized ligand (arising, for example, from the immobilization procedure) is predicted to have no effect on the K-values obtained. A more complex linear equation applicable principally for determination of K under more general conditions was also found. The conditions are defined under which the C50-values (i.e. concns of a series of ligands inhibiting the binding to an immobilized ligand by 50%) can be directly used for comparison of dissociation constants. The use of the s/b vs C plot was tested experimentally: transferrin, several glycoproteins or synthetic carbohydrate-containing copolymers were immobilized by adsorption in the wells of polystyrene microculture plates and thus served as immobilized ligands. Solutions of 125I-labelled ligand-binding proteins (lectins or monoclonal antibodies binding transferrin) were incubated in these ligand-coated wells in the presence of various amounts of soluble ligands (carbohydrates or transferrins): after equilibrium establishment the s/b values were determined and plotted against C and the values of K were obtained as the intercept of the plot with the abscissa. The method appears to be experimentally simple and the K-values of the lectin-sugar and monoclonal antibody-antigen complexes agree well with those determined by other methods.
Antibodies, Monoclonal↗