A monoclonal antibody detecting dipeptidylpeptidase IV in human tissue

Summary Dipeptidylpeptidase IV (DPP IV) occurs among others in exocrine epithelia, hepatocytes, renal tubuli, endothelia, and myofibroblasts of man and laboratory animals. Also Tµ lymphocytes and their varying differentiated neoplastic counterparts reveal this enzyme activity. The present paper describes a new monoclonal antibody recognizing DPP IV.Additional efforts have been taken to detect the subcellular localization of DPP IV and its isoelectric focusing pattern in different tissue types. The monoclonal antibody anti-DPP IV (clone II-19) shows a reaction pattern indistinguishable from the corresponding enzymehistochemical reaction. These findings were further substantiated by immunoblotting analysis. In line with the results of direct enzyme measurements in different subcellular fractions a considerable portion of the enzyme is localized in the membrane fraction.Dedicated to Professor Dr.Dr. h.c. Karl Lennert, Kiel, on the occasion of his 65th birthdayThis study was supported by the Deutsche Forschungsgemeinschaft, SFB 111, program CL1     

A mouse monoclonal antibody detecting the allospecificity HLA-A3

Balb/c mice were immunized with a human B-lymphoblastoid cell line typed HLA-A3, B7. The splenocytes of the immunized mice were fused with a murine myeloma. Supernatants of the cultures were screened against the immunizing cell line in fluorochromasia. Positive cultures were expanded and cloned. One of the clones, X 15.4, was expanded and brought to ascites in Balb/c mice. Monoclonality of the antibody X 15.4, which belongs to the class IgM and immunoprecipites a molecule of 44000 daltons, was demonstrated by isoelectric focusing. By complement dependent citotoxicity the ascites only reacted with the lymphocytes of all HLA-A3 individuals from a panel of 146 donors, showing no crossreactions. X 15.4 appears to be one of the very rare xenomonoclonal antibodies suitable for HLA typing.     

识别人白细胞免疫标志的单克隆抗体

A monoclonal antibody (48)against human Pan-Leucocytes was prepared. This antibody reacted with all haemopoietic cells tested, but not with red cells and platelets by indirect immunofluorescent staining. It was also disclosed that this antibody is only bound to lymphoid tissues or leucocytes scattered in other tissues with immunoperoxidase staining. The reactivities of McAb 48 with large a mount of various target cells were identical with anti-HLE, McAb of CD45 group. Therefore McAb 48 also recognizes T200 antigen and belongs to CD45 group. The value of McAb 48 in differential diagnosis of malignant lymphoma is discussed.     

A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and B17

A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kobler and Milstein [1]. This antibody recognizes a new specifity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.     

A human monoclonal antibody against HLA-Cw1 and a human monoclonal antibody against an HLA-A locus determinant derived from a single uniparous female

Abstract: Two human monoclonal antibodies (HuMAbs) with widely different HLA specificities were raised from a uniparous HLA-seropositive female. Screening against a large panel of serologically HLA-typed lymphocytes in the complement-dependent cytotoxicity test showed that one of these HuMAbs, VP6G3, was specific for HLA-Cwl, thereby constituting the first HuMAb against an HLA-C locus product. The second HuMAb, VP5G3, was directed against an HLA-A-encoded determinant shared by HLA-A11, -A25, -A26 and -A66. The epitopes responsible for binding were determined by comparing the aminoacid sequences and were pinpointed to the 6K/9F combination for HuMAb VP6G3, and 163R with a critical contribution of aminoacids present at positions 166/167 for HuMAb VP5G3.     

A double determinant sandwich immunoassay for quantitation of serum monoclonal anti-I-A antibody

A double-determinant sandwich radioimmunoassay (RIA) is described for the specific detection of anti-I-Ak monoclonal antibody (mAb) in the sera of non-Ighb murine hosts undergoing anti-Ia immunotherapy. This RIA utilizes 2 previously undescribed mAb reagents generated against an Ia.17-specific mAb secreted by the 10-3.6 hybridoma. The first reagent, 7.34, is specific for Ighb-linked allotypic determinants on the Fc portion of IgG2a immunoglobulins as defined by the pattern of reactivity with normal sera from a panel of inbred and Igh recombinant inbred strains. The second reagent, 58.3, is an anti-idiotypic mAb recognizing unique determinants in the combining site of 10-3.6 immunoglobulins, as determined by the specificity of the 58.3 mAb in solid-phase RIA and the capacity of this reagent to inhibit the binding of labeled 10-3.6 mAb to I-Ak-expressing spleen cells. In an RIA procedure using purified 58.3 mAb as substrate and 125I-labeled 7.34 as the detection reagent, serum concentrations of 10-3.6 as low as 1-5 ng/ml can be measured reproducibly after mathematical linearization of the sigmoid standard curve. In the present studies, the serum half-life of 10-3.6 mAb was calculated from assay data to be 3-5 h in I-Ak homo- or heterozygotes and 72 h in non-I-Ak mice. The serum level of 10-3.6 as a function of the mAb treatment protocol was also examined and results are considered with respect to the efficacy of different therapeutic regimens in prolonging transplant survival. Sandwich immunoassays of this type (RIA or ELISA) should provide a highly sensitive and specific means for monitoring serum mAb levels in individuals subjected to antibody immunotherapy for treatment of autoimmune disease, transplant rejection or tumor progression.     

A monoclonal antibody recognizing a determinant common to HLA—A3 and All

Spleen cells from a mouse immunized with an AML cell expressing HLA-A3 produced a hybridoma secreting an anti-HLA-A3, A11 monoclonal antibody, 26D3. By complement-dependent cytotoxicity at dilutions to 1:10(4) the ascites antibody lysed 13/13 A3, 15/15 A11, and 8 other lymphocytes from a panel of 98 donors. The 26 D3 immunoprecipitated a molecule consisting of subunits of 44,000 and 12,000 daltons. Monoclonality of the antibody was demonstrated by isoelectric focusing and protein A affinity chromatography.     

A monoclonal antibody specific for a carbohydrate epitope recognizes an IgE-binding determinant shared by taxonomically unrelated allergenic pollens

Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.     

A monoclonal antibody detecting a possible public specificity of the HLA-C locus

An IgM monoclonal antibody (CC-Cl 11) was produced by fusing myeloma cell line SP2/08 with lymphocytes of a Balb/c mouse previously immunized with peripheral blood lymphocytes of an A2, Bw44, B27, Cw1, Cw7, DR5 donor. Reactivity of CC-Cl 11 on a lymphocyte panel of 172 unrelated donors and lysostripping and absorption experiments have shown that CC-Cl 11 recognizes an antigenic determinant common to HLA-Cw1 and Cw3 positive lymphocytes.     

A monoclonal antibody that detects a polymorphic determinant common to HLA-DR1 and 2

In an attempt to study the gene products of the HLA complex, a monoclonal antibody, named HU-30, was produced by immunizing BALB/c mice with a cultured human B lymphoblastoid cell line, Shi-C3 (Aw24, Aw31, Bw51, Bw52, DR2, DR blank, MT1, MT2, MB3). HU-30 belonged to the IgG2 subclass and was active in complement dependent cytotoxicity. When the serological specificity was evaluated with a panel of 15 cultured human lymphoblastoid cell lines, it was found that HU-30 detected a polymorphic determinant, common to HLA-DR1 and 2, with much stronger cytotoxic activity against HLA-DR2 positive B cell lines. When HU-30 was tested against a panel of B cells from 84 healthy donors at a dilution of 2^-13, it gave positive reactions only with cells typed as HLA-DR2. Furthermore, sequential coprecipitation studies indicated that the HU-30 determinant was borne on the molecules carrying the HLA-DR determinants. Thus, HU-30 appears to be of great value as a tissue typing reagent monospecific for HLA-DR2.     

A monoclonal antibody reacting with a membrane determinant expressed on activated chicken T lymphocytes

A monoclonal antibody (INN-CH-16) was prepared which reacts with a cell surface antigen termed chicken activated T lymphocyte antigen. This antigen is expressed on antigen- or mitogen-activated T lymphocytes and is not present on nonstimulated lymphocytes. It has an apparent molecular mass of 48-50 kDa under reducing conditions. The value of this antibody for the immunohistochemical characterization of infiltrating cells in the thyroid glands from Obese strain chickens with spontaneous thyroiditis is demonstrated.     

A monoclonal mouse anti-rat Ia antibody which cross reacts with a human HLA-DRw determinant

A mouse monoclonal antibody, called MRC OX 3, which detects a polymorphic Ia determinant in the rat and cross reacts with an Ia determinant coded for by the I-A subregion in the mouse, detects a polymorphic determinant on human B cell lines. MRC OX 3 antibody binds to human B cell lines which express HLA-DRw specificities DRw 1, DRw2 and DRw6 but does not bind to those cells which only express other HLA-DRw specificities. No significant binding of MRC OX 3 antibody to either normal or mitogen stimulated human peripheral blood leukocytes, some of which are typed as HLA-DRw1, DRw2 or DRw6, could be detected. The binding of MRC OX 3 antibody to a human B cell line could be completely inhibited by pre-absorbing the antibody with purified rat Ia antigen.     

Identification of a murine monoclonal antibody specific for an allotypic determinant on mouse CD3.

A murine monoclonal antibody (mAb; 7D6) that was mitogenic for T cells was derived from 129/Sv animals immunized with a T helper clone from C57BL/6 origin. Fluoresceinated 7D6 labeled T cells from most common mouse strains but not from 129/Sv and LP/J animals, and this labeling was inhibited by the anti-CD3 epsilon mAb 145-2C11. The mitogenicity of 7D6 for T cells had a similar strain specificity. The antibody immunoprecipitated the T cell receptor (TcR) complex from a T cell hybridoma. After dissociation of this immunoprecipitate with detergents, the CD3 gamma and epsilon chains were retained by the 7D6 antibody. Immunoprecipitation data were also obtained with COS cells transfected with the CD3 gamma, delta or epsilon chains alone, in pairs or together. They confirmed that 7D6 bound the CD3 gamma epsilon pair, suggesting that the antibody recognizes a conformational epitope formed by gamma epsilon pairing, whereas 145-2C11 bound both gamma epsilon and delta epsilon pairs. These results, therefore, add to current information about TcR structure and subunit stoichiometry. We have demonstrated that the 7D6 mAb specifically binds to a CD3 dimer comprised of gamma and epsilon chains. We thus provide additional evidence that indicates that two CD3 epsilon chains are found within the receptor, one linked to CD3 gamma and the other to CD3 delta.     

A monoclonal anti-glycophorin A antibody recognizing the blood group M determinant: studies on the subspecificity

A mouse monoclonal antibody (425/2B, IgM) was obtained which shows specificity for blood group M determinant of glycophorin A. The antibody is pH-dependent. At pH 6-7 it reacted strongly with blood group M antigen, but also cross-reacted distinctly with N antigen. At pH 8.3 the antibody showed moderately decreased reactivity with M antigen, but no interaction with N antigen was detectable by hemagglutination, immunoblotting, or microplate ELISA. The direct binding studies and inhibition of 425/B antibody by untreated or modified blood group M and N glycoproteins or tryptic glycopeptides showed that the binding to the antigens was not affected by acetylation of their amino groups, or removal of amino-terminal amino acid residue. Desialylation of the antigens decreased their reactivity with the antibody and this effect was distinctly stronger at pH 7 than 8.3. The antibody reacted strongly at pH 7 and 8.3 with glycophorin B of Henshaw phenotype, whereas its reactivity with normal glycophorin B was weak or undetectable at these pH values, respectively. The results obtained indicated that anti-M specificity of 425/2B antibody is related to the 5th amino acid residue of glycophorin A (anti-Mgly specificity) and that pH shift from 7 to 8.3 changes the fine specificity of the antibody. At pH 8.3 the reactivity of the antibody is more dependent on glycine residue (higher anti-M specificity) and less dependent on sialic acid residues in the antigen.     

A mouse monoclonal antibody against a polymorphic determinant in a defined subset of DR molecules

The hybridoma technique was used to produce a mouse monoclonal antibody, designated as XI 21.4, which belongs to the IgG2a class. It is active in complement-dependent cytotoxicity and detects a B-cell antigenic determinant associated with DR1, DR4, DRw10, and, possibly, DRw9. Microfingerprinting of the immunoprecipitate from a homozygous DR4 cell line shows a typical alpha DR pattern and a beta pattern coinciding with that of DR4 molecules.     

A cytotoxic monoclonal antibody specific for the private alloantigenic determinant of the HLA-B 13 molecule

A murine monoclonal antibody (TÜ 110) prepared against blast cells of a patient with acute “undifferentiated” leukemia was tested in the microcytotoxicity assay on peripheral blood lymphocytes of 122 normal Caucasian donors. The TÜ 110 reactivity was found to show a correlation coefficient of 1.0 in population analysis for the presence of the HLA-B locus specificity B 13 as defined by alloantisera. Family segregation studies confirmed MHC linked inheritance of the TÜ 110 antigenic determinant strictly on HLA-B 13 positive haplotypes. As the first monoclonal reagent against the private specificity of this HLA-B locus antigen, TÜ 110 provides the possibility to study the structural relationships of sub- and supertypic determinants on this allotype and may help to correlate antigenic domains of HLA-B 13 with definable functional properties.     

A monoclonal antibody to the HLA-DR product recognizes a polymorphic Ia determinant in mice.

A rat monoclonal antibody which recognizes a product of the HLA-DR locus is described. The determinant recognized by this antibody is monomorphic in man but polymorphic in mice. Mapping studies with inbred and recombinant strains of mice show that this antigen as associated with H-2 haplotypes b, d and q and is coded for by genes within the I-A subregion.     

A highly conserved determinant on human rheumatoid factor idiotypes defined by a mouse monoclonal antibody

Different human IgM rheumatoid factor (IgM RF) idiotypes have been described defined by polyclonal rabbit anti-idiotypic antibodies. These antisera do not allow clear genetic analysis of the idiotypic determinants, be they cross-reactive or private. Therefore, we tried to obtain a set of monoclonal anti-idiotypic antibodies directed against RF idiotypes. Purified IgM RF serum from a patient with classical rheumatoid arthritis was used to immunize BALB/c mice. The spleen cells were fused with Sp 2/0 Ag 14, a nonsecreting mouse myeloma cell line, and a hybrid producing monoclonal anti-idiotypic antibody was selected. The mouse antibody, an IgG1 kappa, reacts with an identical or similar determinant located on (or close to) the binding site of all tested monoclonal or polyclonal IgM RF from totally unrelated patients with Waldenstr?ms's macroglobulinemias or rheumatoid arthritis. The monoclonal antibody also reacts with 2 rheumatoid arthritis patients' IgG RF and with a low proportion of normal polyclonal IgM without detectable RF activity. An hypothesis is proposed to explain the existence of a such highly conserved determinant on RF idiotypes.     

An enzyme immunofiltration assay useful for detecting human monoclonal antibody

A microenzyme-linked immunoassay (EIA) utilizing an immunofiltration manifold has been developed which provides a rapid, simple, and sensitive method of detecting human monoclonal antibody class, concentration, and specificity. In this assay either whole cells or soluble antigens were immobilized on glass fiber filters followed by incubating with the test human hybridoma supernatant with subsequent analysis by EIA. A specially designed 96-chamber immunofiltration plate is employed which serves as both an incubation chamber and as a filtration manifold. The assay described is unique in that small volumes of human hybridoma supernatant are required, crude preparation of only a few target cells are needed, labile cell surface antigens are preserved and it can be completed in 3 h. This assay is well suited for the rapid screening of large numbers of human hybridoma supernatants.