Production and characterization of a monoclonal antibody to biotin.

Monoclonal antibodies to biotin have been prepared by using biotin linked to keyhole limpet haemocyanin (KLH) as the antigen. Spleen cells obtained from mice immunized with biotin-KLH were fused with the myeloma cell line NS-1. The resulting hybridomas were screened for the production of antibodies to biotin using an enzyme-linked immunosorbent assay. Clones producing antibodies to biotin were isolated by limiting dilution methods. Four cell lines, each derived originally from a different fusion, were chosen for the production of monoclonal antibodies. The monoclonal antibodies obtained have been characterized with respect to their ability to interact with biotin, biotin-bovine serum albumin, biotin-KLH and biocytin as well as to inhibit biotin-dependent enzymes. They have been used to produce cellular biotin deficiency in vitro for studies of biotin function.     

Purification and characterization of human lipoprotein lipase and hepatic triglyceride lipase. Reactivity with monoclonal antibodies to hepatic triglyceride lipase

Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 +/- 15 and 19 +/- 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded Mr of 60,500 +/- 1,800 and 65,200 +/- 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospecific antibody against antithrombin. In additions, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. Interaction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.     

Production and characterization of a monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol

A monoclonal antibody was obtained by the fusion of mouse myeloma cells with splenocytes isolated from Balb/c mice, which had been immunized with diacetoxyscirpenol-hemiglutarate (DAS-hemiglutarate) and verrucarol-hemiglutarates covalently bound to ethylenediamine-modified bovine serum albumin. The anti-DAS-antibody that could be induced was of the IgM type with kappa-chains. The titer of the monoclonal anti-DAS-antibody in ascites fluid obtained from mice injected the selected cell line was much higher than those of conventional antisera. An enzyme-linked immunosorbent assay based on the competitive binding principle in which the antibody was applied had a sensitivity of 1 ng DAS per assay. The relative cross-reactivity of the monoclonal antibody in the CI-ELISA with the related trichothecenes such as triacetoxyscirpenol, 15-monoacetoxyscirpenol, diacetylverrucarol, 4-monoacetoxyscirpenol and scirpentriol were found to be 1.8, 0.8, 0.15, 0.02 and less than 0.001, respectively. The trichothecenes verrucarol, T-2 toxin, T-2 tetraol, deoxynivalenol, 3-acetyldeoxynivalenol and trichothecin showed no cross-reactivity.     

Production and characterization of a monoclonal antibody to human type III collagen

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.     

Production and characterization of monoclonal antibody to dermatan sulfate proteoglycan

We purified dermatan sulfate proteoglycan (PG) from the capsule of human ovarian fibroma for use as an immunogen. A monoclonal antibody, designated 6B6, was produced which reacts to the intact molecule of dermatan sulfate PG and the chondroitinase AC-treated core molecule on Western-blotted nitrocellulose membrane. Localization of materials showing crossreactivity to this antibody was studied in human tissues by indirect immunohistochemistry. The interstitial elements of almost all tissues examined were positive for the antibody: dermis, submucosal layer of digestive tract, perichondral layer, perivascular connective tissue, perineurium, adventitia of aorta, vessel wall of vein, pleura, and fibrous capsule of kidney and liver. Positive staining was also observed in fibrous elements at post-necrotic foci of cardiac muscle and pancreas, and at atherosclerotic lesions of aorta. The distribution of the antigen, core protein of the dermatan sulfate PG, revealed with 6B6 was compared to that of the dermatan sulfate side chain, which was demonstrated with antibody 9A-2 (Couchman et al.: Nature 307:650, 1984) after treatment with chondroitin sulfate B-lyase. The distribution of both antigens, core protein, and dermatan sulfate side chains showed the same pattern, with minor exceptions. The antibody 6B6 will be a useful tool to study the immunohistochemical localization of dermatan sulfate PG.     

Production and immunochemical characterization of monoclonal antibody to IRR ectodomain

The insulin receptor-related receptor (IRR) is the only known metabotropic sensor of extracellular alkaline medium involved in the regulation of the acid–base balance in the body. IRR is expressed in certain cell populations of the kidney, stomach, and pancreas that can come into contact with the extracellular fluids with alkaline pH. To study IRR structure and function, we obtained a stable hybridoma cell-line-producing antibody to the extracellular portion of the receptor. The monoclonal antibody isolated from ascitic fluids showed a positive reaction with the antigen in the ELISA test. The minimum working concentration of antibodies was 12.5 ng/mL. The ability of the antibodies to specifically recognize the purified ectodomain of IRR and the fulllength receptor was confirmed by western blot, immunoprecipitation, and immunocytochemistry.     

Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells

A murine monoclonal antibody, termed HeFi-1, was produced after immunization with the L428 Hodgkin's disease tissue culture cell line. HeFi-1 selectively stained only the Reed-Sternberg or Hodgkin's cells in 18 of 18 cases of Hodgkin's disease, including the nodular sclerosis, mixed cellularity, and lymphocyte-depleted histologic subtypes. HeFi-1 did not stain any cells in normal lung, brain, salivary gland, thyroid, gall bladder, pancreas, liver, testis, breast, endometrium, or kidney. Rare large cells at the edge of the lymphoid follicles were stained in normal tonsil, colon, and hyperplastic thymus. There was no staining of any cells in 14 cases of B cell non-Hodgkin's lymphoma; however, the malignant cells in three of 11 cases of non-Hodgkin's lymphoma which appeared to express T cell markers were also stained with HeFi-1. Tissue culture cell lines including the T cell acute lymphocytic leukemia lines MOLT4 and CEM, the histiocytic cell line U-937, and the amniotic cell line WISH were not stained. Seven Epstein Barr virus (EBV)-positive lymphoblastoid cell lines were stained with HeFi-1, but there was no staining of three EBV+ African Burkitt's lymphoma cell lines or three EBV- American Burkitt's cell lines. HeFi-1 did not block the ability of the L428 cells to stimulate a mixed lymphocyte reaction or function as accessory cells for mitogen-induced human T cell proliferative responses. Modulation of the HeFi-1 cell surface antigen on the L428 cells was not observed. HeFi-1 specifically immunoprecipitated a cell surface protein of approximately 120,000 daltons from both the L428 and EBV+ lymphoblastoid cell lines. HeFi-1 monoclonal antibody should prove useful not only in the diagnosis, staging, and potential therapy of Hodgkin's disease, but also for determining the cell of origin of the Reed-Sternberg cell.     

Production and characterization of a monoclonal antibody for Pefloxacin and mechanism study of antibody recognition

In this report, an artificial antigen (PFLX–BSA: Pefloxacin connected bovine serum albumin) was successfully prepared. The monoclonal antibody against pefloxacin was produced and characterized using a direct competitive ELISA. The linear range of detection was 0.115–6.564 µg/L. The limit of detection defined as IC₁₅ was 0.170 ± 0.05 µg/L and the IC₅₀ was 0.902 ± 0.03 µg/L. The antibody variable region genes were amplified, assembled, and sequenced. A three–dimensional structural model of the variable region was constructed to study the mechanism of antibody recognition using molecular docking analysis. Three predicted essential amino acids, Thr53, Arg97 of heavy chain and Thr52 of light chain, were mutated to verify the theoretical model. Three mutants lost binding activity signi?cantly against pefloxacin as predicted. These may provide useful insights for studying antigen–antibody interaction mechanisms to improve antibody affinity maturation in vitro.     

Production and characterization of a mouse monoclonal antibody specific for lentil lectin

A murine IgA monoclonal antibody (MoAb) was produced against the widely used glucose/mannose-specific two-chain mitogen from Lens culinaris (lentil) belonging to the Vicieae tribe of the Leguminosae family. The MoAb designated, 98, F-10, was found to be specific for lentil lectin when tested in dot blotting against 22 different native lectins. The antigenic specificity was also tested against subunits of 13 completely sequenced legume lectins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose filters. The MoAb showed a strong reaction only against the lentil heavy subunit. Comparison of the amino-acid sequences revealed 13 amino-acid residues which might be involved in the epitope reactive with this antibody. The MoAb did not react with synthetic peptides from the heavy subunit of lentil.     

Production and characterization of a monoclonal antibody to vacuolar H+ATPase of renal epithelia

A monoclonal antibody to vacuolar H+ATPase isolated from bovine kidney medulla was produced and characterized by immunoprecipitation and immunocytochemistry. The antibody, immobilized on beads, specifically immunoprecipitated both solubilized N-ethylmaleimide-sensitive ATPase activity and proton-transporting vesicles from renal microsomes; control experiments with an "irrelevant" monoclonal antibody showed no immunoprecipitated activity. By fluorescent immunocytochemistry, the antibody stained the membranes of intracellular vacuolar compartments in LLC-PK1 cells. Immunocytochemical staining showed that the monoclonal antibody colocalized partially with N-(3-[2,4-dinitrophenyl)amino)propyl)-N-(3-amino-propyl)methylamine, a probe for acidic compartments, with the endocytic markers dextran and transferrin, with the lysosomal probe alpha 2-macroglobulin, and with clathrin. The anti-vacuolar H+ATPase antibody showed no colocalization with staining for mitochondrial H+ATPase. The anti-vacuolar H+ATPase antibody should serve as a specific probe for examining the distribution and dynamics of the vacuolar proton pump in renal epithelial cells.     

Production and characterization of a monoclonal antibody to the O-acetylated peptidoglycan of Proteus mirabilis.

A monoclonal antibody (PmPG5-3) specific for the O-acetylated peptidoglycan of Proteus mirabilis 19 was produced by an NS-1 myeloma cell line and purified from ascites fluid by a combination of ammonium sulfate precipitation and affinity chromatography. The monoclonal antibody (an immunoglobulin M) was characterized by a competition enzyme-linked immunosorbent assay to be equally specific for both insoluble and soluble O-acetylated peptidoglycan but weakly recognized chemically de-O-acetylated P. mirabilis peptidoglycan, the non-O-acetylated peptidoglycans from Escherichia coli and Bacillus subtilis, and the peptidoglycan monosaccharide precursors N-acetylglucosamine and N-acetylmuramic acid dipeptide. The monoclonal antibody did not react with D-alanine or lipopolysaccharide isolated from P. mirabilis. Based on this evidence, the binding epitope on the P. mirabilis peptidoglycan is predicted to be linear and to comprise the glycan backbone, including both the N- and O-acetyl moieties. Monoclonal antibody PmPG5-3 was used to localize the O acetylation of the P. mirabilis peptidoglycan by immunoelectron microscopy. Murein sacculi of P. mirabilis were heavily and randomly labelled with the immunogold, whereas very little labelling and no labelling were observed on the sacculi isolated from de-O-acetylated P. mirabilis and E. coli, respectively. Based on the apparent pattern of immunogold labelling, a physiological role for peptidoglycan O acetylation in P. mirabilis is proposed.     

Production and characterization of a monoclonal antibody (BBH5) directed to ganglioside lactone

The occurrence of lactones in various ganglioside preparations has been clearly demonstrated, yet the natural occurrence of ganglioside lactones in cells and tissues has been the subject of long debate, since lactones can be formed readily during preparation of gangliosides. We now report the generation of a monoclonal antibody (BBH5) that reacts specifically with lactones of disialogangliosides having the NeuAc2-8NeuAc2-3Gal sequence, but does not crossreact with the parent ganglioside. The specificity of the antibody resides on the first lactone ring between two sialic acid residues but not on the second lactone ring between sialic acid and galactose, as evidenced by reactivity with lactonized GD_1b having the first lactone ring (L1), and by reactivity with lactonized polysialic acid homo-oligomers ([NeuAc2-8] _n NeuAc). The sialic acid carboxyl involved in the lactone ring was unequivocally determined after ammonolysis followed by methylation and fast atom bombardment mass spectrometry. The antibody BBH5 thus provides a novel tool for studies of the natural occurrence of lactones in cells and tissues.Abbreviations BSA bovine serum albumin - CM chloroform-methanol - CMW chloroform-methanol-water - FAB-MS fast atom bombardment mass spectrometry - IHW isopropanol-hexane-water - MAb monoclonal antibody - PBS phosphate-buffered saline - TLC thin layer chromatography     

Purification and characterization of lipoprotein lipase and hepatic triglyceride lipase from human postheparin plasma: production of monospecific antibody to the individual lipase

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were purified to homogeneity from human postheparin plasma. Molecular, catalytic and immunological properties of the purified enzymes were investigated. The native molecular weights of LPL and HTGL were 67,200 and 65,500, respectively, by gel chromatography. The subunit molecular weights of LPL and HTGL were 60,600 and 64,600, respectively, suggesting that these enzymes are catalytically active in a monomeric form. In addition, the purified LPL and HTGL each gave a single protein band when they were detected as glycoproteins with a probe of concanavalin A. The purified enzyme preparations were free of detectable antithrombin III by Western blot analysis. Catalytic properties of the purified enzymes were examined using triolein-gum arabic emulsion and triolein particles stabilized with phospholipid monolayer as substrates. LPL catalyzed the complete hydrolysis of triolein to free oleate and monooleate in the presence of apolipoprotein C-II. Apparent Km values for triolein and apolipoprotein C-II were 1.0 mM and 0.6 microM, and Vmax was 40.7 mmol/h per mg. HTGL hydrolyzed triolein substrate at a rate much slower than LPL, and produced mainly free oleate with little monooleate. Apparent Km and Vmax values were 2.5 mM and 16.1 mmol/h per mg, respectively. Polyclonal antibodies were developed against the purified LPL and HTGL. The purity and specificity of these antisera were ascertained by immunotitration, Ouchterlony double diffusion and Western blot analyses. The anti-human LPL and anti-human HTGL antiserum specifically reacted with the corresponding either native or denaturated enzyme, indicating that two enzymes were immunologically distinct. We developed an assay system for LPL and HTGL in human PHP by selective immunoprecipitation of each enzyme with the corresponding antiserum.     

Production and characterization of a monoclonal antibody cross-reactive with most group A trichothecenes

A monoclonal antibody cross-reactive with most group A trichothecenes was produced by fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with 3-acetyl-neosolaniol-hemisuccinate conjugated to bovine serum albumin. One stable clone, H159B1D5, which produced monoclonal antibody that bound with both T-2 toxin and diacetoxyscirpenol (DAS) was obtained after subcloning. Enzyme-linked immunosorbent assay (ELISA) revealed that the antibody belongs to the immunoglobulin G1 (kappa chain) isotype and had binding constants of 2.81 x 10(9), 1.05 x 10(9), and 1.57 x 10(8) liters per mole for T-2 tetraol tetraacetate, T-2 toxin, and DAS, respectively. The relative cross-reactivities of the antibody with T-2 tetraol tetraacetate, T-2 toxin, and DAS were 200, 100, and 20, respectively, with tritiated T-2 toxin as the marker ligand. The relative cross-reactivities for the above toxins were 667, 100, and 73, respectively, with tritiated DAS as the marker ligand. No cross-reaction with HT-2 and deoxynivalenol triacetate was observed in either system. By using this monoclonal antibody, an indirect ELISA for analysis of T-2 toxin was also developed. The linear portion of the standard curve for analysis of T-2 toxin in each analysis by radioimmunoassay and ELISA was in the range of 0.1 to 2 ng and 0.05 to 1.0 ng, respectively.     

Production and characterization of a monoclonal antibody specific for Salmonella O5-antigen

The present report described the production and characterization of a monoclonal antibody, TMY1, specific for an O-antigen of Salmonella bacilli. The following results were obtained: The slide agglutination test against strains of Salmonella serovars indicated the responsiveness of TMY1. TMY1 was reactive only to strains that also possessed O5-antigen in group O4. The agglutinating ability of TMY1 was absorbed completely with bacilli possessing O5-antigen in group O4. When treated with 1 N HCl, bacilli possessing O5-antigen in group O4 showed no agglutinability in the presence of TMY1. The data indicates that TMY1 was specific for O5-antigen. Strains of Salmonella group O4 were classified by the agglutination test with TMY1 into two subgroups, O5-antigen positive and negative, suggesting the usefulness of TMY1 as an epidemiologic tool for the serotyping of Salmonella.     

Production and characterisation of a monoclonal antibody to Serpulina hyodysenteriae

Abstract A monoclonal antibody (mAb) directed against Serpulina hyodysenteriae , the causative agent of swine dysentery, was produced and characterised. The mAb (BJL/SH1) reacted in Western blots with a protein with a molecular mass of about 30 kDa in outer membrane preparations from a range of S. hyodysenteriae isolates of different serotypes. It did not react with preparations made from a variety of non- S. hyodysenteriae intestinal spirochaetes. Immunogold labelling was used to confirm the location of the reactive epitope on the cell outer membrane. The mAb agglutinated and produced fluorescence only with strains of S. hyodysenteriae , and should prove to be a useful reagent for identification of S. hyodysenteriae .     

Production and immunological characterization of a monoclonal antibody to Trichophyton quinckeanum: interaction with phosphorylcholine-bearing components

A monoclonal antibody (Tq-1) that interacts with the phosphorylcholine (PC)-bearing antigens of Trichophyton quinckeanum was produced by fusion of myeloma cells (JKAg-8) with spleen cells of BALB/c mice immunized with an alum-precipitated fraction of T. quinckeanum cytoplasmic antigen. It was characterized as an IgM class antibody by immunodiffusion using anti-Ig heavy chain specific reagents, ELISA using immunoglobulin-specific peroxidase-conjugated antibodies, and by gel filtration chromatography; it showed high affinity for Staphylococcus aureus protein-A. Interaction of Tq-1 with PC-like antigens of T. quinckeanum was demonstrated by inhibition studies using ELISA, immunoblotting, immunoprecipitation and immuno electron microscopy techniques. The binding activity of Tq-1 antibody with a range of dermatophyte proteins was completely inhibited by prior incubation with PC hapten. Moreover, dermatophyte antigens reacting with the monoclonal antibody reacted strongly with sera from chronically infected mice. Dermatophyte antigens derived from both young (24 h) and old (20 d) cultures reacted with Tq-1 and this binding was inhibited by PC, suggesting that Tq-1 target antigen PC appears at an early stage during fungal growth and remains throughout its life.     

Production and characterization of a monoclonal antibody to dopamine D2 receptor: comparison with a polyclonal antibody to a different epitope.

A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 microM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein.     

Production and characterization of a monoclonal antibody against putative T lymphocytes of Catla catla

Catla catla is the fastest growing Indian major carp and one of the major aquaculture species in South Asia. A monoclonal antibody (MAb) designated B8 MAb was produced against nylon wool-enriched thymus mononuclear cells of C. catla. This MAb did not show reactivity with macrophage and epithelial cell lines derived from catla thymus in cellular ELISA. In flow cytometric analysis of gated lymphocytes, the percentage of B8 positive (B8+) cells in thymus (n?=?10, 500?C600?g) was determined to be 77.7?%. Similarly, the percentage of B8+ cells in kidney, spleen and blood (n?=?5) was 15.08, 1.1 and 32.17?%, respectively. Western blotting of reduced membrane proteins showed that B8 MAb reacted with a polypeptide having a molecular weight of 168.2?kDa. In indirect immunoperoxidase test, B8+ cells appeared to be lymphoid cells with a high nucleus to cytoplasmic ratio. B8 reactive cells were densely packed in central region of thymus whereas, a few cells were found to be positive in kidney and spleen sections. B8 MAb also reacted with a significant population of lymphocytes in blood smears. Considering the economic importance of C. catla, this MAb should be a useful tool for studying immune response of this fish species.     

Production and immunohistochemical characterization of a monoclonal antibody raised to proteoglycan purified from a human yolk sac tumour

Summary A large proteoglycan with chondroitin sulphate and dermatan sulphate side chains has been isolated and purified from a yolk sac tumour of the left ovary from a 23-year-old female. A monoclonal antibody, designated 2B1, was produced which reacted specifically with the intact molecule of the large proteoglycan and the chondroitinase ABC-treated core molecule. The localization of substances showing cross-reactivity to this antibody was studied in a variety of human tissues by means of indirect immunohistochemistry. The interstitial elements of nearly all tissues of a 5-month-old foetus were intensely reactive with the antibody, but in adult tissues structures that gave positive reactions were limited; only the perivascular and perimuscular fibrous elements were reactive, except for the aorta, which reacted extensively. In contrast, the interstitial elements of the carcinoma tissues tested were intensely reactive. Thus antibody 2B1 can be regarded as a useful tool for studies on the immunohistochemical localization of large proteoglycan in various human tissues.