Characterisation of a monoclonal antibody against native human type I collagen

A monoclonal antibody against a pepsin-soluble mammalian type I collagen has been produced. This antibody, subclass IgG1, kappa, was specific for type I collagen and did not cross-react with a range of other collagen types or connective tissue proteins. The epitope recognized by the antibody was dependent upon an intact triple-helical structure for the collagen, and was shown by rotary shadowing and by immunoblotting of collagenase-derived fragments to be near the C-terminal of the pepsin-soluble collagen. Although the antibody had a low affinity, with Kd = 4 x 10(-7) M, it could be used for immunohistology of tissue sections and for studies of collagen produced by cells in culture. The antibody, which was raised against human collagen, also recognized type I collagens from certain other species, including calf, pig, sheep, goat and dog.     

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.     

Development and characterization of monoclonal antibodies to human type III procollagen

Hybridomas which secrete monoclonal antibodies against human type III procollagen have been developed. By an enzyme-linked immunosorbent assay, three of the monoclonal antibodies have been determined to be against non-helical extensions of the molecules while two of the antibodies are against helical portion of the molecules which is sensitive to bacterial collagenase action. These findings have been further confirmed by carrying out immuno-reaction of the pro α-chains transferred on nitrocellulose paper from sodium dodecyl sulfate polyacrylamide gels. These monoclonal antibodies have been found to be suitable reagents for immunohistochemical studies as well as for immunoassays of type III procollagen and collagen.     

Production and characterization of a monoclonal antibody to dog hepatic lipase

Partially purified dog hepatic lipase was used as antigen to produce monoclonal antibodies in mice. In addition to enzyme-linked immunosorbent assay (ELISA), a reliable and efficient procedure for screening antibodies reacting to hepatic lipase has been developed. A method to distinguish antibodies directing to active site or non-active site epitopes has also been described. We obtained three positive clones that survived after subcloning and expansion. All three monoclonal antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. Specificity of monoclonal antibody LDHL No. 537 to dog hepatic lipase was demonstrated by passing post-heparin plasma through its immunoaffinity column. Only dog hepatic lipase was removed by LDHL No. 537 from post-heparin plasma. The immunoaffinity chromatography also demonstrated the co-existence of three enzyme activities (mono- and triacylglycerol lipase and phospholipase A1) on the dog hepatic lipase molecule. The subunit weight of dog hepatic lipase has been estimated at 57500 +/- 600 (n=3) by using immunoaffinity chromatography and the combination of immunoprecipitation and autoradiography methods.     

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 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.     

The localization and secretion of type IV collagen in synovial capillaries by immunohistochemistry using a monoclonal antibody against human type IV collagen

The localization and the secretion of type IV collagen in synovial capillaries have been investigated by detecting the antigenic determinant of the major triple helix of human type IV collagen. Type IV collagen was indicated to be localized mainly in the lamina densa of basement membranes (BM) and to be secreted by both endothelial cells and pericytes. The pericytes secreted this collagen to both surfaces facing endothelial cells and the interstitial connective tissue. On the contrary, the direction of type IV collagen secretion by the endothelial cells was strictly confined to one side, namely towards the surface facing the BM. The absence of the antigenic determinant in rough endoplasmic reticulum and Golgi apparatus of the endothelial cells and pericytes indicated that the major triple helix of type IV collagen is mainly formed in the secretory vesicles after budding from the Golgi apparatus.     

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.     

Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody

The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.     

Production and characterization of a unique monoclonal antibody against human B cells (33.2.1)

A monoclonal antibody specific for a polymorphic antigen on human B cells (33.2.1) was produced and characterized. By flow cytometry, 33.2.1 was found to react with peripheral blood B cells, monocytes, and Epstein-Barr virus (EBV)-transformed B-cell lines, but not with peripheral blood T cells, mitogen-activated T cells, or allo- or autoactivated T cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that 33.2.1 recognizes a noncovalently bound bimolecular complex composed of an alpha chain of about 32 kDa and a beta chain of about 28 kDa. The failure of anti-HLA-DR, anti-Leu-10, and anti-HLA-DC1 to remove the 33.2.1 antigen by sequential immunoprecipitation suggests that 33.2.1 recognizes a distinct molecule rather than a different epitope on either HLA-DR or DS/DC/MB. In T-cell-independent B-cell activation systems, preincubation with 33.2.1 markedly inhibited RNA and DNA synthesis as well as polyclonal Ig production. In contrast, anti-HLA-DR was inhibitory only when it was present throughout the culture, but not when it was used for preincubation. Anti-Leu-10 led to only moderate inhibition. These results suggest that 33.2.1 recognizes a unique Ia-like antigen critical for B-cell activation.     

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.     

Occurrence of collagen and proteoglycan forms of type IX collagen in chick embryo cartilage. Production and characterization of a collagen form-specific antibody.

Type IX collagen from chick embryonic cartilage is a proteoglycan bearing a single chondroitin sulfate chain covalently linked to the alpha 2(IX) polypeptide chain. We have isolated type IX collagen metabolically labeled with [3H]proline using an antibody to type IX collagen and have found that the molecule is synthesized in two forms, a collagen form (COLIX) and a proteoglycan form (PGIX). In cultured chondrocytes, the two forms of type IX collagen showed a different ability to be deposited in the matrix. We have suggested the possibility that both forms may arise from an alternative substitution of a chondroitin sulfate chain to the NC3 domain of the alpha 2(IX) chain. Based on the reported amino acid sequence at the NC3 domain of alpha 2(IX), we have synthesized undecapeptides containing the sequence around the glycosaminoglycan attachment site of the alpha 2(IX) chain. Antibody against the peptide, which was raised in rabbit, only recognized COLIX and made it possible to distinguish COLIX from PGIX. Evidence shows that this could be due to a difference in antigenicity of the NC3 domain of the alpha 2(IX) chain between COLIX and PGIX caused by the substitution of a chondroitin sulfate chain to the serine residue in this domain. Therefore, this antibody may be useful as a probe for studies on the functions of glycosaminoglycan substitution in type IX collagen.     

Preparation and characterization of a human aurora-A kinase monoclonal antibody

We have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an Elisa (Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation.     

Production and characterization of monoclonal antibodies against human type K pyruvate kinase

K-type pyruvate kinase was purified from human kidney by immunoadsorbant chromatography. Monoclonal antibodies secreting hybridomas were made using conventional techniques. Two clones were established which produced antibodies against K-type not cross-reacting with the other pyruvate kinase isoenzymes, named the M, L and R-types. The specificity of the monoclonal antibodies was proven by enzyme-linked immunosorbent assay, immunoprecipitation and immunoblotting experiments. The M- and K-isoenzymes are produced from the same gene probably by alternative splicing, and all differences between both enzymes originate from one exon coding for 45 amino acids (Noguchi et al. J. Biol. Chem. 261, 13807-13812 (1986]. The monoclonal antibodies are specific for K-type under denaturing conditions. Thus, it is likely that these antibodies recognize (a) continuous epitope(s), of which at least some amino acids are coded in the K-specific exon. The monoclonal antibodies could be successfully used in immunohistochemical studies. Neurons and astrocytes in brain, Kupffer cells in liver, connective tissue cells and vascular smooth muscle cells showed immunoreactivity. However, striated muscle cells in skeletal muscle and heart and hepatocytes were not immunoreactive. Other types of glial cells, e.g., oligodendrocytes and microglia, so far studied, showed no reaction either.     

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.     

Isolation and characterization of an anti-peptide monoclonal antibody to human erythropoietin

A site-specific monoclonal antibody to human erythropoietin has been developed. It is secreted by a hybridoma cell line derived from the fusion of murine myeloma cells with the splenocytes of a mouse that had been immunized with a 26-residue synthetic peptide antigen homologous to the amino-terminal sequence of the hormone. The antibody binds specifically to peptide, 125I-erythropoietin, and biologically active erythropoietin. The equilibrium dissociation constants of the antibody-erythropoietin and the antibody-peptide interactions are identical, Kd = 6.7 X 10(-9) M, suggesting strong conformational similarity or identity of the epitope as expressed on the peptide and the hormone. Immune complexes formed between the antibody and either human or rat erythropoietin exhibit full biologic activity. However, the antibody does not recognize the baboon, sheep, or canine hormones, indicating antigenic differences or structural variation among these erythropoietins. These results indicate that the amino-terminal region of erythropoietin is not involved in receptor binding. Furthermore, they form a basis for the study of the structure and function of the hormone using anti-peptide antibodies.     

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 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.