Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans

Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.     

Production and characterization of monoclonal antibodies to two new mosquito Aedes aegypti salivary proteins

Mosquito salivary proteins, which are fundamental to the process of blood feeding, also facilitate disease transmission and cause allergic reactions. The identification and characterisation of these proteins have been hampered by the difficulty of obtaining them in purified form. In this report, we describe the production of mouse monoclonal antibodies (mAbs) against mosquito salivary proteins. BALB/c mice were immunised with Aedes aegypti saliva proteins. Hybridomas were produced by fusion of spleen cells with a mouse myeloma cell line. Positive clones were selected using a saliva-capture ELISA and further identified using immunoblotting. Three mAbs reacted with a 44 kDa protein (Aed a X1) in the saliva-immunoblotting, and did not react with 2 recombinant salivary proteins, rAed a 1 (apyrase) and rAed a 2 (D7), in both immunoblotting and ELISA. Two other mAbs reacted with a 37 kDa protein in saliva-immunoblotting, but failed to react with the 37 kDa rAed a 2 in either immunoblotting or ELISA, suggesting that there is a second 37 kDa protein (Aed a X2) which is recognised by the two mAbs. The 44 kDa and 37 kDa proteins have not been previously identified. These mAbs provide a means to purify proteins, to isolate new genes from the salivary gland cDNA library, and to standardise mosquito extracts, facilitating studies of disease transmission by mosquitoes and of mosquito allergy.     

Production and preliminary characterization of monoclonal antibodies to rat plasma fibronectin

We have produced several monoclonal antibodies which appear to be directed against different antigenic determinants of rat plasma fibronectin. Fibronectin was purified from rat plasma by affinity chromatography on gelatin-Sepharose and arginine-Sepharose columns. Mice were immunized and hybridomas were prepared by fusing spleen cells with Sp2/0-Ag14 myeloma cells using poly(ethylene glycol). Three hybridomas (RFN1, RFN2 and RFN3) were selected for characterization. All are IgG molecules, one is IgG2a, one IgG2b and one IgG1. Titers of ascites fluids produced using these hybridomas range from 102 400 to greater than 409 600. The antibodies cross-reacted to different degrees with human fibronectin. Rat fibronectin was radioactively labeled and cleaved using human polymorphonuclear leukocyte elastase. Four major peptides, Mr approx. 160 000, 140 000, 60 000 and 30 000 were produced. Each of the hybridoma antibodies immunoprecipitated different elastase peptides. RFN1 precipitated the Mr 160 000 peptide, RFN2 precipitated the Mr 160 000 and the Mr 140 000 peptide and RFN3 precipitated the Mr 60 000 peptide as well as low molecular weight material migrating at the buffer front. These antibodies will be useful in studies of structure/function relationships of rat fibronectin.     

Production and characterization of two monoclonal antibodies against human myeloperoxidase

Two monoclonal antibodies against human myeloperoxidase, designated 3-2H3 and 4-2C11, were produced and characterized. Both bound to the native enzyme, but neither bound to the denatured enzyme or to its two denatured subunits. 4-2C11 bound to the three types of leukocyte myeloperoxidase, I, II, and III, as well as to the four types of myeloid leukemia HL-60 cell myeloperoxidase, IA, IB, II, and III. 3-2H3 did not bind to enzyme IB, but bound to the other types of leukocyte and HL-60 cell enzymes. On incubation with myeloperoxidase III, 4-2C11 inhibited the enzyme activity, but 3-2H3 did not. Both antibodies belong to the IgG1 subclass.     

Production and characterization of monoclonal antibodies to Aeromonas sobria surface antigens

A panel of eight murine monoclonal antibodies (Mabs) was raised against the surface antigens of Aeromonas sobria isolated from a patient with diarrhoea. Antibodies to the LPS core and O-antigen and to protein antigens were generated. Three Mabs of the IgM isotype against either protein or LPS agglutinated 34/75 Aeromonas isolates from clinical and environmental sources. Similar numbers of A. hydrophila, A. sobria and A. caviae isolates were agglutinated. The Mabs were screened for their ability to inhibit A. sobria adhesion to HEp-2 cells, and haemagglutination (HA). Two Mabs directed against conformationally dependent epitopes on a 43-kDa protein blocked both functions. Of the anti-LPS Mabs one blocked adhesion only, and another blocked HA but not adhesion. Immunoprecipitation studies suggested that LPS-protein complexes may be involved in these potential virulence functions of A. sobria.     

Production and characterization of monoclonal antibodies to Aeromanas sobria surface antigens

Abstract A panel of eight murine monoclonal antibodies (Mabs) was raised against the surface antigens of Aeromonas sobria isolated from a patient with diarrhoea. Antibodies to the LPS core and O-antigen and to protein antigens were generated. Three Mabs of the IgM isotype against either protein or LPS agglutinated 34/75 Aeromonas isolates from clinical and environmental sources. Similar numbers of A. hydrophila, A. sobria and A. caviae isolates were agglutinated. The Mabs were screened for their ability to inhibit A. sobria adhesion to HEp-2 cells, and haemagglutination (HA). Two Mabs directed against conformationally dependent epitopes on a 43-kDa protein blocked both functions. Of the anti-LPS Mabs one blocked adhesion only, and another blocked HA but not adhesion. Immunoprecipitation studies suggested that LPS-protein complexes may be involved in these potential virulence functions of A. sobria .     

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

Production and characterization of two monoclonal antibodies specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.     

Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase.

A library of monoclonal antibodies directed against individual proteins of the rabbit poxvirus (RPV) virion within a complex immunogenic mixture has been generated through the use of in vivo and in vitro immunization regimens. The relative efficacies of the two procedures were compared. Based on immunoblot analysis, the in vitro immunization regimen led both to a wider variety of monoclonal antibodies to different proteins and to a larger number of antibodies directed against proteins of higher molecular weights. Each method, however, has advantages, and the two procedures appear to be complementary. A simple method to recognize antibodies directed against the virion DNA-directed RNA polymerase was developed. Monoclonal antibodies directed against two subunits (137 and 34 kilodaltons [kDa]) of the RNA polymerase were identified and used to study the biogenesis of the enzyme and to map the two corresponding genes within the viral genome by using an RPV DNA library cloned into the lambda gtll expression vector. Both proteins are synthesized late in the infectious cycle and are restricted totally to the cytoplasm. Preliminary mapping data place the genes encoding the 137-kDa protein within the HindIII H fragment, whereas the gene for the 34-kDa protein is located within the left most region of the HindIII A fragment.     

Generation of monoclonal antibodies directed against organ-specific endothelial cell surface determinants.

Organ-specific determinants expressed on the luminal surface of vascular endothelia are often unstable when cells are removed from their normal tissue environment and grown in culture. Unspecific endothelial cells of large vessel origin [e.g., bovine aorta (BAEC)] can be modulated to express and preserve such determinants when they are grown on the extracellular matrix of the desired organ. Lung matrix-modulated BAEC were used here to generate MAb against lung-specific vascular endothelia. Immunization was accomplished with outside-out membrane vesicles obtained by incubating BAEC monolayers grown on lung matrix with a low-strength paraformaldehyde solution. In four of the six fusions performed, this active immunization was preceded by passive immunization with mouse antiserum directed against membrane vesicles from BAEC grown on plastic. Among the growing hybrids, 7.6% secreted MAb that bound efficiently to both BAEC grown on lung-derived matrix and BAEC grown on plastic, while 3.5% (50) secreted MAb that bound primarily to BAEC grown on lung matrix. The fusion data show that only a passive/active immunization protocol yielded MAb directed against lung-specific endothelia. For example, MAb 6D3 and 5F5 selectively recognized endothelia from small- and medium-sized venules of bovine lungs, but failed to react with endothelial cells in other organs and tissues.     

Production and characterization of monoclonal antibodies to N-acetyl-aspartyl-glutamate

N-acetyl-aspartyl-glutamate (NAAG) is a putative neuromodulator/neurotransmitter in the mammalian nervous system. Immunohistochemical studies with polyclonal NAAG antisera have revealed immunoreactive neurons and processes in several brain regions. However, these antisera crossreact to some degree with N-acetyl-aspartate (NAA), which is present in mM concentrations in brain, prompting the development of monoclonal antibodies (MAb) more specific for NAAG. By fusing spleen lymphocytes obtained from BALB/c mice pre-immunized with NAAG covalently linked to bovine serum albumin by carbodiimide with SP2/0-Ag 14 mouse myeloma cells, we produced three IgG2a (kappa) MAb which specifically reacted with NAAG. These MAb exhibited negligible crossreactivity with NAA or with structurally similar peptides, as shown by solid-phase radioimmunoassay. Antibody activity was absorbed out selectively by both NAAG-thyroglobulin conjugate and free NAAG. These MAb stained many nuclei of the medulla-pons and midbrain, mitral cells in the olfactory bulb, pyramidal neurons in sensorimotor cortex, locus ceruleus, and several cholinergic cranial nuclei. The staining pattern strongly correlated with NAAG levels determined by HPLC. Monoclonal antibodies significantly enhanced sensitivity of staining, allowing visualization of dorsal horn neurons in spinal cord, which were not readily detectable with polyclonal antiserum. Availability of these MAb now facilitates further clarification of the role of NAAG in the brain.     

Production and characterization of monoclonal antibodies against aflatoxin M1.

By using an indirect enzyme-linked immunosorbent assay, four monoclonal antibodies were selected after fusion of mouse P3-NS1-Ag4-1 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin M1 (AFM1) conjugated to bovine serum albumin. Two of these antibodies were found to be specific for AFM1 and were designated AMW-1 and AMW-4. The specificities of AMW-1, which had higher affinity to AFM1, were determined by a competitive direct enzyme-linked immunosorbent assay with peroxidase-AFM1 as the marker. The relative cross-reactivity of each toxin (relative to AFM1) with AMW-1, as determined by the amount of aflatoxin necessary to cause 50% inhibition of enzyme activity, was 12, greater than 40, 12, and greater than 40 for B1, B2, G1, and G2, respectively.     

Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen.

Several recently isolated monoclonal antibodies which reacted with simian virus 40 T antigens also reacted with proteins found in uninfected and untransformed cells. The proteins were different from each other, PAb419 reacting with a 35,000-molecular-weight protein, PAb427 reacting with a 75,000-molecular-weight phosphoprotein, PAb405 reacting with a 150,000-molecular-weight phosphoprotein, and PAb204 reacting with a 68,000-molecular-weight protein. It is suggested that although some of these cross-reactions may be fortuitous, they may, as an alternative, reflect similarities of shape and perhaps function between domains of the viral T antigen and the relevant host proteins.     

Purification and preliminary characterization of sucrose-phosphate synthase using monoclonal antibodies

Monoclonal antibodies specific for sucrose phosphate synthase (SPS; EC 2.4.1.14) have been obtained for the first time. Three independent clones have been isolated which inhibited spinach (Spinacia oleracea L.) leaf SPS activity and facilitated the enzyme purification by immunoprecipitation. All three clones were specific for the spinach enzyme but neither inhibited nor precipitated the SPS present in tissue extracts of maize (Zea mays L.), barley (Hordeum vulgare L.), soybean (Glycine max L.), and sugar beet (Beta vulgaris L.). The inhibition of SPS activity by all three clones was reversible in the presence of UDPG, suggesting the presence of an epitope at the substrate-binding site. Immunoprecipitates of active enzyme preparations consistently revealed the presence of a 120 kilodalton polypeptide, indicating that the enzyme may be a homotetramer with a native molecular weight of about 480 kilodaltons. The occasional appearance of a 52 kilodalton polypeptide in the immunoprecipitates of some enzyme preparations was not the result of proteolysis, was not necessary for enzyme activity, and did not contain an antigenic site as revealed by Western blotting experiments. All three antibodies bind weakly to the SDS denatured 120 kilodalton subunit bound to nitrocellulose. The specific activity of the purified spinach enzyme was determined for the first time to be approximately 150 units per milligram SPS protein (pH 7.5 and 25°C) based on quantitative immunoprecipitation of the enzyme.     

Production and characterization of monoclonal antibodies against the two subunits proteins B1 and B2 of Escherichia coli ribonucleotide reductase

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, named protein B1 (170 000) and protein B2 (87 000). We purified and characterized five monoclonal antibodies against B1 and three against B2 from hybridomas obtained by fusion of spleen cells from immunized mice and the myeloma cell line P3-X63Ag8. All are of the IgG1 class with a high affinity for the antigen with dissociation constants in the nanomolar range. Four of the anti-B1 monoclonals and all three anti-B2 monoclonals neutralize reductase activity while one anti-B1 monoclonal binds tightly to B1 without affecting its activity. Fab fragments prepared from three anti-B1 monoclonals had similar dissociation constants. The anti-B1 monoclonals interacted with separate epitopes while two of the anti-B2 monoclonals appeared to react with the same epitope. In the case of B1, various allosteric states of the protein induced by binding of effectors had no apparent effect on the interaction with monoclonals, nor did their binding prevent subsequent binding of effectors. With B2, binding of monoclonals did not affect the typical electron paramagnetic resonance spectrum of the protein and thus did not involve either the tyrosyl free radical or the iron center of B2. All neutralizing antibodies interfered with the interaction between the two subunits, explaining their effect on enzyme activity, since active ribonucleotide reductase consists of a B1-B2 complex.     

Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid involved in multiple physiological processes. Importantly, dysregulated S1P levels are associated with several pathologies, including cardiovascular and inflammatory diseases and cancer. This report describes the successful production and characterization of a murine monoclonal antibody, LT1002, directed against S1P, using novel immunization and screening methods applied to bioactive lipids. We also report the successful generation of LT1009, the humanized variant of LT1002, for potential clinical use. Both LT1002 and LT1009 have high affinity and specificity for S1P and do not cross-react with structurally related lipids. Using an in vitro bioassay, LT1002 and LT1009 were effective in blocking S1P-mediated release of the pro-angiogenic and prometastatic cytokine, interleukin-8, from human ovarian carcinoma cells, showing that both antibodies can out-compete S1P receptors in binding to S1P. In vivo anti-angiogenic activity of all antibody variants was demonstrated using the murine choroidal neovascularization model. Importantly, intravenous administration of the antibodies showed a marked effect on lymphocyte trafficking. The resulting lead candidate, LT1009, has been formulated for Phase 1 clinical trials in cancer and age-related macular degeneration. The anti-S1P antibody shows promise as a novel, first-in-class therapeutic acting as a “molecular sponge” to selectively deplete S1P from blood and other compartments where pathological S1P levels have been implicated in disease progression or in disorders where immune modulation may be beneficial.