Identification of human alpha-synuclein specific single chain antibodies

Parkinson's disease (PD) is a common neurodegenerative disease of unknown etiology. Evidence suggests a role for protein misfolding in disease pathogenesis. One pathologic feature observed in dopaminergic neurons is the intracytoplasmic eosinophilic inclusions known as Lewy bodies. One component of Lewy bodies, the presynaptic protein, alpha-synuclein forms oligomers and higher order aggregates and is proposed to be involved in dopaminergic neuronal death. In an effort to discriminate between alpha-synuclein conformational forms as well as design potential disruptors of pathogenic misfolding we panned a human phage antibody library for anti-synuclein single chain antibodies (scFvs). We identified six scFvs which recognize different conformers of alpha-synuclein in both an ELISA and Western blot analysis. These scFvs may further our understanding of alpha-synuclein's role in PD.     

Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor

We have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block alpha-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit alpha-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds alpha-BuTx with a forward rate constant of 0.98 X 10(5) M-1 s-1, d-tubocurarine (dTC) with a KD of (6.8 +/- 0.3) X 10(-8) M, and pancuronium with a KD of (1.9 +/- 1.0) X 10(-9) M. The site recognized by B mAbs binds alpha-BuTx with a forward rate constant of 9.3 X 10(5) M-1 s-1, dTC with a KD of (4.6 +/- 0.3) X 10(-6) M, and pancuronium with a KD of (9.3 +/- 0.8) X 10(-6) M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by alpha-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site.     

Recombinant single chain Fv antibodies specific for glycoprotein D of equid herpesvirus 1

Single chain Fv (scFv) molecules generated by phage-display technology represent a new and efficient tool in the research and diagnostics of infectious diseases. The recombinant glycoprotein D of Equid herpesvirus 1 was successfully expressed in E. coli cells. The protein was produced predominantly in soluble fraction and was then purified on a nickel-agarose column. The scFv antibodies against the glycoprotein were selected and several clones of glycoprotein D-specific scFv-antibodies were identified; t of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and used as reagent in an immunofluorescence test.     

Monoclonal antibodies specific for potato mop-top virus, and some properties of the coat protein

A method was devised which gave consistent yields (1–2 mg/kg leaves) of potato mop-top furovirus (PMTV) particles. Monoclonal antibodies (MAbs) were produced and some properties of 10 of them were studied. Four MAbs readily detected PMTV isolates from six countries in Northern Europe and Japan when the isolates were trapped with polyclonal antibody; and diagnostic tests based solely on MAbs (SCR 68 to coat plates and biotin- or enzyme-labelled SCR 69 to detect trapped virus) were devised. The pattern of reactions of the MAbs in ELISA and immunoblots suggested that they react with at least five different epitopes. PMTV coat protein preparations were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Three bands of 23.9 kd, 21.5 kd and 20.5 kd were visible in silver-stained gels and all three reacted with PMTV specific MAbs. The relative amounts of the three bands varied between different virus preparations, but the 21.5 kd band was usually the most abundant. The three bands were probably not produced by anomalous behaviour in SDS-PAGE. Moreover PMTV protein was readily degraded by trypsin treatment giving a band of 20.5 kd. Therefore the results suggest that PMTV coat protein sub-units are sensitive to degradation by plant proteases. At least two degraded forms were found when purified preparations were analysed by SDS-PAGE, and the undegraded protein was estimated to be 23.9 kd. The PMTV MAbs did not react in immunoblots with SDS-treated coat protein preparations of beet necrotic yellow vein furovirus or Indian peanut clump furovirus.     

Monoclonal antibodies reactive with the T cell receptor zeta chain: production and characterization using a new method

We have developed a novel method for the production and characterization of monoclonal antibodies reactive with lineage-restricted intracellular Ag. Using this technique, we have produced a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells. One of these antibodies, designated TIA-2, was found to react with greater than 98% of peripheral blood T lymphocytes. Immunoblotting experiments showed TIA-2 to recognize a 32 kd protein that was reduced to 16 kDa in the presence of 2-mercaptoethanol. Immunoprecipitates analyzed on non-reducing/reducing diagonal polyacrylamide gels showed the homodimeric structure recognized by TIA-2 to be associated with additional structures whose pattern closely resembled that of the T cell receptor complex. When immunoprecipitates formed using antibodies reactive with CD3 epsilon were immunoblotted with TIA-2, the homodimeric TCR zeta chain was specifically recognized. Using TIA-2 as a TCR zeta specific reagent, we show that whole cell expression of this TCR subunit is dramatically reduced following exposure to mAb reactive with CD3. mAb reactive with activating epitopes of CD2 were also capable of down-modulating the expression of TCR zeta, but to a lesser degree. Exposure to Con A or IL-2, on the other hand, did not reduce the whole cell expression of TCR zeta. Given the central importance of TCR zeta in the expression of a functionally competent Ag receptor, its reduced expression in response to certain activating stimuli is likely to play an important role in regulating T cell responsiveness.     

Monoclonal antibodies of IgA isotype specific for lipopolysaccharide of Salmonella enteritidis: production, purification, characterization and application as serotyping reagents

Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains.     

Monoclonal antibodies for the analysis and purification of isopentenyladenine cytokinins

A series of monoclonal antibodies (McAb) have been generated which show a high affinity for the cytokinin, isopentenyladenosine (2iPA). The McAb show different specificities to a range of cytokinins in a radioimmunoassay and are suitable for the analysis of isopentenyladenine (2iP) and related compounds in plant extracts. One McAb, MAC 160, has a high cross-reactivity with several cytokinins and is a good candidate for use in group separation or the analysis of a number of cytokinins simultaneously after separation. When coupled to Sepharose 4B affinity support, the McAb can also be used as immunoaffinity matrices to purify and resolve cytokinins from plant extracts. The McAb have been used to demonstrate the presence of concentrations of 2iP in tissues of the moss, Physcomitrella patens which exceed those in its culture medium.     

Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.     

Monoclonal antibodies specific for bovine pancreatic asparagine synthetase. Production and use in structural studies

Thirteen stable hybridoma cell lines producing monoclonal antibodies specific for asparagine synthetase were established and one monoclonal antibody was chosen to produce an immunoaffinity resin for the purification of asparagine synthetase. Bovine pancreatic asparagine synthetase was purified to a specific activity of 395 nmol of Asn produced/min/mg. Electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 54,000 polypeptide. Prior cross-linking with dimethyl suberimidate resulted in a band at Mr = 52,500 (monomer) and two additional bands at Mr = 97,000 and 98,000 (dimers), suggesting the possibility of a heterogeneous enzyme population with slight differences in subunit composition. The ratio of Gln-dependent and NH3-dependent asparagine synthetase activities was constant for immunoaffinity-purified enzyme, but the ratios of glutaminase activity to synthetase activities varied, suggesting separate aspartate and glutamine binding sites. The monoclonal antibodies were tested as inhibitors of the Gln-dependent and NH3-dependent asparagine synthetase activities as well as for inhibition of the glutaminase activity of the enzyme. Two antibodies inhibited Gln- and NH3-dependent synthesis of asparagine, but did not affect the glutaminase activity of immunoaffinity-purified asparagine synthetase. A third monoclonal antibody inhibited Gln-dependent synthesis of asparagine and glutaminase activity, but activated NH3-dependent asparagine synthetase activity. These data are discussed in terms of multiple substrate binding domains within the asparagine synthetase molecule.     

Effects of anti-idiotype vaccine on tumour growth and on production of soluble factors modulating cell-mediated immunity in vitro

Summary The previous observation, that single i.p. doses of a monoclonal anti-idiotypic antibody (MAIA) injected into BALB/c mice induced suppressor factors, was extended to multiple i.v. doses. These induced enhancing factors, which were produced in spleen cell cultures, required L3T4⁺ cells for their formation, lacked the IJ marker, and bound to anti-immunoglobulin, showing them to be antibodies. Selective immunoabsorption demonstrated two separate enhancing antibodies; both bound to MAIA but they had different affinities for specific and non-specific tumour antigens. Subsequently, single and multiple MAIA doses were tested in vivo for their effects on tumour growth. The single doses had variable effects depending on time of administration, and these effects were tumour-specific; the multiple doses strongly inhibited tumour growth when given before tumour challenge, but also had non-specific effects on another tumour as anticipated from the in vitro results.     

Monoclonal antibodies specific for high molecular weight form of human epidermal growth factor

Hybridomas that secrete monoclonal antibodies specific for the high molecular weight (HMW) form of human epidermal growth factor (hEGF) were established by fusing spleen cells obtained from mice immunized with purified urinary HMW-hEGF with myeloma P3 x 63Ag8.653. The resulting monoclonal antibodies were characterized basically into two groups. One group recognized both EGF and HMW-hEGF, while the other recognized HMW-hEGF specifically on radio immunoprecipitation. Surprisingly, the majority of the isolates was positive by western blotting. Utilizing these monoclonal antibodies for affinity chromatography, we purified HMW-hEGF successfully from urine. These antibodies may be an extraordinarily powerful tool for histological study related to both forms of EGF.     

Immunological properties of monoclonal antibodies specific for meningococcal polysaccharides: the protective capacity of IgM antibodies specific for polysaccharide group B

Two IgM monoclonal antibodies, MB32 and MB34 specific for meningococcal polysaccharide group B have been raised. Both were detectable by radioimmunoassay and agglutination, but only MB34 was effective in counter immunoelectrophoresis and complement fixation. MB34 was also far more potent than MB32 when tested for passive protection of mice infected with either Neisseria meningitidis group B or Escherichia coli K1. These data demonstrate that group B-specific antibodies do play a protective role in mice infected with these bacteria.     

Ribosome display: next-generation display technologies for production of antibodies in vitro

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.     

Ribosome display: next-generation display technologies for production of antibodies in vitro

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.     

Application of phage-displayed single chain antibodies in western blot

A phage display single chain fragment variable library constructed on pⅢ protein of M13 filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules. After four rounds of panning, there was a remarkable enrichment in the titer of bound phages. Twenty phage clones were selected from the last round and screened by means of phage-ELISA. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. The dilutions of antibody phages depend on the affinity between antibody-displayed phage particles and antigens.     

Characterization of two monoclonal antibodies specific for lacto-series type 1 chain Gal beta 1----3GlcNAc-terminal structures

Murine monoclonal antibodies, TE-1 and TE-3, generated by immunization with a biosynthetic reaction product containing a terminal Gal beta 1----3GlcNAc structure have been produced and found to react specifically with underivatized type 1 chain lacto-series carbohydrate structures. Detailed analysis of these antibodies, both IgM, indicates two differing classes of epitope specificity. Antibody TE-1 was found to bind preferentially to longer chain carbohydrate structures containing a terminal Gal beta 1----3GlcNAc disaccharide, indicating that optimal antibody binding involved more than recognition of this disaccharide. In contrast, antibody TE-3 was found to bind strongly carbohydrate structures containing terminal Gal beta 1----3GlcNAc structures irrespective of chain length. Modification of core chain structures by addition of fucose and/or sialic acid residues completely abolished antibody binding with either antibody. TLC immunostaining of neutral glycolipids isolated from a variety of human colonic adenocarcinoma cell lines indicated intensely stained bands, particularly with antibody TE-3, which correlated with the level of expression of type 1 chain based glycolipid derivatives. These antibodies are applied to the detailed study of the regulation of synthesis of lacto-series type 1 chain based carbohydrate structures.     

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.