Monoclonal antibodies to queuine

Monoclonal antibodies specific for queuine have been prepared. Synthetic 9-(5-carboxypentyl)queuine (cp9Q) was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), and the conjugate was used to immunize BALB/c mice by intraperitoneal and subcutaneous injection. Monoclonal antibodies were subsequently obtained by fusion of spleen cells and the mouse myeloma cell line X63Ag8U1. An enzyme-linked immunoabsorbent assay (ELISA) using o-phenylenediamine as peroxidase substrate was used for screening of clones and characterization of antibodies. Inhibition experiments with various homologous nucleosides revealed that the monoclonal antibody designated as 2D8E6 has no cross-reactivity with guanosine, adenosine or 7-methylguanosine.     

Monoclonal antibodies to mammalian neurofilaments

Hybrid cell lines secreting monoclonal antibodies against mammalian neurofilaments have been prepared. An improved protocol for the preparation of neurofilaments and methods for the identification and isolation of such hybridomas are presented. The antibodies produced specifically stain only neuronal cell types in both cerebellar sections and culture.     

Monoclonal antibodies to leucine enkephalin

Monoclonal antibodies to leucine enkephalin have been produced after fusion of mouse myeloma cells with spleen cells from hyper-immune mice. Hybrid clones 2D1 and SL1 were characterised using radioimmunoassay and an enzyme-linked immunosorbent assay. The antibody 2D1 was of low affinity and showed a maximum sensitivity of 0.1ng. The antibody binds equally well to the sulphated leucine enkephalin and to methionine enkephalin. It does not cross-react with dynorphin, methionine enkephalin-arg-phe or oxidised methionine enkephalin. The hybrid clone SL1 appears to be specific for leucine enkephalin. Preliminary immunocytochemical studies have shown that both antibodies bind specifically to leucine enkephalin in defined areas of the central nervous system.     

Monoclonal antibodies

The hybridoma technology developed by K?hler and Milstein has initiated a new era in biological sciences. In the last decade the possibility of generating limited amounts of monoclonal antibodies of predefined specificity has become a routine method in many laboratories throughout the world. The constant quality of various antibody preparations from 1 hybridoma cell line represents another important advantage of this method. Apart from the use for several purposes, e.g. HLA-typing, differentiation of lymphocyte subpopulations and blood group antigens, monoclonal antibodies play an important role in the determination of various tumor markers. In most modern immunoassays monoclonal antibodies are used. Furthermore, there is nowadays a limited experience concerning the in vivo use of monoclonal antibodies in malignant disease. Radiolabelled antibody immunodetection has been applied e.g. in colorectal and testicular tumors for the detection of tumor metastases. The therapeutic use of monoclonal antibodies has been reported in some patients with tumors of the hemopoietic system. The production of new murine and human monoclonal antibodies against various tumor types is subject of current investigations. The aim of these efforts is the development of monoclonal antibodies suitable for in vitro tumor diagnosis and application in vivo.     

Monoclonal antibodies to yeast catalase T

Hybridoma cell lines secreting antibodies directed against $\text{Saccharomyces cerevisiae}$ catalase T were constructed by fusing spleen cells of mice immunized with catalase T with P3×63Ag8 mouse myeloma cells. Culture supernatants were assayed for specific antibodies by incubation with ³⁵S-labelled yeast extracts, adsorption of the immune complexes to Protein A — carrying $\text{Staphylococus aureus}$ cells and analysis of the adsorbed yeast proteins by sodium dodecylsulfate gel electrophoresis. Two hybrid clones were isolated mediating adsorption of a protein with electrophoretic mobility of catalase T; one of them, showing considerably higher activity, was characterized further. Anti-bodies produced by this clone belong to the IgG class of immuniglobulins; they can be used for immunoadsorption, but not for direct immunoprecipitation and recognize authentic catalase T as well as catalase T apoprotein.     

Monoclonal antibodies to beet soil-borne virus

Four monoclonal antibodies (MCA) were obtained to the ‘Ahlum’ serotype of beet soil-borne virus (BSBV). On ELISA plates which had been precoated with polyclonal antibodies (PCA) all four MCA detected this serotype with a higher sensitivity than alkaline phosphatase-labelled PCA. Three of the MCA were specific for the ‘Ahlum’ serotype, but a fourth one also detected the distantly related ‘Wierthe’ serotype. Cross-reactions with wheat soil-borne or oat golden stripe furoviruses were not observed. One of the MCA reacted with an epitope which is exposed along the entire length of the BSBV particles, whereas two others were specific for epitopes which are exposed on one particle extremity only. Although these latter two epitopes occur apparently on the same extremity of the particles, they seem to be different, because one is found only on the particles of the ‘Ahlum’ serotype, whereas the other one is present also on the particles of the ‘Wierthe’ serotype. The fourth MCA is specific for a cryptotope which is not exposed on the intact virus particles, but presumably on some degradation product or precursor of the viral coat protein present in crude sap preparations. All four epitopes are SDS-labile; they are not detected on denatured viral coat protein on Western blots.     

Monoclonal antibodies to p-coumarate

p-Coumaric acid is one of the predominant phenolic acids acylating the cell walls of grasses; p-coumarates are mainly esterified by lignins and arabinoxylans. Here we describe the production and characterisation of two monoclonal antibodies against p-coumarates.The 5-O-pCou-Ara(1 → 4)Xyl was chemically synthesized and conjugated to a carrier protein. Two interesting antibodies were obtained, hereinafter named INRA-COU1 and INRA-COU2. The specificity of these monoclonal antibodies has been evaluated using competitive-inhibition assays with different oligosaccharides and phenolic compounds. INRA-COU1, recognized free p-coumaric acid or p-coumarate esters. INRA-COU1 did not react with any of the other hydroxycinnamic acids and related compounds found in plants. INRA-COU2, only recognizes esterified p-coumarate. These antibodies were used to study the localization of p-coumarates in the cell walls of grasses. Immunocytochemical analyses indicated noticeable amounts of p-coumarate in the cell walls of the aleurone layer of wheat grain, in the epiderm of cereal straw, and in the exoderm of wheat root.The use of these antibodies will contribute to a better understanding of the organisation and developmental dynamics of cell walls in Graminaceae.     

Monoclonal antibodies to Legionella pneumophila cytolysin

The fusion of spleen cells, taken from BALB/c mice immunized with the purified preparation of L. pneumophila cytolysin, with cells Sp2/0 and NP has been carried out. As a result, hybridoma cells producing IgG1, IgG3 and IgM antibodies to this protein have been obtained. All monoclonal antibodies (McAb) thus obtained react with L. pneumophila strain lysates in the precipitation test, while IgG3 and IgM antibodies react with erythrocyte diagnostic agents prepared from the lysate of L. pneumophila cells in the hemagglutination test. In the Western blot assay, McAb react with the 37 KD protein (cytolysin) and a number of other proteins from L. pneumophila cultures and L. pneumophila cell lysate, but do not react with the species-specific protein with a molecular weight of 29 KD, contained in the outer membrane of L. pneumophila, as well as with other species: L. bozemanii, L. dumoffii, L. longbeachae, L. micdadei. The possibility of using these McAb conjugated with FITC and peroxidase for the rapid diagnosis of Legionella infection is shown.     

Monoclonal antibodies specific to plant tubulin

Summary Tubulin was isolated from mung bean seedling by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4 B) and ion exchange (DEAE-Sephacel) chromatography. Using SDS-PAGE together with blotting with subunit-specific antitubulins, mung bean tubulin has been shown to consist of two -tubulin subunits, MBT₂ and MBT₃, of which MBT₃ is a minor component, and one -tubulin, MBT₁.Monoclonal antibodies were produced by fusing mouse myeloma cells and spleen cells from a Balb/c mouse immunized with mung bean tubulin. Antibody producing cell lines were identified by an ELISA assay and immunofluorescence microscopy and subsequently cloned by limiting dilution.The properties of monoclonal antibody (K₄E₇G₃) were examined by Western blot analysis and indirect immunofluorescence studies. K₄E₇G₃ reacts with MBT₂ and MBT₃ -tubulin subunits of mung bean tubulin, but not with MBT₁ -tubulin nor with the - and -subunits of sheep brain tubulin. Peptide fragments transferred onto nitrocellulose papers were treated with K₄E₇G₃ and with other monoclonal antibodies that are known to be specific to the -subunit of yeast tubulin and - or -subunit of mammalian brain tubulin. MBT₂ and MBT₃ are shown to be similar but not identical and are quite different from MBT₁ and the -subunit of sheep brain tubulin. K₄E₇G₃ reacts with peptide fragments in MBT₂ and MBT₃ that are not found in digests of brain tubulin, and that are either not reactive or only weakly reactive to the antibodies to yeast and brain -tubulin. It is concluded that K₄E₇G₃ and another monoclonal antibody, K₂D₇B₈, which has similar properties, are relatively specific for plant -tubulin.In indirect immunofluorescence studies on a wide range of plant cells, the epitopes recognised by these monoclonal antibodies are shown to be present in all types of microtubule array that were investigated. The spindle, preprophase band, phragmoplast and interphase microtubules were clearly observed in onion and mung bean root tip cells. Reactions with spindle microtubules ofFunaria spore mother cells and with the blepharoplast and flagella microtubules of fern spermatozoa are also seen. However, studies using several animal cell lines have shown that K₄E₇G₃ and K₂D₇B₈ do not give positive immunofluorescent localization of animal microtubules, correlating with the inability of K₄E₇G₃ to react with brain tubulin subunits on Western blot analysis.     

Monoclonal antibodies to tissue-specific chromatin proteins

Antisera raised in mice to chromatins from different tissues of the chicken reacted preferentially with the chromatin type that was used for immunization. This tissue specificity was also evident in the spectrum of monoclonal antibodies generated when mice were immunized with erythrocyte chromatin. Three erythroid-specific antigens and one antigen that was present in a number of chicken tissues were characterized in further detail. These antigens, which comprised less than 0.1% of the erythrocyte chromatin proteins, were nuclear localized although three were also detected in the cytoplasm. Two of the erythroid-specific antigens existed as multiple polypeptides in isolated chromatin. The multiple chromatin forms of one antigen were derived from a precursor protein that was selectively cleaved within 1 min after erythrocyte lysis. Analysis of this antigen in extracts from erythrocytes and reticulocytes indicated that the cleavage of the precursor protein was developmentally regulated in vivo.     

Monoclonal anti-idiotypic antibodies to opioid receptors

Two monoclonal anti-idiotypic antibodies (anti-Id-135 and anti-Id-14, both of the IgM class) which interact with the binding site of opioid receptors were generated. A monoclonal anti-beta-endorphin antibody (3-E7) which displays binding characteristics for opioid ligands similar to opioid receptors served as the antigen (Gramsch, C., Meo, T., Riethmüller, G., and Herz, A., (1983) J. Neurochem. 40, 1220-1226; Meo, T., Gramsch, C., Inan, R., H?llt, V., Weber, E., Herz, A., and Riethmüller, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4048-4088) and the hybridomas obtained were screened for anti-idiotypic antibodies with Fab fragments of 3-E7. The anti-idiotypes were then screened for opioid binding to rat brain membrane receptors, yielding several positive clones two of which were more intensively studied. Both anti-idiotypic antibodies were about equally potent in displacing the mu- and delta-opioid receptor ligands [3H]dihydromorphine, 125I-labeled beta-endorphin, [D-Ala2, D-Leu5-3H]enkephalin and [3H]naloxone from rat brain membrane opioid receptors; no interaction was observed with the kappa-ligands [3H]ethylketazocine or [3H]bremazocine. The anti-idiotypic antibodies were able to precipitate [3H] diprenorphine binding sites from solubilized opioid receptor preparations. In addition, both antibodies showed opioid antagonistic properties as demonstrated by their abilities to block the inhibitory effect of [D-Ala2, D-Leu5-3H]enkephalin on prostaglandin E1-stimulated cAMP accumulation in NG 108-15 hybrid cells. Our findings demonstrate the successful generation of monoclonal antibodies interacting with membrane-bound and solubilized opioid receptors of the mu- and delta-type.     

Monoclonal antibodies to human urinary thrombopoietin

Monoclonal antibodies (MA) to a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) were obtained from hybridomas derived from the fusion of P3 X 63/Ag 8 cells and spleen cells from TSF-immunized BALB/c mice. The immunizing protein was a partially purified TSF-rich preparation from the urine of a thrombocytopenic patient, and was shown to stimulate platelet production in rebound-thrombocytotic mice; i.e., platelet counts of recipient mice were increased to 133% of control and the values for percentage 35S incorporation into platelets were elevated to 225% of control. Media from several hybrid cultures were tested in a microantibody detection technique that measured the binding of MA to a 125I-purified TSF preparation from human embryonic kidney (HEK) cells. The immune complex was precipitated by the addition of goat anti-mouse IgG serum and centrifugation. One clone gave 25% binding of 125I-TSF after a sevenfold dilution of the medium. This cell line was recloned and four of the subclones produced MA that gave even greater binding capacities. Hybridized cells were injected into "pristane-primed" mice and the antibodies produced in the ascites fluid were also shown to bind the 125I-TSF. Compared to the results of normal mouse serum, ascites fluid containing MA was shown to bind the unlabeled TSF from HEK cells. The TSF activity was significantly reduced in the supernatant fluid after precipitating the TSF-anti-TSF immune complex by a second antibody when tested in an immunothrombocythemic mouse assay. After SDS-PAGE, the precipitate from this TSF-MA conjugate showed that the antiserum bound a single 32,000 mol wt component, indicating the monospecificity of the MA. MA directed toward human TSF will allow studies that were not previously possible.     

Monoclonal antibodies to cruciform DNA structures

Two monoclonal antibodies, 2D3 and 4B4, have been raised against a cruciform structure in a heteroduplex DNA molecule. Antibody binding to DNA fragments was determined by a radioimmunoassay in which DNA--antibody complexes were separated from unbound DNA by acrylamide gel electrophoresis. These antibodies seem to recognize conformational determinants specific to cruciform structures. 2D3 and 4B4 antibodies do not bind to linear double-stranded homoduplex DNA fragments, linear single-stranded DNA or single-stranded simian virus 40 DNA containing a stem--loop structure, but do bind to the original cruciform and to a different cruciform with one shortened arm. 2D3 also bound to a T-shaped double-stranded DNA molecule, while 4B4 binding to this structure was weak. The monoclonal antibodies 2D3 and 4B4 were found to be immunoglobulin G1 and immunoglobulin M, respectively.     

Monoclonal antibodies to rhamnogalacturonan I backbone

Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides—BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [→2)-α-l-rhamnosep-(1→4)-α-d-galacturonic acid p-(1→]₇ structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose–galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples.     

Monoclonal antibodies to neurospora adenylate cyclase

A monoclonal antibody against $\text{Neurospora}$ soluble adenylate cyclase was obtained. The antibody inhibits cyclase activities from several lower eucaryotic organisms but not activities associated to testicular cytosol or turkey erythrocyte membranes.     

Monoclonal antibodies to plant plasma-membrane antigens

Murine monoclonal antibodies to membrane antigens were generated by immunization with a crude cellular membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. From a panel of thirteen monoclonal antibodies, seven were found to be directed against antigens present on the plasma-membrane by immunofluorescence visualization of antibody binding to the surface of isolated protoplasts. The corresponding set of plasma-membrane antigen(s) were present in root, shoot and leaf tissue and some but not all of these antigens were of wide species distribution, being found in Nicotiana tabacum L., N. plumbaginifolia L., Glycine max L., Phaseolus vulgaris L. and Triticum aestivum L. Topologically specific labeling of intact protoplasts with a monoclonal antibody reactive with an epitope present on the plasma-membrane specifically labeled a membrane fraction which equilibrated at a density of 1.14 kg/l following centrifugation in a sucrose gradient. In addition to use as biochemical markers for fractionation and molecular characterization of plasma-membranes, these monoclonal antibodies provide the basis for new selection tools in plant cell and gene manipulations.