Production of monoclonal antibodies specific for Eimeria tenella microgametocytes

A panel of 4 monoclonal antibodies specific for Eimeria tenella, the causative agent of cecal coccidiosis of birds in the genus Gallus, was produced by standard techniques. The indirect fluorescent antibody (IFA) test demonstrated specificity of these 4 antibodies for the microgametocytes. Hybridoma TIA3B9 secreted a monoclonal antibody of subisotype IgG2b that was used throughout the course of this study. Immunologic potency of this antibody was demonstrated by in vitro experiments that revealed a greater than 50% reduction in oocyst production, indicating an apparent inhibition of fertilization.     

Production and characterization of monoclonal antibodies specific for Shewanella colwelliana exopolysaccharide.

Six monoclonal antibodies were produced to whole cells of Shewanella colwelliana (Aco1 to Aco6) and two (Aco22 to Aco23) to purified exopolysaccharide (EPS). Aco1, -4 to -6, -22, and -23 bound to both the cell surface and the purified EPS, while Aco2 and -3 bound to cells only. The EPS of S. colwelliana was antigenically unique from those of nine other species of marine bacteria that were tested. Mapping studies revealed that all of the EPS-specific monoclonal antibodies bound to the same epitope. This EPS epitope was sensitive to cleavage of ester bonds, but neither pyruvate, acetate, nor terminal nonreducing sugars were required for antigenicity. When S. colwelliana was grown on rich media, most of its EPS was loosely associated with the cell surface.     

Production and characterization of monoclonal antibodies specific for Mycobacterium bovis

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.     

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.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Production of monoclonal antibodies specific for ganglioside GD3.

Four kinds of anti-GD3 monoclonal antibodies, DSG-1, -2, -3, and -4, of the IgM class were obtained by the immunization of BALB/c mice with enzootic bovine leukosis tumor tissue-derived ganglioside GD3 inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay and by enzyme immunostaining on thin-layer chromatography. The reactivities of the monoclonal antibodies obtained to four ganglioside GD3 variants [GD3(NeuAc-NeuAc), GD3(NeuAc-NeuGc), GD3(NeuGc-NeuAc), and GD3(NeuGc-NeuGc)] were tested. All of the monoclonal antibodies were found to react with GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc) but not with GD3(NeuGc-NeuAc) or GD3(NeuGc-NeuGc). Furthermore, various purified glycosphingolipids were used to determine the specificity of these monoclonal antibodies. All 4 antibodies reacted only with ganglioside GD3 [GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc)], but not with several gangliosides linking the GalNAc, Gal beta 1-3GalNAc, NeuAc alpha 2-3Gal beta 1-3GalNAc, or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-3GalNAc residue to the Gal moiety of ganglioside GD3 (GD2, GD1b, GT1b, or GQ1b, respectively), ganglioside GT1a having the same terminal NeuAc alpha 2-8NeuAc alpha 2-3Gal residue as ganglioside GD3, other gangliosides, and neutral glycosphingolipids. These findings suggest that the 4 monoclonal antibodies obtained may be specific for the epitope of NeuAc-alpha 2-8Sia alpha 2-3Gal beta 1-4Glc residue of ganglioside GD3.     

Production and partial characterization of monoclonal antibodies for detection of entomopoxvirus from Melanoplus sanguinipes

Entomopoxviruses (EPV) are currently being considered as candidate grasshopper (Orthoptera: Acrididae) microbial control agents. Classical techniques for diagnosing infections in grasshoppers are laborious, time consuming, and sometimes inaccurate. Specific murine monoclonal antibodies were developed against an EPV from Melanoplus sanguinipes (Fab) for use in a nitrocellulose-based enzyme-linked immunoassay (dot-blot). An IgG_2b monoclonal antibody was used to diagnose infections in grasshoppers 13 days following injection with virions. Of 25 grasshoppers that had patent infections microscopically, 22 produced positive results on the dot-blot. In a second test, 39 patently infected grasshoppers all produced positive results. Seven additional grasshoppers in the first test and 2 in the second test gave positive reactions in the dot-blot method but virus was not detected upon microscopic examination. The monoclonal antibody did not cross-react with other commonly occurring grasshopper pathogens. The dot-blot method detected as few as 2.5×10⁶ purified EPV virions. The improvement over existing detection techniques should facilitate evaluation of EPV for field use.

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Prodution et caractérisation partielle d'anticorps monoclonaux pour la détection d'entomopoxyvirus de Melanoplus sanguinipes

Résumé Les criquets sont très nuisibles aux pâturages de l'Ouest des USA et du Canada. Les méthodes classiques de protection sont basées sur les traitements chimiques lors des pullulations. Un entomopoxvirus (EPV) extrait de M. sanguinipes est généralement considéré comme un outil, pour le contrôle des populations sur une longue période, en vue de la suppression des criquets. Les méthodes actuelles d'isolement de EPV sont longues, pénibles et peu fiables. Les tests d'adsorption des antigènes sur l'anticorps fixé et le dosage per l'anticorps enzymatiquement marqué sont efficaces, sûrs et donnent à temps des résultats pour déceler des entomopathogènes. Nous avons produit des anticorps monoclonaux de souris contre EPV de M. sanguinipes, et les avons utilisés dans des dosages immunoenzymatiques d'extraits protéiques adsorbés sur nitrocellulose. Les EPV sont recherchés sur des criquets injectés de virions 13 jours avant. Sur 25 criquets qui présentaient des infections nettes lors d'examens microscopiques, 22 ont donné des résultats positifs par sérologie. Dans un second test, sur 38 criquets nettement contaminés, tous ont donné des résultats positifs; 7 criquets suppl'ementaires mentaires dans le premier test et 2 dans le second test ont donné des résultats positifs en sérologie alors que l'examen microsopique n]avait pas test ont donné des résultats positifs en sérologie alors que l'examen microsopique n'avait pas révélé de virus. Ceci montre que ce type de détection est plus sûr que la méthode ordinaire. Les anticorps monoclonaux ne donnent pas de réactions avec les autres pathogènes courants des criquets. La méthode sérologique a permis de détecter même une concentration de 2,5×10⁶ virions EPV.

     

Development and characterization of monoclonal antibodies specific for the genus Listeria

Abstract Monoclonal antibodies were obtained by the classic hybridoma technique with lymphocytes of BALB/c mice immunized with formalin killed Listeria monocytogenes cells. Among 1000 hybridomas issued from the fusion, four monoclonal antibodies (mAbs A6 A E4, C10 A F7, G4 A D6, G7 A D5) gave interesting results. By Western-blot analysis with various soluble extracts of different Listeria species, the four mAbs reacted with two major antigens of 38 and 41 kDa, with all Listeria species tested. The mAb A6 A E4 is an IgG2b with κ light chains and reacted only with Listeria antigens without any cross reaction with other organisms tested by ELISA, dot-blotting and Western-blotting. With the same conditions, the three other mAbs reacted with Listeria and with other genus extracts, particularly with Streptococcus and Enterococcus . mAb A6 A E4-reactive antigens are proteins, and glycoprotein immunoassay indicated that the epitope is devoid of carbohydrate moiety. This mAb A6 A E4-reactive protein was neither expressed on cell surface nor released outside the bacteria; immunogold electron microscopy showed that these antigens were localized in the cytoplasma area.     

Production and characterization of murine monoclonal antibodies specific for baboon IgG heavy and light chain epitopes

We have generated a panel of murine monoclonal antibodies (MAbs) that recognize baboon IgG epitopes. The reactivity of the MAbs with IgG from other primate species was also examined. Specificity for IgG heavy (H) or light (L) chain epitopes was determined by Western blot analysis. The H chain-specific MAbs were analyzed for IgG subclass specificity and the L chain-specific MAbs for reactivity with baboon IgM and polymeric sIgA. Finally, an ELISA was developed to demonstrate the utility of the MAbs in analysis of humoral immune responses in baboons.     

Production and characterization of monoclonal antibodies specific for the functional domains of poly(ADP-ribose) polymerase

Monoclonal antibodies were developed against poly(ADP-ribose) polymerase and analyzed for their reactivity against the NAD+- and DNA-binding fragments. Two fusions were performed to obtain hybridomas and the resulting anti-poly(ADP-ribose) polymerase antibodies were further screened by characterization of their immunoglobulin light chains. Five different hybridomas were isolated which produced different immunoglobulin light chains, all of which were specific for poly(ADP-ribose) polymerase. The specificities of these antibodies were determined by immunoblotting against the purified poly(ADP-ribose) polymerase, its autodegradation fragments, and the fragments prepared by limited proteolysis with chymotrypsin and papain. These fragments have been suggested to contain the NAD+-binding site, the DNA-binding site, and the automodification site, respectively. All the monoclonal antibodies reacted with the 116 kdalton (kDa) band corresponding to the purified enzyme. Four antibodies reacted exclusively with antigenic site(s) on the 46-kDa fragment which contains the DNA-binding site. A fifth antibody reacted exclusively with a clearly different antigenic site on the 74- and 54-kDa fragments which possess the NAD+ (substrate) binding site. The immunoreactivity with the major autodegradation products (69- and 46-kDa fragments) of the purified enzyme confirms this difference between the two groups of antibodies. The 22-kDa fragment corresponding to the auto-modification site does not show any immunoreactivity with the antibodies.     

Development of hybridoma-produced antibodies directed against Eimeria tenella and E. mitis

Hybridoma cell lines, which secreted antibodies directed against two different strains of Eimeria tenella and one strain of E. mitis, were produced by fusion of spleen cells from sporozoite-immunized Balb/cByJ mice with P3-X63-Ag8 myeloma cells. The antibodies demonstrated at least eight different binding patterns on or in air-dried sporozoites as determined by the indirect immunofluorescent antibody (IFA) test. These patterns varied from a general internal fluorescence similar to that seen with sporozoites exposed to hyperimmune chicken serum, to fluorescence observed on the tip, pellicle, and refractile body of the parasite. Five cloned, antibody-secreting cell lines were successfully established. Four of these clones produced antibody that reacted only with various strains of E. tenella and cross-reacted with no other species of coccidia. The fifth clone produced antibody directed against only E. mitis and did not react with any other coccidial species.     

Production and characterization of monoclonal antibodies specific for the transmembrane protein of simian immunodeficiency virus from the African green monkey.

Mouse monoclonal antibodies were produced against simian immunodeficiency virus (SIV) from the African green monkey (SIVAGM). The antibodies reacted with the transmembrane protein of all five SIVAGM isolates but not with those of SIVs from the rhesus macaque and mandrill or of human immunodeficiency virus type 1 or type 2, indicating that they recognize a species-specific epitope strongly conserved in SIVAGM. The transmembrane proteins of several SIVAGM isolates were found to vary in molecular size, even in the deglycosylated form after N-glycanase treatment, indicating heterogeneity of the SIVAGM isolates.     

Production and characterization of monoclonal antibodies for aflatoxin B1

Hybridomas that secreted antibodies for aflatoxin B1 were selected using two immunization protocols referred to as A and B. Protocol A is a standard immunization method and resulted in the selection of only two clones that produced monoclonal antibodies against aflatoxin B1. In protocol B a unique immunization schedule which resulted in the generation of 10 hybridomas is described. Of the 10, one antibody was highly specific to B1, four antibodies reacted equally strongly with B1, G1 and weakly with B2. Another four reacted strongly with B1 and weakly with B2 and G1. One clone reacted equally strongly with B1, G1 and B2. Interestingly all the 10 antibodies showed little or no cross-reaction with G2.     

Generation and characterization of monoclonal antibodies specific for the human IFN-gamma receptor

Purified preparations of the human IFN-gamma R derived from placental membranes were used to produce receptor-specific murine mAb. Supernatants from growth-positive wells were screened for their ability to block binding of 125I-IFN-gamma to human placental membranes. Ten inhibitory cultures were identified. Two of these (GIR-208 and GIR-301) abrogated all binding of radioligand to either intact placental membranes or soluble, purified IFN-gamma R. Three others (GIR-72, 76 and 94) showed moderate blocking activity (65, 59, and 49%, respectively) whereas the remaining five (GIR-57, 67, 83, 109, and 153) blocked binding to a low but significant extent (20 to 40%). Specificity experiments demonstrated that the antibodies reacted with the receptor and not the ligand (IFN-gamma). None of the antibodies reacted with IFN-gamma by ELISA. Moreover, GIR-208 and GIR-301, but not isotype-matched controls, identified the receptor by Western blot analysis. GIR-208 and GIR-301 also completely abrogated binding of 125I-IFN-gamma to either mononuclear phagocytes (U937) or human fibroblasts (WISH). Competition experiments revealed that GIR-208 and GIR-301 recognized similar epitopes on the IFN-gamma R and that these (or this) epitopes were identical to or linked to the ligand binding site of the receptor. In addition, both antibodies inhibited development of IFN-gamma-dependent anti-viral activity in WISH cells in a dose-dependent fashion. These data thus indicate that the IFN-gamma R expressed on human placental cells, mononuclear phagocytes, and fibroblasts are similar.     

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 of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons.

Choline acetyltransferase (ChAT) has been purified from pig brain to greater than 95% homogeneity (purification factor: 646 000, specific activity of the purified enzyme: 128 mumol acetylcholine formed/min/mg). Gel electrophoresis of the purified enzyme in the presence of sodium dodecylsulphate and beta-mercaptoethanol revealed a single protein band at 68 000 daltons. Immunoprecipitation and double immunodiffusion tests showed that antisera raised against this protein specifically recognize ChAT. A monoclonal antibody prepared against the enzyme specifically binds a protein from crude pig brain supernatants which has a mol. wt. of 68 000 and a specific activity of 153 mumol/min/mg. This antibody shows no species cross-reactivity. The specificity of the immunohistochemical localization of ChAT has been established by comparing the labeling of pig retina using the antiserum with that obtained using the monoclonal antibody. Both probes specifically identify the same retinal structures: labeled cell bodies are found in the inner nuclear layer and the ganglion cell layer, while a double band is stained in the inner plexiform layer. In rat spinal cord, the antiserum labels the motoneurons and the preganglionic sympathetic neurons, located in the intermedio-lateral nucleus, the intercalated region, and the central autonomic area.     

Production and properties of novel human thyroid cancer specific monoclonal antibodies.

Monoclonal antibodies (TCM-7, -9 and -12) against human thyroid differentiated cancers were established by screening with human thyroid cancers, normal and benign thyroid tissue, and normal human serum protein. A monoclonal antibody (TCM-9) with strong specificity for human thyroid cancer but not for Graves' disease, adenoma or normal thyroid, was shown to recognize a 300 K protein but not to bind to native or mature human thyroglobulin. When TCM-9 was used in immunohistochemical staining tests on more than 30 types of non-thyroid lesions, no reactivity of TCM-9 was observed except with skin immature teratoma, lip squamous carcinoma and stomach adenocarcinoma, which revealed weak reactivities. TCM-9 also showed strong reactivity with two undifferentiated thyroid cancer cell lines and one tissue specimen. Thus TCM-9 is a novel monoclonal antibody against the thyroid cancer.