Antigenic determinants distinctive of Methanospirillum hungatei and Methanogenium cariaci identified by monoclonal antibodies

Monoclonal antibodies were prepared against two species of Methanomicrobiaceae. Antibody 1A is specific for Methanospirillum hungatei strain JF1 and the determinant it recognizes is expressed on the surface of JF1 cells, where it is exposed and accessible to antibody. The determinant is found in a polypeptide (MW<12,000) in the sheath that covers the bacterial cell; it is not present in Methanospirillum hungatei strain GP1; and it is not expressed on the surface of whole cells of the other 24 methanogenic bacteria tested. It is therefore a marker of strain JF1, consequently, antibody 1A is potentially useful for tracking JF1 and fragments thereof in a variety of samples. Antibody 7A is specific for Methanogenium cariaci JR1c. It did not react with any other methanogen tested, not even with Mg. marisnigri or Ms. hungatei JF1, although these cross-react with Mg. cariaci if tested with polyclonal antisera. Therefore antibody 7A recognizes specifically a marker of Mg. cariaci JR1c.Abbreviations SIA slide immunoenzymatic assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Monoclonal antibodies to surface and cytoskeletal components of the spermatozoid ofPteridium aquilinum

Summary Detergent extracted spermatozoids of the fernPteridium aquilinum were used as mixed antigen preparations for raising monoclonal antibodies in order to obtain reagents for detecting as yet uncharacterized components of the plant cytoskeleton. Selected antibodies were studied by immunofluorescence microscopy of developing spermatids and mature spermatozoids. Some reacted directly with fixed cells, others required permeabilization treatments with cold methanol or Triton X-100. AntibodiesPas2D9 andPas6D7 bind to glycoprotein antigenic determinants that are exposed on the surface of the plasma membrane. Several antibodies interact with cytoskeletal components.Pas1D3,Pas5D8 andPas5F4 bind to the cytoskeleton of permeabilized cells including the flagella. Three react specifically with the flagellar band or associated components:Pas2G6 reacts with the whole flagellar band but shows a prominent binding to basal bodies,Pas5E2 binds exclusively to basal bodies, andPas5E7 detects mitochondria associated with the flagellar band. Cross-reactions to wheat root tip cells at different stages of the cell cycle are described inMarc andGunning (1988).Abbreviations MLS multilayered structure - MT microtubule - MAb monoclonal antibody - MAP microtubule associated protein     

Identification of a second distinct strain of beet mild yellowing luteovirus using monoclonal antibodies and transmission studies

The variation among isolates of beet mild yellowing luteovirus (BMYV), collected from commercial crops of sugar beet during 1990, 1992 and 1993, was studied using monoclonal antibodies and transmissions to indicator species. The common strain of BMYV, which occurs throughout the sugar-beet root growing area, reacts with monoclonal antibodies MAFF 24, BWYV-BC-510H and BYDV-PAV-IL-1, and infects Capsella bursa-pastoris. A second strain, which failed to react with monoclonal antibody BYDV-PAV-IL-1 and which did not infect C. bursa-pastoris, was identified in 11% of sampled infected plants. The implications of the properties of this strain for the epidemiology of BMYV are discussed.     

Two monoclonal antibodies highly specific for the blood group N determinant

Two monoclonal IgM antibodies, 179K and 35/5F, obtained following immunization of mice with A₂,MN or O,MN human erythrocytes, agglutinate NN and MN red cells strongly, and MM erythrocytes weakly. As shown by hemagglutination inhibition and solid phase ELISA, both antibodies are highly specific for the blood group N determinant. They react with N glycoprotein, its amino-terminal glycopeptides and with Ss glycoprotein (glycophorin B), which carries the blood group N determinant. They fail to react with M glycoprotein, M glycoprotein-derived glycopeptides, or with internal glycopeptides derived from N glycoprotein. Reaction of the antibodies with N glycoprotein is abolished by desialylation, periodate oxidation/borohydride reduction, orN-acetylation of the glycoprotein. Thus, the antibodies are specific for an epitope which includes sialylated oligosaccharide chain(s) and is located in the region of the amino-terminal leucine residue of N glycoprotein. MMU^– erythrocytes, lacking both blood group N and Ss glycophorin are non-reactive. Agglutination of MMU⁺ erythrocytes by the anti-N antibodies occursvia interaction with glycophorin B and correlates with the Ss phenotype of red cells MM,S erythrocytes are usually more strongly, agglutinated than MM,ss cells. The agglutination of MM erythrocytes decreases markedly as the pH is increased from 6 to 8, while agglutination of NN red cells is much less affected by shifts in pH over this range. As a result, both monoclonal antibodies are highly anti-N specific typing reagents when the agglutination assay is carried out at pH 8.     

Identification of Borrelia burgdorferi Isolated in Korea Using Outer Surface Protein A (OspA) Serotyping System

Two characteristic strains (935T, 934U) of B. burgdorferi isolated from Ixodes persulcatus and a wild rodent (Apodemus agrarius) in Korea were selected and analyzed by an immunoblot method using the monoclonal antibodies directed to different epitopes of outer surface protein A (OspA). The reactive pattern of strain 934U with these monoclonal antibodies was identical to that of strains belonging to B. afzelii and that of strain 935T was different from other isolates. Monoclonal antibody (5TEE3) which is specific to strain 935T did not react with any other Western and Japanese isolates. So, it was suggested that there exist at least two groups of B. burgdorferi in Korea. One could be classified as B. afzelii and the other is a divergent group from three known species of B. burgdorferi sensu stricto, B. garinii and B. afzelii.     

Monoclonal antibodies for a comparative serological study of strains of Vibrio anguillarum

Murine monoclonal antibodies against characteristic determinants of heat stable somatic antigens of typical serovar I (820/15/8 Kop) and serovar II (134/82/1 Kiel) of European Vibrio anguillarum strains were produced. Three stable hybridoma cell lines, called aVaI/1F4, aVaI/6F4 and aVaI/2H5, produced monoclonal antibodies each against a typical serovar I determinant of strain 820/15/8 Kop. One hybridoma cell line (aVaII/5A4) producing monoclonal antibodies against Vibrio anguillarum strain 134/82/1 Kiel (serovar. II) was established. Fourteen Vibrio strains and Aeromonas salmoniáda strains were serologically compared by Enzyme-Linked Immunosorbent Assay (Elisa ).     

Immunoenzymatic techniques for the detection of Phoma exigua in infected potato tissues

Antisera to Phoma exigua var. foveata and var. exigua were prepared by injecting rabbits and mice with protein solutions from mycelium. Specific antibodies were isolated and immunoenzymatic techniques (double antibody sandwich ELISA and indirect ELISA) were used to test for the fungus in inoculated tubers and sprouts and in stems grown from these tubers. The fungus was detected in these different tissues, with var. foveata being more aggressive, demonstrating the applicability and sensitivity of the techniques. The antibodies produced to the two varieties of the fungus were not specific to their own varieties. They also reacted with Phoma tracheiphila but did not react with several other common potato pathogens. Preliminary results obtained with antibodies from mouse ascite liquid suggest the possibility of producing specific monoclonal antibodies.     

Immunochemical characterization of a polysaccharide antigen isolated from the oral microorganismEubacterium saburreum,strain 02/725

A serologically highly active polysaccharide antigen (PS 02/725) consisting of equimolar amounts ofd-glycero-d-galacto-heptose and a 6-deoxyheptose was isolated fromEubacterium saburreum, strain 02/725, by trypsin digestion and subsequent gel filtration and ion-exchange chromatography. Whole bacterial cells injected intravenously stimulated rabbits to produce precipitins and complement-binding antibodies of the IgG class specific for PS 02/725.     

Antigenic determinants of measles virus hemagglutinin associated with neurovirulence.

The biological activity of monoclonal antibodies specific for the hemagglutinin protein of measles virus strain CAM recognizing six epitope groups according to their binding properties to measles virus strain CAM/R401 was investigated in vivo in our rat model of measles encephalitis. When injected intraperitoneally into measles virus-infected suckling rats, some monoclonal antibodies modified the disease process and prevented the necrotizing encephalopathy seen in untreated animals. The analysis of measles virus brain isolates revealed emergence of variants that resisted neutralization with the passively transferred selecting monoclonal antibody but not with other monoclonal antibodies. Monoclonal antibody escape mutants were also isolated in vitro, and their neurovirulence varied in the animal model. Sequence data from the hemagglutinin gene of measles virus localize a major antigenic surface determinant of the hemagglutinin protein between amino acid residues 368 and 396, which may be functionally important for neurovirulence. The data indicate that the interaction of antibodies with the measles virus H protein plays an important role in the selection of neurovirulent variants. These variants have biological properties different from those of the parent CAM virus.     

Chicken developmental antigens: analysis of erythroid populations with monoclonal antibodies

Fusions were performed between the mouse PAI myeloma cell line and spleen cells from Balb/c mice immunized with intact erythrocytes from 1-day Cornell K-strain White Leghorn chickens. Following single cell cloning, four hybridoma clones were found to secrete erythroid specific monoclonal antibodies. Based on its pattern of reactivity, the antibody (IgG2a, kappa) secreted by clone 10C6 detects a specific avian oncodevelopmental antigen associated with the hematopoietic system: chicken fetal antigen (CFA). Two other clones, designated as 3F12 and 4C2, produced antibodies (IgM, kappa) that recognize another avian developmental antigen: chicken adult antigen (CAA). A fourth clone, 9F9, produced an antibody (IgM, kappa) that reacts with all peripheral erythrocytes from both Japanese quail and chicken regardless of age. Clone 10C6 antibody apparently detects an erythrocyte specific (ES) determinant of CFA associated with determinant #8 while antibodies of clones 3F12 and 4C2 recognize a chicken specific determinant of CAA. Analysis by complement mediated microcytotoxicity indicated that the epitopes detected by 10C6 vs 3F12 and 4C2 antibodies were expressed on erythrocytes in a reciprocal fashion during development. Furthermore, strain variations in the incidence of erythrocytes carrying these epitopes were observed. The usefulness of these monoclonal antibodies for the study of erythroid populations is discussed.     

Antigenic Heterogeneity in the 115,000 Mr Major Surface Antigen of Trichomonas vaginalis1

ABSTRACT The 115,000-molecular-weight antigen of Trichomonas vaginalis was characterized using monoclonal antibodies developed to three different strains of T. vaginalis and one strain of Tritrichomonas foetus. The antigen was found to be present on all strains or isolates of T. vaginalis examined and was demonstrated to be located on the external surface plasma membrane by agglutination assays and complement-mediated lysis assays. Characteristics of the antigen were assessed with a proteolytic enzyme and periodate oxidation. Periodate treatment of whole T. vaginalis abrogated binding for eight antibodies while use of pronase-treated antigen resulted in loss of antibody binding for two different antibodies. Screening of 19 axenized clinical isolates of T. vaginalis and one strain each of T. foetus and Giardia lamblia with type-specific antibodies delineated three major groups of T. vaginalis based on antigenic specificities (epitope distributions) within the 115,000-molecular-weight antigen. In addition, one epitope of the 115,000-molecular-weight antigen was found only on the immunizing strain. Two epitopes were present on all T. vaginalis isolates as well as T. foetus and G. lamblia. One epitope was common to all T. vaginalis except one. A minimum of six different epitopes of the 115,000-molecular-weight antigen were identified. Antigens purified with type-specific or “common” monoclonal antibodies shared the same partial peptide maps demonstrating relatedness.     

Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A broad spectrum monoclonal antibody to alpha-tubulin does not recognize all protozoan tubulins

Summary Monoclonal antibodies able to recognize single antigenic determinants are a powerful tool for the study of immunological heterogeneity of antigens. In this paper we have used a monoclonal antibody against the -subunit of pig brain tubulin (TU-01) to investigate the immunoreactivity of tubulins from mammals, avians, amphibia, echinodermata, plathelmints, slime moulds and protozoa. Immunoreactivity was detected using immunoblotting and indirect immunofluorescence of isolated cells. Our results show that the antigenic determinant recognized by the TU-01 antibody is present in all metazoan tubulin tested and among the eukaryotic microorganisms only in the flagellateTrichomonas vaginalis. Indirect immunofluorescence also reveals that not allTrichomonas microtubules are stained by TU-01 antibody indicating the presence of different tubulins within a single cell. This results are consistent with the multitubulin hypothesis (Fulton andSimpson 1976).     

Differential toxinogenesis in the genusPichia detected by an anti-yeast killer toxin monoclonal antibody

The differential toxinogenesis of 25 isolates belonging to species of the potential yeast killer genusPichia that were previously classified in the genusHansenula was comparatively demonstrated by two serologic techniques (indirect immunofluorescence and double immunodiffusion) by using a monoclonal antibody against a yeast killer toxin produced by a selected strain ofPichia anomala (UCSC 25F). The killer phenotypes of thePichia isolates were evaluated by their ability to kill each other. The results, although of insufficient taxonomic value for a reliable separation of either species or genera, attest to the genomic heterogeneity for the killer character in the genusPichia as well as the presumptive dual killer/sensitive identity for each single isolate.     

Antigenic variation among Sendai virus strains detected by monoclonal antibodies

Thirteen strains of Sendai virus isolated from various sources in the 1950's and after 1976 were compared for their reactivities with monoclonal antibodies prepared against the prototype strain MN of Sendai virus. Results revealed that while the 5 strains isolated in the 1950's reacted with all the monoclonal antibodies as the prototype strain did, the 2 strains isolated in 1976 and 1978 did not react with an F-specific monoclonal antibody, and the other 6 strains isolated after 1978 lacked reactivity with an HN-specific monoclonal antibody.     

Phylogenetic analysis of Methanobrevibacter isolated from feces of humans and other animals

Comparative 16S rRNA gene sequence and genomic DNA reassociation analyses were used to assess the phylogenetic relationships of Methanobrevibacter fecal isolates. The 16S rRNA gene sequences of Methanobrevibacter smithii strain PS and the human fecal isolates B181 and ALI were essentially identical, and their genomic DNA reassociated at values greater than 94%. The analysis of 16S rRNA sequences of the horse, pig, cow, rat, and goose fecal isolates confirm that they are members of the genus Methanobrevibacter. They had a high degree of sequence similarity (97–98%) with the 16S rRNA gene of M. smithii, indicating that they share a common line of descent. The 16S rRNA genes of the horse and pig isolates had 99.3% sequence similarity. Sequence analysis of the 16S rRNA gene of the sheep fecal isolate showed that it formed a separate line of descent in the genus Methanobrevibacter. Genomic DNA reassociation studies indicate that the horse, pig, cow, and goose fecal isolates represent at least three new species. The horse and pig isolates were the only animal isolates that had > 70% genomic DNA reassociation and represent strains of a single species. The cow, goose, and sheep isolates had little or no genomic DNA reassociation with M. smithii or with each other. The relationship of the rat isolate to the other animal isolates was not determined. An evaluation of the relationship of 16S rRNA gene sequence similarity and genomic DNA reassociation of Methanobrevibacter and other methanogenic archaea indicated that genomic DNA reassociation studies are necessary to establish that two methanogenic organisms belong to the same species. Received: 17 November 1997 / Accepted: 16 January 1998     

Idiotypic determinants of monoclonal antibodies that bind to 3-fucosyllactosamine

Many monoclonal antibodies that react with the lacto-N-fucopentaose III (LNF III) antigenic determinant, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc, have been described recently. The terminal trisaccharide of this determinant, fucosyllactosamine, is present on glycolipids and glycoproteins and on the surface of granulocytes, monocytes, and other cells. To study the structural and genetic diversity of these antibodies, syngeneic anti-idiotypic monoclonal antibodies were produced in BALB/c mice against PMN 6, a monoclonal antibody directed against this sequence. Anti-idiotypic antibodies 6B1 and 6C4 reacted with 50% of a panel of 20 anti-LNF III monoclonal antibodies, whereas 6A3 reacted strongly only with PMN 6. This indicates that the determinants recognized by 6C4 and 6B1 represent major cross-reactive idiotopes of this family of antibodies. The binding of idiotypic antibodies to a glycolipid bearing this antigenic determinant was completely inhibited by the three anti-idiotypic antibodies, 6A3, 6B1, and 6C4. The idiotopes could be demonstrated on the heavy chain of the monoclonal antibodies by an antibody transfer technique when mild reducing conditions were employed, but a high concentration of reducing agent destroyed the idiotypic determinants. This suggests that the anti-idiotypic antibodies recognize conformational structures expressed on the heavy chain molecules. The binding of 18 monoclonal antibodies to two glycolipid antigens and to a fucosyllactosamine-bovine serum albumin conjugate was compared. Antibodies that possessed the 6C4 cross-reactive idiotope bound to fucosyllactosamine-bovine serum albumin more weakly than idiotype-negative antibodies (p = 0.001). This suggests that the 6C4-positive antibodies might represent germline structures.     

Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome

Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the α5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.     

The monoclonal antibody,anti-asialoglycophorin from human erythrocytes,specific forβ-d-Gal-1-3-α-d-GalNAc-chains (Thomsen-Friedenreich receptors)

The monoclonal antibody 22.19 of IgM class obtained after immunization of BALB/c mice with asialoglycophorin of human erythrocyte membranes is described. The specificity of this antibody for -d-Gal-1-3--d-GalNAc- disaccharide chains (Thomsen-Friedenreich receptors) was established by studying its reactivity against various erythrocytes, glycoproteins and oligosaccharides and by comparison with two lectins, peanut agglutinin andVicia graminea lectin, which recognize these disaccharide chains.Abbreviations PNA peanut agglutinin - VgL Vicia graminea lectin - TF Thomsen-Friedenreich - HSA human serum albumin - MoAb monoclonal antibody     

Monoclonal antibodies to rabbit and pig zonae pellucidae distinguish species-specific and shared antigenic determinants

Monoclonal antibodies against rabbit or porcine zonae pellucidae (ZP) demonstrate species-specific and shared antigenic determinants. In addition, these antibodies are used to characterize the biochemical nature of these determinants. All of six monoclonal antibodies developed against porcine ZP react with porcine but not with rabbit ZP. Only one of seven monoclonal antibodies developed against rabbit ZP cross-reacts with porcine ZP. None of these antibodies recognized antigens associated with other tissues tested. High-resolution, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by immunoblotting was used to demonstrate that the cross-reactive antibody recognizes an antigenic determinant which is associated with the major low molecular weight glycoprotein of both the pig and rabbit ZP. Since this antibody recognizes all charge species of this glycoprotein, it is apparent that the antigenic determinant recognized by this antibody involves protein. Further studies demonstrate that proteolytic digestion of ZP will destroy the antigenic determinant while glycosidic digestion of ZP has no effect on antibody binding. Although polyclonal antibodies to this glycoprotein inhibit sperm from binding to the zona pellucida, this monoclonal antibody does not affect sperm binding. None of the species-specific antibodies recognize ZP glycoproteins following 2D-PAGE. This is a property typical of antibodies directed against conformational antigenic determinants. The presence of common as well as unique zona antigenic determinants could explain why ZP proteins induce heteroantibodies which result in infertility while alloimmunization has no effect on fertility.