Monoclonal antibodies to the species-specific and group antigens of Rickettsia prowazekii]

A number of hybridomas to different R. prowazekii determinants were obtained by the hybridization of spleen cells of BALB/c mice immunized with R. prowazekii corpuscular and soluble antigens. Some of the monoclonal antibodies (McAb) reacted with R. prowazekii thermolabile species-specific protein and did not react with R. typhi antigens (McAb of batches B4/4 and A-D3). McAb C5/2 and A3/2 reacted with the group thermostable antigen, common for R. prowazekii and R. typhi. McAb to the species-specific thermolabile antigen belonged to IgG2a. The McAb thus obtained permit the identification of R. prowazekii and R. typhi and the solution of the problem of the intragroup differentiation of rickettsiae belonging to the typhus group.     

Monoclonal antibodies against rat Leydig cell surface antigens

Monoclonal antibodies (MAbs) directed against the Leydig cell surface may be used to identify this cell in testicular preparations. Collagenase-dispersed adult rat interstitial cells were fractionated on Percoll density gradients, and Leydig cell-enriched fractions were used to prepare MAbs. Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIF) on isolated testicular cells and immunocytochemical localization on paraffin sections of adult testes. In density gradient fractions, immunoglobulin (Ig) M MAbs (LC-1C6 and LC-6H6) labeled the surface of cells possessing the morphological characteristics of Leydig cells. The density gradient profiles of MAb-binding activity observed by IIF and ELISA were parallel with the Leydig cell distribution as determined by [125I]-human chorionic gonadotropin (hCG) binding, testosterone response to hCG in vitro, 3 beta-hydroxysteroid dehydrogenase histochemistry and electron microscopy. The MAbs prominently labeled most interstitial cells in sections, but there was little or no labeling of connective tissue, endothelial or seminiferous tubule cells. Both MAbs recognized components of Mr 58,000 in Western blots of Leydig cell-enriched extracts. The results indicate that LC-1C6 and LC-6H6 recognize antigens on the Leydig cell surface that are not present on other isolated testicular cells from the adult rat. These MAbs are specific markers of the Leydig cell in situ and in vitro.     

Monoclonal antibodies to bull sperm surface antigens

Four monoclonal antibodies were generated during the present investigation. Mice were immunized with washed ejaculated bull spermatozoa. Spleen cells from the immunized mice were fused with myeloma cells (SP2/0) and four different hybridoma clones were obtained, producing specific monoclonal antibodies. These antibodies were designated as SP₂A₅, SP₁A₂, SP₁C₄ and SP₁C₆ respectively. All belonged to the IgG sub-class 1. The specificity of these monoclonal antibodies was tested using both ELISA (enzyme-linked immunosorbent assay) and indirect immunofluorescence staining techniques. Quantitative estimation of antibody-antigen reaction was done by optical density measurements. Ejaculated bull spermatozoa were always reactive for each ELISA procedure. Other test cells including spleen, testicular, ovarian, uterine and pancreatic cells from both bull and rabbit were non-reactive. Bull testicular spermatozoa were also non-reactive. However, seminal fluid (without sperm) was reactive. All four monoclonal antibodies were reactive to the midpiece of the ejaculated bull spermatozoa. In addition, SP₂A₅ was also reactive to the acrosomal area and SP₁A₂ was reactive to the acrosomal and the post-acrosomal area. Cytoplasmic droplets of ejaculated spermatozoa from bull and human were also possibly reactive to one antibody (SP₁C₆). The results clearly suggest the heterogeneous nature of the sperm cell plasma membrane and precise molecular alteration in the plasma membrane components (antigens) as the sperm cells differentiate and mature during their transit through the epididymis.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

Monoclonal antibodies recognizing cell surface antigens in Drosophila melanogaster

Monoclonal antibodies have been made against cell surface antigens from Drosophila melanogaster, as probes for “differentiation antigens.” The immunogens used were 0–16 hr embryonic cells and mass isolated imaginal discs. The tissue distributions of the antigens recognized by three antiembryonic antibodies and two antiimaginal disc antibodies have been defined by indirect immunofluorescent (IIF) assays on differentiated embryonic cell cultures and on dissected third instar larval organs. These antigens fall into two major categories being either ubiquitous or tissue-limited in distribution. In indirect radioimmunoassays against 12 Drosophila cell lines the antigens showing ubiquitous tissue distributions were detectable on all cell lines whereas the tissue-limited antigens were absent from some cell lines. Such a screen against cell lines therefore provides a straightforward means of identifying antibodies against nonubiquitous antigens. One antibody recognizing a tissue-limited antigen was detected as muscle-specific by IIF assays on differentiated embryonic cell cultures. The second tissue-limited antigen was found to label basement membranes, by IIF assays against third instar larval organs. The temporal distribution of the antigens during embryogenesis (3–21 hr) has been studied by indirect radioimmunoassay. In this respect the antigens fall into three classes: (1) abundant throughout embryogenesis (a ubiquitous antigen), (2) present throughout embryogenesis but increasing markedly in abundance between 12 and 15 hr (two ubiquitous antigens and the muscle-specific antigen), and (3) absent early in development but appearing at about 12 hr postfertilization (the tissue-limited, basement membrane antigen).     

Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development. Received: 8 February 1999 / Accepted: 26 July 1999     

Monoclonal antibodies to Mycoplasma hyorhinis surface antigens: tools for analyzing mycoplasma-lymphoid cell interactions

A library has been constructed of approximately 50 monoclonal antibodies that recognize antigens of Mycoplasma hyorhinis. Characteristics of six antibodies are discussed. Each reacts with a discrete determinant borne on a protein-containing molecule of distinct molecular size. Three of these respective antigens are expressed at the surface of mycoplasmas colonizing lymphoblastoid cells in culture. Of these three surface antigens, two are selectively expressed on strain GDL but not on strain BTS-7 of M. hyorhinis, thus defining strain-restricted immunological specificities within these species. One monoclonal antibody of the IgM class (mu, kappa) mediated marked complement-dependent growth inhibition of M. hyorhinis in broth culture. These monoclonal reagents should facilitate analysis of mycoplasma surface architecture, and the molecular interactions of these organisms with the host cell surface.     

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     

Monoclonal antibodies to dynein subunits reveal the existence of cytoplasmic antigens in sea urchin egg

Monoclonal antibodies directed against subunits of a sea urchin flagellar dynein were used to test for the presence of cytoplasmic antigens in preparations of fertilized eggs and mitotic apparati . A 9-10 S complex composed of 330,000-, 134,000-, and 126,000-mol-wt subunits was isolated from outer arms of Strongylocentrotus purpuratus sperm flagella and used to characterize the antibodies. Seven monospecific antibodies to the 330,000 subunit and two against the 134,000 subunit of the 9-10 S complex were identified by binding to nitrocellulose blots of electrophoretograms resolving polypeptides from different dynein preparations. The antibodies were applied also to blots of polypeptides from fertilized sea urchin egg at the first metaphase and a cellular fraction of mitotic apparati . Three of the antibodies to the 330,000 subunit bound to a cytoplasmic polypeptide of approximately the same molecular weight and the two antibodies to the smaller subunits recognized a polypeptide of 124,000 apparent molecular weight. Both antigens appeared to be enriched in the fraction containing mitotic apparati . These results indicate that polypeptides similar to two subunits of the 9-10 S complex are present in eggs at metaphase, and they are apparently associated with the mitotic apparatus.     

Monoclonal antibodies to cell surface antigens involved in sex pheromone induced mating in Streptococcus faecalis

Hybridoma cell lines producing monoclonal antibodies to Streptococcus faecalis cell surface antigens were constructed. Some of the antibodies isolated were directed against surface components involved in pheromone-induced mating. This paper describes the use of the monoclonal antibodies to identify antigenically related surface components detected by immunoprecipitation and Western blotting, their use in pheromone response assays, and their use as functional inhibitors in mating experiments.     

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.     

Monoclonal antibodies specific for tight-binding human chromatin antigens reveal structural rearrangements within the nucleus during the cell cycle

The class of nonhistone chromosomal proteins that remains bound to DNA in chromatin in the presence of 2.5 M NaCl-5 M urea has proven refractile to biochemical analysis. In order to study its role in chromatin organization, we have produced monoclonal antibodies that are specific for the HeLa DNA-protein complex that remains after extraction of chromatin with high salt and urea. The antibody-producing clones were identified with an ELISA assay. Of the six clones selected, five were stabilized by limiting dilution. All clones are IgG producers. None cross-react significantly with native DNA, core histones, or the high-mobility group nonhistone proteins. All antibodies are specific for nuclear or juxtanuclear antigens. Indirect immunofluorescence shows that three antibodies, which are nonidentical, stain three different nuclear networks. Available evidence indicates that two of these networks are the nuclear matrix. A fourth antibody reveals structures reminiscent of chromocenters. A fifth antibody, AhNA-1, binds to interphase HeLa chromatin and specifically decorates metaphase chromosomes. AhNA-1 similarly recognizes rat chromosomes. Each of these monoclonal antibodies also reveals a changing pattern of nuclear staining as cells progress through the cell cycle. Presumably, this reflects the rearrangement of the cognate antigens.     

Monoclonal antibodies to bovine blood group antigens

Hybridomas were made by fusing mouse myeloma cells with spleen cells from mice immunized with bovine red cells. Sixteen cloned lines which secreted haemolytic monoclonal antibodies reacting with antigens in the A, B, F, Z and S blood group systems were established; one of the antibodies identified a new factor in the B system. Extensive tests on red cells from 1000 animals indicated that several of the antibodies are suitable for use in routine blood typing; others are of potential use for genetic studies of the bovine blood group systems.     

Monoclonal antibodies to Mycoplasma pneumoniae antigens]

Approaches to obtaining stable mouse hybridomas, capable of producing monoclonal antibodies (McAb) to M. pneumoniae key antigens, were developed. As the result of hybridization experiments, 7 clones were obtained; of these, 4 clones stably synthesized IgG McAb. Clones H1/H9 and H9/B2 synthesized antibodies to thermolabile, proteinase-sensitive K protein, produced by cytoplasmic membranes of M. pneumoniae cells. The molecular weight of this protein was found to be 90 kD. McAb of clone H1/H9, labeled with horse-radish peroxidase and fluorescein isothiocyanate, specifically reacted with M. pneumoniae antigens in the immunofluorescence test and the enzyme immunoassay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data are prerequisites for the development of diagnostic test systems for the detection of M. pneumoniae antigens in different biological substances obtained from patients with respiratory pathology.     

Monoclonal antibodies as neural cell surface markers

A comparison is made of the immunohistochemistry at the ultrastructural level of three monoclonal antibodies directed against surface components of CNS cells. Hybridomas secreting these antibodies were obtained from two cell fusions of a rat myeloma cell line and immune splenocytes derived from rats immunized either with primary mouse brain cultured cells or membrane components. In cultures one antibody, anti-BSP-2 (Brain Surface Protein-2), was preferentially directed against neurones while another, anti-BSP-3 (Brain Surface Protein-3), preferentially labeled astrocytes. In mouse cerebellar sections, both labeled the surface of Purkinje cells, granule cells and astrocytes. In addition a cytoplasm localization was apparent in granule cells and astrocytes. Another antibody anti-MESA-1 (Mouse Endothelial Surface Antigen-1) reacted exclusively with the surface of endothelial cells lining blood vessels. These data are discussed with reference to the biochemical nature of the corresponding antigens and to known glycoproteins of neural cell membranes.