Monoclonal antibodies specific for the amastigote stage of Leishmania pifanoi. I. Characterization of antigens associated with stage- and species-specific determinants

Eight mAb were produced against membrane-enriched preparations of Leishmania pifanoi amastigotes either grown in axenic culture (P-1 through P-6) or isolated from macrophage cell culture (P-7 and P-8). Two mAb produced against promastigote membranes (P-9 and P-10) were found to be specific against this stage. Antibodies P-1 through P-8 on analysis by radioimmune binding only reacted with determinants present on amastigotes. mAb P-2, P-4, and P-8 also reacted with Leishmania amazonensis amastigotes but not promastigotes. No cross-reactions were found on any other species of Leishmania or with membranes of Trypanosoma cruzi epimastigotes or amastigotes. An indirect immunofluorescence assay using mAb P-1 through P-8 confirmed the stage specificity and binding to L. pifanoi axenically grown amastigotes, amastigotes within infected hamster tissue, and amastigotes within J774.1 macrophages. When Western blot analysis of amastigote membranes was conducted, one distinct group of molecules associated with L. pifanoi-specific determinants was identified. mAb P-1, P-3, P-5, P-7, and P-8 bound to molecules Mr 43 and 34 kDa. Promastigote-specific mAb P-9 recognized a diffuse pattern from 88 to greater than 200 kDa, and mAb P-10 localized a second class of proteins with Mr53 kDa. On immunoprecipitation of solubilized [35S]methionine-labeled amastigotes, mAb P-2 recognized a doublet of Mr 35 and 33 kDa and another doublet at Mr 17.5 and 13.5 kDa. mAb P-4 and P-7 each precipitated a band at Mr 34 kDa. These studies indicate that antigenically the axenically cultured amastigote is closely related to macrophage-derived amastigote. These mAb and/or purified protein Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of New World leishmaniasis.     

Leishmania tropica: characterization of a lipophosphoglycan-like antigen recognized by species-specific monoclonal antibodies.

Species-specific monoclonal antibodies to Leishmania tropica, T11 and T13-15, recognize membranal and secreted antigens. The membrane form of the antigen migrates on sodium dodecyl sulfate-polyacrylamide gels with a diffuse molecular weight from 15 to 50 kDa and can be labeled with palmitic acid, myoinositol, galactose, glucosamine, and inorganic phosphate. Both phosphate and sugar-labeled material were isolated from metabolically labeled promastigotes by affinity chromatography on antibodies coupled to Sepharose 4B. No binding to Ricinus communis agglutinin was observed. This material behaves like lipophosphoglycans from other Leishmania but contains unique species-specific epitopes. It is susceptible to cleavage by phospholipase C and after digestion no longer partitions into the detergent phase following a Triton X-114 extraction. All four monoclonal antibodies appear to recognize a carbohydrate epitope on the lipophosphoglycan since periodate treatment of this material bound to nitrocellulose essentially eliminated antibody binding. In addition, T15 binding could be blocked by 5 mM mannose-6-PO4 and fructose-1- or 6-PO4, but not by mannose, glucose, fructose, or the additional PO4 derivatives examined. The antibodies recognize a similar but not identical epitope, as demonstrated by a competitive radioimmunoassay using 125I-labeled T11, T13, and T15. Expression of surface antigen is elevated during the promastigote stationary phase.     

Subspecies- and species-specific antigens of Leishmania mexicana characterized by monoclonal antibodies

Monoclonal antibodies have been produced that are specific for the reference stocks of Leishmania mexicana species and subspecies L. mexicana mexicana(L11, M379), L. mexicana amazonensis (WR303, H6, LV72), and L. mexicana pifanoi (L20). The specificities of these antibodies were confirmed by analyses employing an indirect radioimmune binding assay and 107 stocks of New World Leishmania. The molecules associated with these species- and subspecies-specific determinants have been characterized by Western blot analysis and consist of mainly low m.w. (11,000 to 50,000) membrane-associated components.     

Species-specific antibodies to Leishmania tropica (minor) recognize somatic antigens and exometabolites

Monoclonal antibodies were raised against membranes of Leishmania tropica (formerly L. tropica minor). These antibodies do not react with membranes or homogenates prepared from any other species of Leishmania examined. The antigens identified by all four monoclonal antibodies reside on the external surface of the promastigote and are released into the culture medium. Immunoprecipitation of metabolically ([3H]glucose, -glucosamine, or [35S]methionine)-labeled, [125I]lactoperoxidase-labeled, or Western blot analysis of the parasites identified species-specific determinants residing on several different molecules with Mr between 12,000 and 140,000. All of the monoclonal antibodies appear to recognize determinants on a common [3H]sugar-labeled glycoconjugate, Mr = 32,000 to 44,000, but also react with additional components of 55,000, 80,000, 92,000, and 130,000 m.w. Antibody binding to the parasites is very sensitive to mild oxidation with periodate, suggesting that the species-specific determinants are carbohydrate in nature.     

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.     

Cell antigens recognized by rabbit antibodies specific for oligomannosyl determinants

Rabbit antibodies to cell wall mannans of various microbial strains and their mutants were found to be cross-reactive to cell carbohydrates of mammalian sperm and 4–6-day-old blastocysts. Immunochemical studies indicate that oligomers of α1→2, α1→3, α1→6, and probably also α→4 linked mannose residues of sperm carbohydrates are available for antibody binding. At least 80% of binding activity of a yeast mannan antibody to sperm can be effectively inhibited by specific haptens or digestion with exo-α-D-mannosidase, an enzyme activity highest in testicular tissue. In order to determine the role of this enzyme in the metabolism of the cross-reactive mannan antigens of sperm, the relative amount of a specific α-linked oligomannosyl determinant of bovine sperm from homozygous normals was compared to that of heterozygous carriers of α-mannosidase deficiency. Extensive cross-reactivity between the microbial and mammalian oligomannosyl determinants suggest that these are conserved structures in cell carbohydrates, although the organization of these units in the microbial cell wall lipopolysaccharide has very little similarity to the carbohydrate moieties of mammalian glycoproteins.     

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.     

Monoclonal antibodies against defined Leishmania antigens protect mice against infection by different species of Leishmania

Mice monoclonal antibodies (IgG) have been raised against Leishmania infantum promastigotes by fusing SP 2/0 myeloma cells and immunized mice splenic cells. The monoclonal antibodies have been detected by indirect immunofluorescence. In vivo tests showed that some of them could inhibit the life cycle of several Leishmania species from the Old and the New World. Studies of these protective monoclonals by the western blot technique showed the presence of three constant antigens (40 kD, 70 kD and 113 kD) amongst the Leishmania species studied.     

Monoclonal antibodies specific for v-abl- and c-abl-encoded molecules.

Monoclonal antibodies specific for regions of the transforming protein of Abelson murine leukemia virus were prepared. Antibodies directed against the kinase domain inhibited the autophosphorylation of v-abl proteins, and all of the antibodies reacted with the products of the murine and human c-abl loci.     

Procyclic tsetse fly midgut forms and culture forms of African trypanosomes share stage- and species-specific surface antigens identified by monoclonal antibodies

Procyclic culture form (PCF) trypanosomes were established from a bloodstream form population of cloned Trypanosoma brucei rhodesiense and were used to immunize mice for hybridoma production. Indirect immunofluorescence was used to select 10 hybridomas which secreted antibodies that bound to the surface of homologous living PCF. The antibodies reacted with PCF of several clones of T.b. brucei, T.b. gambiense, and T.b. rhodesiense, but not with PCF of T. congolense or T. vivax, or with promastigotes of several species of Leishmania parasites. The antigens were not detectable in ethanol/acetic acid-fixed bloodstream forms or in lysates of bloodstream forms of any of the T. brucei subspecies, and are thus species-specific and stage-specific markers. Selected monoclonal antibodies bound to procyclic trypanosomes taken directly from the midgut of infected tsetse flies, and to immature epimastigote forms in salivary probes, and may therefore be useful in epidemiologic investigations.     

Monoclonal antibodies to cell surface components of zoospores and cysts of the fungusPythium aphanidermatum reveal species-specific antigens

A panel of monoclonal antibodies (MAbs) designated PA1 to PA8 has been raised against cell surface components of zoospores and cysts of the pathogenic fungusPythium aphanidermatum. The antibodies were selected on the basis of binding assays using indirect immunofluorescence. Four binding patterns were observed: PA1 labeled the entire zoospore surface including both flagella, PA2 binding was restricted to the anterior flagellum, PA3–PA6 bound to the adhesive cell coat secreted by zoospores during encystment, and PA7 and PA8 labeled zoospores and the cyst cell wall. Electron microscopic immunogold labeling of zoospores showed that PA2 bound to the mastigonemes on the anterior flagellum. The MAbs were tested for binding to zoospores and cysts of several isolates ofP. aphanidermatum, and to zoospores and cysts of several species ofPythium, Phystophthora, Aphanomyces, andSaprolegnia. The results showed that the antigens recognized by MAbs PA1–PA6 were restricted toP. aphanidermatum, whereas those recognized by PA7 and PA8 occurred on all species tested.     

Diseases associated with specific HL-A antigens.

Significantly increased prevalences of particular HL-A antigens have been reported for many human diseases. The correlation is particularly striking in ankylosing spondylitis, Reiter''s syndrome, psoriasis and some immunopathic disorders, so that HL-A typing may be of great value in diagnosis. The possible mechanisms whereby these associations may occur suggest the cause of certain disorders, and further investiatation will likely help in the understanding of the pathogenesis of many diseases.     

Production and characterization of species-specific monoclonal antibodies against Leishmania donovani for immunodiagnosis

Sixteen species-specific monoclonal antibodies were produced against membranes of Leishmania donovani. These antibodies only reacted with determinants present on L. donovani. No cross-reactions were found with any other species of Leishmania or with membranes of Trypanosoma cruzi. An extensive analysis of the binding specificities of selected antibodies was carried out by using whole promastigote homogenates as antigen. Monoclonal antibodies D-1, D-2, D-3, and D-4 correctly identified all 44 L. donovani stocks from a cross-panel of 84 New and Old World Leishmania stocks. Antibodies D-1 and D-2 were also useful for species classification by immunofluorescence. No cross-reactions were observed with any other Leishmania species examined. Based on either Western blot and/or radioimmunoprecipitation analyses, five distinct groups of molecules associated with L. donovani-specific antigenic determinants were identified. These molecules range in m.w. from 18 to 84 kilodaltons. The antigenic molecules recognized by antibodies D-2, D-10, and D-13 are also recognized by antibodies present in sera from patients with visceral leishmaniasis (kala-azar). Kala-azar sera obtained from cases in both the Old and New World specifically compete with these monoclonal antibodies for the appropriate antigenic determinants in Western blot analysis. These monoclonal antibodies and/or the purified protein antigens may be useful in the development of a serologic assay for the clinical diagnosis of visceral leishmaniasis caused by L. donovani and in epidemiologic studies of leishmaniasis.     

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder

Summary Spleen cells from BALB/c mice immunized with cells derived from transitional cell carcinomas (TCC) of the human urinary bladder were fused with mouse myeloma Sp 2/0 Ag14 cells. Monoclonal antibodies from six established hybridomas were investigated for specificity in a cell ELISA and in indirect immunofluorescence against a large panel of fixed intact cells. Three of the antibodies reacted with half or more of the eight bladder tumors and with a few unrelated tumors. They did not react at all with malignant or normal cells of hematopoietic origin. A fourth antibody reacted with seven of eight bladder tumors. It also reacted weakly with a prostatic carcinoma, with five of six malignant or transformed B cell lines, and with a subpopulation of normal lymphocytes, but not with any of the other cells on the test panel. These four antibodies did not react with cells derived from normal urothelium. The results suggest that these antibodies might recognize cell-type-restricted antigens associated with malignancy. Another antibody reacted with almost all urothelium-derived cells. It also reacted with three of three melanomas but not with any other cells on the panel. The sixth antibody reacted with 32 of the 37 cells tested. The spectrum of reactivities displayed by the antibody suggested that it recognizes HLA antigens. Abbreviations used in this paper: TCC, transitional cell carcinoma of the urinary bladder; TAA, tumor-associated antigens; ELISA, enzyme-linked immunosorbent assay; NBCS, newborn calf serum; PBS, phosphate-buffered saline, pH 7.2; ALP, alkaline phosphatase; GDA, glutardialdehyde; BSA, bovine serum albumin; IF, indirect immunofluorescence; LCL, lymphoblastoid cell lines: B-lymphocytes transformed in vitro with Epstein-Barr virus     

Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica.

Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa.     

Monoclonal antibodies specific for keratan sulfate detect epithelial-associated carbohydrates.

Monoclonal antibodies that specifically recognize epitopes on keratan sulfate glycosaminoglycans were used in this study to identify carbohydrate epitopes associated with many, but not all, types of epithelial cells. Immunoreactive cells included: keratinocytes, sebaceous gland cells, eccrine sweat gland duct cells, salivary gland excretory duct cells, colon adenocarcinoma cells, embryonic chick lung epithelial cells, embryonic chick mesonephric and metanephric kidney epithelial cells, and selected embryonic chick neural tube cells. Depending upon the type of epithelium, epitopes were located either within the cytoplasm or were located on cell surfaces. These epitopes were shared by cells from both human and chick tissues, indicating the absence of species specificity. Not all anti-keratan sulfate antibodies were equally effective in identifying epithelial-associated epitopes. One of the seven antibodies employed in this study failed to detect epitopes in almost all epithelial tissues studied. Of the remaining six antibodies, three were more effective than the others in recognizing epithelial-associated epitopes. These data indicate that carbohydrates that are typically associated with extracellular matrix can also be associated with epithelial cells, but in a form that is not necessarily related to extracellular matrix. These antibodies should prove to be useful in studies of the development of epithelial cells and tissues.     

Monoclonal antibodies specific for African swine fever virus proteins.

We have obtained 60 stable hybridomas which produced immunoglobulins that recognized 12 proteins from African swine fever virus particles and African swine fever virus-infected cells. Most of the monoclonal antibodies were specific for the three major structural proteins p150, p72, and p12. The specificity of some monoclonal antibodies for the structural proteins p150 and p37 and the nonstructural proteins p220 and p60 indicated that proteins p150 and p220 are antigenically related to proteins p37 and p60. The association of some viral antigens to specific subcellular components was determined by immunofluorescence and analysis of the binding of monoclonal antibodies to infected cells. A host protein (p24) seemed to be associated with the virus particles.     

Nucleosomes as antigens. Characterization of determinants and cross-reactivity

Antibodies raised against chicken erythrocyte nucleosomes were characterized in terms of their binding to individual chromatin components and tested for cross-reactivity with chromatin from other species. Most of the proteins present in the immunogen elicited antibodies. Of the histones, H5 elicited the strongest response, followed by H2B, H2A, H3 and H4. In addition, antibodies specific for several non-histone chromosomal proteins (NHCPs) were present, especially those NHCPs which remain bound to DNA in 5.0 M urea, 2.5 M NaCl, at pH 5.0. No antibodies to native DNA could be detected. Strong cross-reactions were observed between the antinucleosome sera, and nuclei, chromosomes, or nucleosomes from other vertebrate (calf, African green monkey), insect (Drosophila melanogaster) and higher plant (Haemanthus katherinae) species. In the case of Drosophila the cross-reaction was shown to be mainly due to the C-terminal (63–128) portion of H2B with smaller contributions from H3 and H4. These results are discussed in relation to exposed antigenic sites on nucleosomes, and the extent to which they have been conserved during evolution. Also, parallels between the immunological response to nucleosomes and the anti-nuclear antibodies characteristics of systemic lupus erythematosus are considered.     

Monoclonal antibodies against a specific nonhistone chromosomal protein of Drosophila associated with active genes

Hybridomas secreting monoclonal antibodies have been produced by fusion of NS-1 mouse myeloma cells with the spleen cells of mice inoculated with a 60-65,000-mol wt fraction of proteins released from Drosophila embryo nuclei treated with DNase I. The antibodies secreted by the hybridomas were examined with polytene chromosomes of formaldehyde- fixed salivary gland squashes by an immunofluorescence assay. Most of the clonal antibodies obtained resulted in specific staining of the chromosomes relative to the cytoplasmic debris. In the case of clone 28, the antibodies showed a preferential association with sites of gene activity, both puffs and loci identified as puffing at some time during the third instar and prepupal period. In larvae that were heat shocked (exposed to 35 degrees C for 15 min before removal and fixation of the glands), the antibodies of clone 28 stained preferentially the induced heat-shock loci while continuing to stain most of the normal set of loci. The antigen for clone 28 was identified as a single protein of approximately 62,000 mol wt by using the antibodies followed by 125I- rabbit anti-mouse Ig to stain nitrocellulose replicas of SDS polyacrylamide gels of total chromosomal proteins. This study demonstrates that monoclonal antibodies can be used successfully in immunofluorescence staining of formaldehyde-fixed polytene chromosomes. The results verify the hypothesis that a specific nonhistone chromosomal protein is preferentially associated with the set of loci that includes both active sites and those scheduled to be active at some time in this developmental program. Such proteins may play a general role in the mechanisms of cell determination and gene activation.