Immunochemical distinction between two different chains of type IV collagen.

Antisera against mouse and human basement-membrane type IV collagen showed in radioimmunoassays distinct binding with large pepsin fragments obtained from the C-terminal portions of alpha 1 (IV)- and alpha 2 (IV)-chains. These reactions were specific for each constituent polypeptide chain. The data were confirmed by immunoadsorption, allowing the separation of antibodies with restricted chain specificity. Inhibition assays with CNBr peptides demonstrated the different localizations of antigenic determinants, which were either species-specific or shared by the human and mouse antigens.     

Studies of murine malaria antigens using monoclonal antibodies. Production, selection, and characterization of antibodies

A panel of ten monoclonal antibodies made against Plasmodium chabaudi and Plasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs to P. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted with P. chabaudi antigens. Of the MAbs to P. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.     

Separation of antigens from Sarcocystis species using chromatofocusing

Crude antigen preparations from bradyzoites of Sarcocystis species exhibit a high degree of cross-reactivity with antisera against heterologous Sarcocystis species, preventing the development of a species-specific immunological test for sarcocystiosis. In this study, we fractionated bradyzoite-derived protein extracts from Sarcocystis tenella, Sarcocystis arieticanis, Sarcocystis gigantea, and Sarcocystis muris by chromatofocusing and obtained distinct protein elution profiles for each species. We then examined the isolated protein fractions for antigenicity with homologous and heterologous reference sera in an enzyme-linked immunosorbent assay. Whereas some antigenic fractions of bradyzoite proteins had equally high reactivity with the homologous and heterologous sera, the reactivity of other fractions was 3-38 times higher with homologous serum than with heterologous sera. Mice immunized with less cross-reactive protein fractions of S. gigantea and S. muris bradyzoites produced a specific immune serum. Thus, it is possible to isolate species-specific antigens from crude mixtures of bradyzoite-derived Sarcocystis antigens for development of species-specific immunological tests for sarcocystiosis.     

Detection of cell-bound immunoglobulins by a radioisotopic micro-mixed hemadsorption reaction with technetium-99m-labeled erythrocytes.

The mixed hemadsorption (MHA) reaction detects antibodies reactive with cell surface antigens by means of antiglobulin-coated indicator erythrocytes. We have developed a radioisotopic modification which employs sheep erythrocytes (SRBC) that have been prelabeled with technetium-99m (99mTc), a high specific acitivity metastable gamma-emitter of short half life. The 99mTc MHA reaction was performed on human and murine cells cultured in Micro-test II plated with six replicate wells per serum dilution. Antibody activity in species-specific xenoantisera and mono- and polyspecific alloantisera was detected in high titer. The sensitivity of 99mTc micro-mixed hemadsorption was 2 times that of the visual assessment of mixed hemadsorption, 100 to 200 times that of the 125-I-mixed antiglobulin reaction and 500 to 1000 times more sensitive than indirect immunofluorescence. The assay system was applied successfully to confirm the species of origin of a panel of previously karyotyped human and mouse cell lines. Our results indicate that the 99mTc micro-mixed hemadsorption method is a rapid, sensitive, quantitative test for the detection of cell surface antigens and membrane reactive antibodies.     

Immunoglobulin production by a human-mouse somatic cell hybrid

The fusion of human lymphocytes and TEPC-15 mouse myeloma cells, which had not been adapted to culture, resulted in the establishment of in vitro hybrid cell cultures. Ten clones of this somatic cell hybrid were examined. There was preferential exclusion of human chromosomes: between two and five human chromosomes were identified in the hybrid clones by Giemsa banding. All of the clones had the mouse parental histocompatibility antigens, but only four clones also retained the human parental histocompatibility antigens. Secretion of parental immunoglobulin was determined by SDS-gel electrophoresis of species-specific immune precipitates. Synthesis of parental immunoglobulin by individual hybrid cells was determined by double label fluorescent antibody staining. Individual cells from six of the clones secreted and synthesized both human and mouse parental immunoglobulins. Three clones secreted only one parental immunoglobulin. Cells from one of these clones secreted and synthesized only human immunoglobulin. Cells from the remaining two clones secreted only one parental species of immunoglobulin but synthesized both human and mouse immunoglobulins. Finally, one clone did not secrete immunoglobulin, yet the individual cells synthesized both human and mouse parental species of immunoglobulin.     

Inhibitory effect of monoclonal antibodies on the growth of Babesia caballi

Monoclonal antibodies (mAbs) were produced against Babesia caballi (USDA strain) to define a species-specific antigen for use in diagnosis and vaccine development. Eight positive clones of B. caballi mAbs determined by indirect immunofluorescent antibody test were selected for purification and further characterisation. Confocal laser microscopy showed that the antigens recognised by the mAbs were located on the surface/cytoplasm, central part, and/or anterior end of B. caballi parasites, with five different reactive patterns. These mAbs seemed to be species-specific, since they did not cross-react with Babesia equi-infected erythrocytes or uninfected erythrocytes. In Western blotting analysis, 18, 20, 34, 36, 48, and 155 kDa proteins of B. caballi merozoites were recognised by six different mAbs. When added to in vitro cultures, four of the mAbs significantly inhibited the in vitro growth of B. caballi parasites. These results provide a rationale for evaluating antigens for the development of diagnostic methods or vaccines.     

The high background immune reactivity of mice to polymorphic determinants on xenogeneic erythrocytes: theoretical and practical implications

Background responses have been assessed by fusing lipopolysaccharide- (LPS) stimulated spleen cells from unimmunized mice with MOPC 315.43 myeloma cells and screening the hybrids for the production of antibody against chicken red blood cells (CRBC). Clones specific for CRBC represented about 1% of total hybrid clones (1000 to 5000 clones were obtained per mouse). The majority of the anti-CRBC clones (greater than 95%) secreted antibody against polymorphic CRBC determinants (present on CRBC from some but not all chickens) rather than species-specific determinants present on all CRBC. Some of the polymorphic determinants were linked to the B locus (the MHC of the chicken) and some were non-B antigens. The relative amount of these 2 categories varied slightly according to the mouse strain. These results agree well with the specificities of natural mouse antibody and rosette-forming spleen cells. The response of immunized mice against CRBC and human RBC was also selective for polymorphic determinants. These results have considerable importance for the use of xenogeneic RBC as "standard" antigens, and are interpreted in terms of a model for the advantages of genetic polymorphism as a protection against antigen mimicry by parasites.     

The host antigen phenomenon in experimental murine schistosomiasis: The transfer of 3-week old Schistosoma mansoni between two inbred strains of mice

A surgical technique for the transfer of 3-week old Schistosoma mansoni to the mesenteric veins of mice is described. Parasites grown in a donor C3H/StCrl strain survive on transfer to recipient C57BL/10J mice despite prior immunization with C3H/StCrl cells or skin grafts. The findings indicate that the ‘species-specific’ mouse host antigens associated with adult worms in xenogeneic transfers do not have an allogeneic basis in these two strains.     

Immunocontraception in mice using repeated, multi-antigen peptides: immunization with purified recombinant antigens

Two immunocontraceptive antigens (AgE and AgF) were constructed that included different combinations of highly species-specific peptides from the mouse reproductive antigens SP56, ZP3, ZP2, and ZP1 in the form of multi-antigen peptides (MAPs). Both AgE and AgF contained three tandem repeats each of ZP2 and ZP3 peptide epitopes and a single copy of a ZP1 peptide sequence all of which had previously been demonstrated to individually have immunodominant or contraceptive effects. In addition, AgF contained a single contraceptive peptide derived from SP56, the putative ZP3 receptor protein on sperm. The antigens were expressed and affinity purified as recombinant repeated multi-antigen (polyepitope) peptides using an Escherichia coli maltose binding protein (MBP) expression system. Female BALB/c mice actively immunized with these antigens in Freund's adjuvants produced variable serum antibody responses to the component peptides. Fertility rates for animals immunized with AgE (40%) and AgF (20%) were significantly reduced compared to MBP immunized mice (90%), but the reduction in fertility did not correlate with peptide-specific serum antibody levels. Ovaries from all immunized mice appeared histologically normal with no evidence of oophoritis. These results demonstrate that high levels of immunocontraception can be achieved in mice, without apparent side-effects, using species-specific immunogens that include repeated peptides from proteins involved in fertilization.     

Comparative immunoreactivity of mouse and hamster sperm proteins recognized by an anti-P26h hamster sperm protein

The binding of the spermatozoon to the zona pellucida is a species-specific phenomenon. We have previously shown that the binding of hamster sperm to the homologous zona pellucida involves a sperm 26-kDa glycoprotein, the P26h, originating in the epididymis. In order to establish to what extent this sperm protein is involved in the species-specific recognition of the egg's extracellular coat, we have compared the inhibitory properties of anti-P26h antibodies in a sperm-zona pellucida assay using hamster and mouse gametes. Anti-P26h IgGs inhibit, in a dose-dependent manner, gamete interactions in both species, although in a less efficient manner in the mouse than in the hamster. While anti-26kDa Fab fragments are as efficient as the intact IgG to inhibit hamster sperm-zona pellucida binding, they have no effect on mouse gamete interaction. ELISA, Western blot, and immunohistochemical experiments have been performed in order to characterize the mouse antigen(s) recognized by the anti-P26h antiserum. ELISA and Western blots showed that this antiserum recognized two proteins on mouse spermatozoa that are less reactive than the hamster P26h. These antigens are localized in the acrosomal region of epididymal spermatozoa of both species. These results indicate that the hamster P26H involved in zona pellucida interaction has certain unique epitopes, while others are common to the sperm of both species. © 1995 Wiley-Liss, Inc.     

Use of the Mycoplasma hominis 102-116 kD proteins as antigen in an enzyme-linked immunosorbent assay

Mycoplasma hominis surface structures involved in human immune response and in the pathogenesis of this bacterial infection are inadequately defined. Attempts have been made to identify M. hominis surface proteins, to determine the antigenicity of these polypeptides, and to examine antigens which could lead to the development of species-specific diagnostic tests. By means of Western blotting, using a pool of sera from patients with culturally proven vaginal infection, most antigens recognized were surface exposed. Among these proteins, antigens of molecular weights between 102 and 116 kD were most consistently revealed. These polypeptides were recovered by electroelution and assayed in an IgG-ELISA. The electroeluted antigen specificity was examined by ELISA and immunoblotting with different mycoplasma species. Electroeluted proteins may be effective and specific for establishing a reliable diagnosis test.     

Specific double diffusion microtechnique for the diagnosis of aspergillosis and paracoccidioidomycosis using monospecific antisera.

Using experimental reference sera against species-specific antigens of Aspergillus fumigatus and Paracoccidioides brasiliensis in a microdouble diffusion technique, a simple and specific test for the immunodiagnosis of aspergillosis and paracoccidioidomycosis has been developed. Only sera that produced lines of identity with either one of the bands formed by the anti-C2 or the anti-E2 reference sera were considered positive for aspergillosis or paracoccidioidomycosis, respectively. The sensitivity of the diagnostic test was similar to those of the classical double diffusion and the immunoelectrophoresis test. No false positives were found in sera obtained from patients affected by other mycoses, nor from healthy controls. The amount of reagents for the specific test was ten fold less than that required by the classical double diffusion test.     

Erythrocyte reagents in the study of Erysipelothrix antigens

The activity of species-specific and type-specific antigens in various preparations isolated from the bacterial mass of standard strains of Erysipelothrix, and also in bacterial cells was studied by means of prepared erysipeloid erythrocyte antigen (species-specific and with general type- and species-specificity) and antibody (species-specific, with general type- and species-specificity as also with type-specificity only) diagnosticums. It has been demonstrated that the activity of these antigens differs in preparations from different strains, depending on the method of extraction. An efficient method of serotyping of Erysipelothrix, based on agglutination of erythrocyte antibody diagnosticums, was proposed.     

Differential expression of mouse embryonic antigens TEC-1 and TEC-2 in the epididymis of four rodent species

The expression, properties and relationship of two mouse embryonic antigens (TEC-1 and TEC-2), which are defined by monoclonal antibodies, were investigated in the epididymis of four rodent species. Absorption analysis, indirect immunofluorescence microscopy and immunohistochemistry revealed that all the species studied contained in their epididymides, but not in testes, either TEC-1 (Chinese hamster), TEC-2 (guinea pigs, rats) or both TEC-1 and TEC-2 (mice) antigens. In an indirect immunofluorescence assay, the antigens were found on spermatozoa isolated from caudae epididymides of guinea pigs, rats and Chinese hamsters but not mice. On the other hand, the TEC-2 antigen, which is expressed on mouse eggs, was not detected on eggs from the other species studied. Immunolabeling of epididymal extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that both epididymal antigens have apparent molecular weights of greater than 200,000. In guinea pigs, rats and mice, the antigens were detected by a two-site sandwich radioantibody-binding assay in which the antigen is immobilized and detected with the same antibody; this indicates that several antigenic determinants were present on the same carrier. In mice, some carriers seem to express both TEC-1 and TEC-2 epitopes. In Chinese hamsters, TEC-1 antigen was only detected by the solid-phase assay, suggesting that in this species there are markedly fewer antigenic determinants per carrier molecule. Interspecies differences in the activities of epididymal glycosyltransferases and/or glycosidases appear to be the biochemical mechanism of the species-specific expression of these antigens.     

Characterization of Candida antigens by crossed-immunoaffinoelectrophoresis

The antigens of three Candida albicans strains (3153 A, 3156 B and CBS 1905) and one C. tropicalis strain were studied by means of crossed-immunoaffinoelectrophoresis with the corresponding polyvalent antisera. Most antigens (from 63.8% to 77.7% depending on the strain) were bound to concanavalin A-sepharose and about 20% to blue cibacron-sepharose for all the strains tested. Free concanavalin A, wheat germ lectin-sepharose and Helix pomatia lectin-sepharose revealed differences between C. albicans 3153 A and C. albicans CBS 1905 on the one hand and C. albicans 3156 B and C. tropicalis on the other, since affinity percentages were from 4.2 to 10.2 and from 14.2 to 20.0 respectively. Among 10 previously described species-specific antigens of C. albicans, 4 were never bound and 5 were bound to concanavalin A-sepharose which was considered an unsuitable agent for antigen purification since it retained 77% of C. albicans antigens. One important species-specific antigen was bound to blue cibacron sepharose and the corresponding purification could be undertaken. Similar results were found for 12 species-specific antigens of C. tropicalis. Blue cibacron-sepharose as well as wheat germ lectin or Helix pomatia lectin-sepharose were found suitable agents for purification of some of them.     

Study of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> antigenic structure to obtain a sensitin for design of species-specific diagnostic kits

A method for isolation of outer membrane proteins from Yersinia pseudotuberculosis, which are perspective for further application as sensitin for design of species-specific pseudotuberculosis antigenic diagnostic kits, has been modified. Common species-specific antigens of nine Y. pseudotuberculosis serovars (with molecular weight from 80.62 to 12.2 kDa) were detected by SDS-PAAGE electrophoresis and immunoblotting of the outer-membrane protein preparations. These antigens react with neither the rabbit experimental antiplague antiserum nor antiserum to 39 Y. enterocolitica serovars and normal rabbit serum.     

A dot-ELISA mimicry western blot test for the detection of swine trichinellosis

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody affinity chromatography was developed for detecting Trichinella spiralis infection in swine. The test was as sensitive as an ELISA using excretory-secretory products as antigen and western blot analysis, and nearly as specific as the western blot. The dot-ELISA detected all of 20 low infections (0.08-4.74 larvae per gram of diaphragm), most of them by 5-6 wk postinfection. Sera from 1,960 farm-reared swine were tested by conventional ELISA, dot-ELISA, and western blot. Of the 1,960 sera, 262 (13.4%) were considered positive on conventional ELISA, 16 (0.82%) by dot-ELISA, and 15 (0.77%) by western blot. The improved specificity was achieved by employing species-specific denatured antigens. More importantly, the dot-ELISA was much simpler to perform than western blot analysis. The principles employed in this test can be adapted to other infectious diseases, such as AIDS.     

Close Association of Virus-Specific Cell Surface (S) and H-2 Antigens in Ad12-Infected and -Transformed Mouse Cells

A virus-specific cell surface (S) antigen in adenovirus type 12 (Ad12)-transformed mouse cells has been assumed to be a direct target for cytotoxic thymus-derived lymphocytes (CTL). In this study, the spatial proximity between the S and H-2 antigens was determined by three different methods, the proximity and co-capping tests, and the test for blocking of CTL-mediated lysis by anti-H-2 serum. In the proximity test with Ad12-infected thymic and splenic lymphocytes, and an Ad12-transformed line of C3H/He (H-2^k) mouse cells, anti-H-2^k and anti-S sera reciprocally inhibited fluorescent-antibody staining of the opposite antigens. By contrast, anti-Thy-1, 2 serum as well as anti-Ia and anti-Ig sera failed to show any appreciable effect in this test, when paired with anti-S serum. In addition, the S and H-2 antigens co-capped in the infected thymic lymphocytes, and CTL-mediated lysis of the transformed cells was abrogated equally by treatment of cells with anti-S and anti-H-2 sera. These results clearly demonstrate that there is a close proximity between the S and H-2 antigens on the surface of Ad12-infected and -transformed mouse cells.     

Chromosomally depleted interspecific hybrid cell clones selected with cytotoxic antisera: Utilization in the study of control of murine leukemia virus host-range

A chromosomally stable mouse-Chinese hamster hybrid cell line was subjected to five rounds of selection with cytotoxic antisera raised in rabbits against either the parental mouse 3T3 cells or the parental Chinese hamster Wg-1 cells. Routine karyological analysis of clones isolated at each stage of serum selection revealed that treatment with either serum resulted in a limited loss of chromosomes (compared to the untreated hybrid cell cultured in parallel) and that the pattern of chromosome loss could not be correlated with the particular antiserum used for selection. However, more detailed analysis with the SSC-formamide C-banding technique, which identifies chromosomes containing a mouse centromere region, demonstrated that while large-scale chromosome loss was not achieved as a result of antiserum selection, the limited loss of chromosomes did, in fact, reflect a specific depletion of chromosomes in response to treatment with cytotoxic antiserum. Specific chromosomal elimination was shown to occur as early as the first round of antiserum treatment. Antigenic analysis of the serum-selected clones revealed a quantitative decrease in the expression of the species-specific surface antigens selected against, but no qualitative loss of antigens was detected. The results suggest that treatment with cytotoxic antiserum may select for clones that have lost specific chromosomes bearing genes regulating the expression of species-specific surface antigens, rather than for those demonstrating large-scale depletion of chromosomes bearing the corresponding structural genes. Some of these chromosomally depleted hybrid cell clones have been used (along with pseudotype viruses containing the genome of vesicular stomatitis virus within the envelope of murine leukemia virus, VSV [MuLV]), to study the mechanisms regulating MuLV replication in Chinese hamster cells. The results indicate that the restriction of MuLV replication in Chinese hamster cells operates at two levels: (a) an inability to adsorb to or penetrate Chinese hamster cells; and (b) an additional intracellular block which is dominant in the mouse-Chinese hamster hybrid cell clones examined. This latter block is presently under study.     

NK recognition of target structures: is the transferrin receptor the NK target structure?

That the transferrin receptor acts as a target antigen for human NK cells has previously been suggested. In this study we used two models to examine the hypothesis that the transferrin receptor is recognized by NK cells. In the first model, we employed mouse cloned NK cells in conjunction with the species-specific monoclonal antibody R17 217, which binds to the murine transferrin receptor. We show that there is no correlation between the amount of transferrin receptor expressed on targets and the susceptibility of these targets to NK lysis or NK binding in cold target competition assays. In the second model, we used human NK cells and transferrin receptor-positive transformants as targets. These transformants were derived from mouse L cells transfected with human DNA and selected for the presence of human transferrin receptor. Results show that, in contrast to the mouse system, there is a correlation between the expression of the human transferrin receptor on targets and the ability of these targets to competitively inhibit the lysis of K562 by NK cells. However, because inhibition is not complete, other cell surface antigens probably play a role in human NK-target interactions.