Antigenic make-up of Trypanosoma cruzi culture forms: identification of a specific component.

A comparison of Trypanosoma cruzi water soluble antigens with those of stercorarian and salivarian trypanosomes, and Leishmania using immunoprecipitation in gels and immunoelectrophoresis, with the aid of hyperimmune rabbit serum and heterologous adsorptions showed the following. 1) There is a high complexity of soluble antigens of T. cruzi and T. rangeli. 2) At the intraspecific level our results demonstrated the antigenic stability of T. cruzi when maintained in vitro, and that there was quantitative antigenic consistency of the culture forms of different strains of T. cruzi from diverse geographic and parasite sources. At the interspecific level, the antigenic relationships between T. cruzi and the other Trypanosomatidae were established, as follows: 6/10ths of the antigens are shared by stercorarian species (T. dionisii, T. rangeli); 4/10ths by a salivarian trypanosome (T. brucei); and 3/10ths by Leishmania (L. donovani, L. mexicana). 3) Among the 4/10ths of antigenic components specific to T. cruzi, one component was characterized by its antigenicity and immunogenicity in natural and experimental infections, and in immunization experiments; this component was specific to T. cruzi when compared to the other Trypanosomatidae antigens.     

Antigenic analysis of three strains of Mycoplasma arginini by two-dimensional immunoelectrophoresis.

The occurrence of species-specific and strain-specific antigens in three strains of Mycoplasma arginini (G-230, leonis and 23243) was studied by two-dimensional immunoelectrophoresis. Approximately 20 antigenic components could be detected in each strain. It was possible to analyze 6 to 7 major and distinct components from each strain by two techniques: "enhancement" where antigen to an additional strain is added to the first phase of the electrophoresis which increases the size of common peaks and "suppression" where antiserum to an additional strain is incorporated in the second phase whereby peak size of components to which both sera have antibody are decreased. A total of 10 distinct antigens were recognized. Electrophoretic mobilities relative to bovine albumin ranged from 0.2 to 1.08. Three components were common to all strains; two of these represented major amounts of material. Four components represented strain-specific components. Unique fast components were found both in strains 23243 and G-230. Three antigens were distributed into only two of the three strains. The electrophoretic mobilities of some common antigens were quite different between strains.     

Immunoelectrophoretic analysis of erythrocytic stages of Plasmodium yoelii and P. chabaudi

Triton X-100 extracts of erythrocytes infected with Plasmodium chabaudi and P. yoelii were analysed in crossed immunoelectrophoresis with rabbit antisera. The parasite origin of the antigens detected was assessed by metabolic radiolabelling of the parasites with 35S-methionine. About 12 immunoprecipitates were obtained with both extracts and their homologous antiserum. Cross-tests showed that the two parasite strains were very similar antigenically. Species-specific antigens could, however, also be demonstrated. Two antigens, present on both P. yoelii- and P. chabaudi-infected erythrocytes, were located on the surface of the host cell membrane as judged from 125I-labellings with lactoperoxidase. Experiments with phenyl-Sepharose showed that most of the antigens detected were hydrophilic and none of them reacted with concanavalin A.     

Antigenic variability of Aspergillus fumigatus strains.

The effect of culture media, temperature of incubation, and continuous shaking of cultures, on the reactivity and yield of antigens of Aspergillus fumigatus were evaluated. It was found that AOAC medium was superior to Czapek medium and shake cultures yielded better results than stationary cultures. However, antigens from stationary cultures in AOAC medium incubated at 30 degrees C for 3 weeks were equally as good as antigens obtained from 2-week-old shake cultures. Antigens from 11 selected strains of Aspergillus fumigatus were used to test antibody activity in 33 sera from patients with various forms of aspergillosis and 35 normal controls by the agar gel double-diffusion method. The results showed that the reactivity of individual antigens varied from 42 to 87%, indicating that antigens from more than one strain of Aspergillus fumigatus may be used. The cross-reactivity between strains were studied by two-dimensional immunoelectrophoresis. The use of polyacrylamide gel electrophoresis and crossed immunoelectrophoresis in the quality assurance of Aspergillus antigens is discussed.     

Sex differences in the pattern of esterase-active antigens in microsomes from various rat strains

Esterase-active antigens present in male and female liver microsomes isolated from three different rat strains (Sprague-Dawley, Wistar and Dark Agouti) were characterized in crossed immunoelectrophoresis in combination with a zymogram method for esterase activity. No qualitative but some quantitative differencies were encountered between the sexes. Thus, two out of ten antigens were present in significantly lower and one in significantly higher concentrations in male than in female microsomes, demonstrating that although the overall esterase activity in liver may be similar for males and females, the concentration of the individual antigens does vary between the sexes. No qualitative or quantitative differences in the pattern of esterase-active antigens were found between the different strains.     

Immunoelectrophoresis of mycobacterial antigens

Polyvalent antiserum to culture filtrate of H37 Ra M. tuberculosis was raised in rabbits. Monospecific antiserum was raised against M. tuberculosis antigen-5, prepared from the culture filtrates by immunoabsorbent affinity chromatography. On immunoelectrophoresis, antigen-5 demonstrated single precipitin arc against polyvalent and monospecific antisera. The culture filtrate antigen demonstrated multiple precipitin arcs against polyvalent antiserum and single precipitin are against monospecific antiserum. Antigen-5 could be isolated and characterized from the culture filtrate of H37 Ra M. tuberculosis. Immunoelectrophoresis could be one of the method to characterize the mycobacterial antigens prepared in the laboratory.     

Partial immunochemical identity between (Na+ + K+)-ATPase and a membrane component extractable with chloroform: methanol

An IgG fraction prepared from an antiserum against a holoenzyme preparation of (Na+ + K+)-ATPase precipitated a single antigen when samples of holoenzyme were subjected to crossed immunoelectrophoresis but precipitated an additional, immunochemically-related antigen when a plasma membrane-enriched fraction was subjected to crossed immunoelectrophoresis under the same conditions. The immunochemically-related antigen could be extracted from the plasma membrane fraction with CHCl3:CH3OH.     

Wall-associated protein antigens of Streptococcus mutans.

When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29 000, while B had a molecular weight of 190 000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue.     

CHARACTERIZATION OF POLLEN ANTIGENS FROM AMBROSIA L. (COMPOSITAE) AND RELATED TAXA BY IMMUNOELECTROPHORESIS AND RADIAL IMMUNODIFFUSION

The pollen antigens of various Ambrosia and related species were studied to learn whether substances closely related to antigen E (the major allergen of Ambrosia artemisiifolia) were present. After conventional immunoelectrophoresis, pollen extracts from six Ambrosia species each produced at least one pronounced precipitin line with antiserum for purified antigen E. Electrophoretic mobility was the same for several species (A. artemisiifolia, A. bidentata, A. psilostachya, and A. trifida) but was relatively lower for A. acanthicarpa and A. ambrosioides. Precipitin rings were also produced when pollen extracts of the various Ambrosia species were subjected to radial immunodiffusion in agarose which contained antiserum for purified antigen E. There was great variation among the Ambrosia species with respect to precipitin ring diameters. The variation may be due to differences among species in content of the antigen E-like substances or to altered interaction with the immobilized antibody. Crossed (2-dimensional) immunoelectrophoresis was shown to be useful for characterizing Ambrosia pollen antigens. Pollen extracts from A. artemisiifolia produced eight pronounced precipitin bands and at least eight faint, relatively fast-moving bands after crossed immunoelectrophoresis with antiserum against a whole pollen extract from the same species. One of the pronounced bands contained antigen E.     

Arylamidases of rat liver and chemically induced hepatomas. II. Preparation and characterization of monospecific antisera against two distinct arylamidase-active antigens.

Monospecific antisera were prepared against the most prominent arylamidase (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) active antigen in plasma membranes (the plasma membrane arylamidase) and lysomal content (the lysosomal content arylamidase), respectively. Plasma membrane extract and lysosomal content were allowed to react in crossed immunoelectrophoresis against their homologous antisera. The electrophoretic plates were washed extensively, dried and subsequently stained for arylamidase activity.The particular immunoprecipitates were thus identified and could be excised to be used for immunizations. The two resulting antisera precipitated the arylamidase used for immunization, but failed to be monospecific as they precipitated additional antigens. These antisera with restricted specificity against some plasma membrane and lysosomal content antigens, respectively, were used to produce immunoprecipitates intended for new attempts to prepare monospecific antisera by a second cycle of immunizations. A monospecific antiserum against the plasma membrane arylamidase was thus obtained, while a third cycle of immunizations was needed to get a monospecific anti-lysosomal content antiserum. The plasma membrane arylamidase showed ATPase activity also after precipitation with the monospecific antiserum, thus still retaining its characteristics as a multienzyme complex.     

Immunoblot analysis of trypomastigote excreted-secreted antigens as a tool for the characterization of Trypanosoma cruzi strains and isolates

An analysis of antibody recognition of Trypanosoma cruzi exoantigens by immunoblotting revealed a unique banding pattern that seems to be characteristic of each strain or isolate. Trypomastigote excreted-secreted antigens (TESA) present in supernatants of LLC-MK2 cells infected with 5 strains and 10 isolates of T. cruzi produced 13 different immunoblotting patterns. The same bands were observed when probed with acute-phase Chagas' disease serum or with serum from a rabbit immunized with the repetitive domain of T. cruzi transialidase recombinant protein (anti-shed acute-phase antigens). Three similar patterns were observed with TESA from 3 human isolates that probably belong to the same T. cruzi strain. When clone CL Brener, clone CL-14, and CL parental strain were analyzed, the same bands were observed, although they presented different biological behavior. These results suggest that immunoblotting analysis of TESA may be a useful tool for characterization of T. cruzi strains and isolates.     

Schistosoma mansoni: reduced worm burdens in mice immunized with isolated Fasciola hepatica antigens.

Mice immunized with Fasciola hepatica antigens are protected to a challenge exposure with Schistosoma mansoni cercariae. This protection is manifested in a 28–54% reduction in worm burdens of the immunized mice over controls. The protective antigens could be isolated by antibody affinity chromatography and react with an antiserum to S. mansoni. These antigens, when used to immunize mice, result in 50–60% reduction in worm burdens over controls. One protective antigen has been isolated which when used alone or in combination with a B-cell adjuvant such as polyadenylic-polyuridylic acid (poly (AU)) results in 56–81% reduction in worm burdens over controls. The complexity of the F. hepatica adult worm antigens was demonstrated by Laurell crossed immunoelectrophoresis. Crossreactivity with antisera to S. mansoni and S. japonicum and the presence of one common antigen between the two genera have been demonstrated.     

Synthesis of thiostatins (major acute-phase alpha 1 proteins) in different strains of Rattus norvegicus

1. Two different thiostatin proteins were detected by crossed immunoelectrophoresis in plasma of the following inbred strains of Rattus norvegicus; Wistar, Sprague Dawley, Hooded Wistar, Lewis, Porton-Albino, ACI, Long Evans, and Katholiek, a mutant strain of the Brown Norway. 2. Only one thiostatin protein was detected using crossed immunoelectrophoresis in plasma from the Buffalo rat. 3. Comparison of partially purified thiostatins from Buffalo, Wistar, and Sprague Dawley rats in polyacrylamide gels showed that the thiostatin protein in Buffalo rat plasma corresponded to thiostatin 1 of the two thiostatins of the Wistar and Sprague Dawley rats. 4. Thiostatin 1 mRNA and thiostatin 2 mRNA, of approximately 1.7 kilobases, were both demonstrated in RNA from Buffalo rat liver.     

Soluble and membrane-bound enzyme-active antigens of rat-liver lysosomes.

Secondary lysosomes were isolated from rat liver and separated into a soluble and a membrane fraction. Plasma membranes and microsomes were also isolated and antisera against the various fractions were prepared in rabbits. Lysosomal content and detergent-solubilized membrane fractions were analysed in two-dimensional immunoelectrophoresis (crossed immunoelectrophoresis). The immunoprecipitates were stained by histochemical procedures for different enzyme activities such as phosphatases, non-specific esterase, arylsulphatase, glycosidases and L-leucyl-beta-naphthylamidase. When lysosomal content was tested against its corresponding antiserum, 17 different precipitates could be seen. Most of the enzyme activities tested were shown to reside separately in one or a few precipitates each. In contrast, when the membrane extracts were investigated, a more polymorphic pattern of enzyme-active precipitates appeared. Thus, when lysosomal membrane extracts were reacted with homologous antiserum 11 precipitates with acid phosphatase activity were obtained. Several of the antigens were electrophoretically different and immunologically non-identical. As expected from the biology of secondary lysosomes, many of their antigens were also found in microsomes and/or plasma membranes, but several antigens unique for lysosomes were detected concomitantly. Closer analysis of these results indicated that several seemingly identical enzyme-active proteins occurred both in soluble and membrane-associated forms. However, while many of the membrane antigens expressed 2-4 different enzyme activities, only one activity was detected in individual precipitates of the lysosomal content. Thus, acid phosphatase activity was found together with esterase activity in three membrane-associated antigens. The precipitates formed by two of these also stained for arylsulphatase and nucleoside tri-, di- and monophosphatase activities. L-Leucyl-beta-naphthylamidase activity was found in one additional acid-phosphatase-active precipitate.     

Relevance of Antigenicity of Candida Albicans Growth Phases to Diagnosis of Systemic Candidiasis

The antigenicity of the two growth phases of Candida albicans has been compared by using crossed immunoelectrophoresis and double-diffusion techniques. Qualitative and quantitative differences in antigenic composition between the phases have been revealed and moreover a specific mycelial antigenic component demonstrated.It is postulated that the use of specific mycelial antigens for routine diagnostic precipitin testing in patients with suspected systemic candidiasis would give more reliable results than at present obtained with yeast cell antigens.     

An immunologic study of the secretory products of rat Clara cells

Lungs of adult rats were lavaged with normal saline containing 0.25 mM phenylmethylsulfonyl fluoride. The surfactant pellet was removed by centrifugation and serum proteins in the lavage were removed by affinity chromatography using rabbit anti-rat whole serum antiserum. The residual proteins, thought to represent products of secretory cells, were used as the immunogen to inject rabbits. The resulting antiserum was absorbed with affinity columns of rat serum and rat liver extract. The gamma globulin fraction of the unbound antiserum was found to react with two proteins in the lavage by immunodiffusion and crossed immunoelectrophoresis. The antiserum specifically stained, by the immunoperoxidase method, a subpopulation of cells consistent in morphology with Clara cells lining the bronchioles and bronchi. The antigens were detectable, by immunohistochemistry, in rat fetus at 19 days of gestation, a progressive increase in the antigen content was noted with increasing gestational age and an adult pattern was noted at 2 weeks of age. In adult animals the intracellular content of the antigens appears to be about twofold greater than their content in the lavage fluid.     

ANTIGEN—ANTIBODY CROSSED ELECTROPHORESIS OF WATER-SOLUBLE RAT BRAIN ANTIGENS

A method of separation by antigen-antibody crossed electrophoresis was applied to the study of water-soluble rat brain antigens. Five different rat brain preparations were used as antigens for immunization. The extract from Triton X-100 treated brains gave the best antibody response. An antiserum containing 27 precipitating antibodies was obtained. The preparation of antigens for immunoelectrophoresis was investigated. Treatment with demineralized water gave the highest number of antigen-antibody precipitates. Treatment with detergents and sonication gave a higher total protein yield, but the number of precipitates was unaffected.     

Specific recognition of the neuronal cell surface by an antiserum raised plasma membrane preparations of immature rat cerebellum

A brain specific antiserum was prepared by immunizing rabbits with a crude membrane fraction from 8-day old rat cerebella. In immunofluorescence studies the antiserum labeled the perikarya and processes of cultured cerebellar neurones. In contrast, other cell types, encountered in cerebellar cultures including astrocytes, endothelial cells and fibroblasts, were consistently unstained. The antiserum when used in crossed immunoelectrophoresis with Triton X-100 solubilized brain extracts reacted predominantly with one antigen that could be identified as the D2 protein.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Immunological aspects of infection of 6 inbred strains of mice by 3 different strains of Trypanosoma cruzi

We evaluated humoral and cellular immune responses in 6 inbred mouse strains (BALB/c, B-10, C3H, A/J, AKR and DBA) infected with 3 Trypanosoma cruzi strains (Peruvian, 21 SF and Colombian), which are the standards for the 3 strains Types of Andrade's classification. Negative delayed-type hipersensitivity reactions to parasite antigens were evidence of suppressed cell-mediated immunity. An early drop of IgG1 and rise of IgM levels were observed in almost all mouse strains infected by any T. cruzi strain. Elevation of IgG2a and/or IgG2b levels was higher in resistant mouse strains. Anti-T. cruzi antibody levels (Indirect immunofluorescence and ELISA) did not correlate with survival. Despite some differences among mouse strains there was a definition of an overall pattern of host response and the maintenance of biological standards which characterize the basic types of T. cruzi strains.     

Characteristics of a high molecular weight extracellular protein of Streptococcus mutans

A high molecular weight protein antigen, designated P1, has been isolated from the culture fluid of chemostat-grown Streptococcus mutans strain Ingbritt and shown to be free of other antigens including glucosyltransferase. Antiserum against the protein was used in rocket immunoelectrophoresis to confirm and extend the previous observation that there were major differences in the amount of the protein produced under different growth conditions. Physico-chemical and serological studies indicated that protein P1 was indistinguishable from antigens B, I/II and IF isolated in other laboratories. Mammalian tissue cross-reactivity of protein P1 was demonstrated by binding of antiserum to P1 to sections of normal rabbit tissues, particularly heart. There was also a statistically significant increase in the number of mononuclear leucocytes in heart tissue of rabbits which had been injected with protein P1, when compared with the levels in control uninjected rabbits; injection with whole cells of S. mutans Ingbritt did not produce this effect.