Serological Comparison of Two Races of Fusarium oxysporum f. sp. lupini

Water-soluble mycelial antigens from two physiological races (2 and 3) of Fusarium oxysporum L. sp. lupini were compared by tandem-crossed immunoelectrophoresis. When antiserum against race 3 was tested some 50 antigens were detected. The two races had apparently almost identical antigenic patterns differing only in one antigen specific to race 3. This specific antigen might be related to the virulence of this fungus.     

Partial purification and characterization of mold antigens commonly found in foods.

Rapid methods are needed for detection of molds in foods; therefore, an enzyme-linked immunosorbent assay was developed. The extracellular and mycelial antigens for Mucor, Aspergillus, Cladosporium, and Geotrichum species were partially purified and characterized. The molecular masses of the mycelial and extracellular antigens, as determined by size exclusion chromatography, ranged from 4.5 x 10(5) to 6.7 x 10(5) Da. There was only one main antigenic peak separated by Sepharose CL-4B and concanavalin A-Sepharose columns for Mucor, Cladosporium, and Geotrichum mycelial and extracellular antigens, but there were two for Aspergillus mycelial antigens and three for Aspergillus extracellular antigens. These antigens contained 10 to 50% protein which was part of the active site since protease digestion significantly decreased antigenic activity. Neutral sugars, ranging from 13 to 75%, made up the rest of the active site, and < 1% phosphate was detected in mycelial antigens. Geotrichum, Cladosporium, and Aspergillus antigens contained mainly glucose, galactose, and mannose. Mucor antigens contained these sugars plus fucose. The percentage of sugars differed between the mycelia and extracellular antigens. Enzymatic digestion and competitive inhibition tests using different sugar derivatives showed that galactosyl residues with beta linkages were immunodominant for Aspergillus, Geotrichum, and Cladosporium antigens and mannosyl residues with alpha linkages were immunodominant for Mucor antigens.     

Immune responses to yeast and mycelial forms of Candida albicans in intraperitoneally infected mice

Candida albicans E-139 produced pure mycelial and yeast cultures in a low sulphate medium at different temperatures. The influence of the morphological phase, dose and viability of the fungi on the kinetic of delayed-type hypersensitivity (DTH) and anti-mycelial and anti-yeast antibodies have been studied in mice injected intraperitoneally. The mycelial form elicited higher DTH levels than the yeast phase. This effect seems to be related to its antigenic properties. The effect of dose on the immune response depends on the viability of the fungus. The mycelial cytoplasmic antigens were more effective than the yeast ones in detecting antibodies induced during the experiments, particularly during the later stages of the observation periods, suggesting that such antigens may be useful in the serodiagnosis of Candida infections.     

Factors Affecting the Antigenicity of Trichophyton rubrum

Nitrogen determinations, performed upon the mycelia of Trichophyton rubrum, indicated that both the total nitrogen to mycelial weight ratio and the protein nitrogen to mycelial weight ratio decreased as the age of the mycelia increased. An increase in nitrogen concentration in the medium produced an increase in the total nitrogen to mycelia weight ratio, but did not necessarily increase the protein nitrogen to mycelial weight ratio. The optimal nitrogen source concentration which produced the highest protein nitrogen to mycelium ratio was found to be considerably less than that recommended in most standard Sabouraud medium formulations. Antisera to antigen preparations, grown on low concentrations of Multipeptone, produced more lines in the gel diffusion reaction than did antisera to antigens grown on standard concentrations of Multipeptone. Antisera to antigenic preparations from 2-week-old mycelia exhibited better and sometimes more lines than those of antigens prepared from 1- or 3-week-old mycelia, regardless of the nitrogen concentration in the medium. Dialysis and storage of the antigen produced no change in the quality of the precipitin lines, even though both processes involved considerable loss of Lowry protein. Immunofluorescence studies showed that young mycelia were more antigenic than the old mycelia, since a substantial degree of cell wall fluorescence was exhibited by the young mycelia, especially at the hyphal tips. Older mycelia lacked this fluorescence. An extracellular antigen was also found to be associated with the young mycelia, but cytoplasmic fluorescence was not observed.     

Immunobiological characteristic of antigens from the parasitic phase of Coccidiodes immitis

The possibility of obtaining serologically active antigens from tissue spherules extracted from the lungs of albino mice infected with C. immitis has been demonstrated. Unlike the method of obtaining spherules in vitro, the accumulation of sufficient quantities of tissue spherules does not involve special difficulties. To obtain more pure and specific antigens from crude tissue spherules material, chromatographic separation on a column with Sephadex G-100 is recommended. Antigens obtained from the spherules in vivo have been found to be more specific and active in serological and allergic reactions in comparison with the corresponding antigens from the mycelial form or from spherules in vitro and can be used in the diagnosis of coccidioidosis as well as in the study of the mechanism of cellular immunity.     

Modulation of antigenicity of mycelial antigens during developmental cycle of Karnal bunt (Tilletia indica) of wheat

Indirect enzyme linked immunosorbent assays (ELISA) were developed using polyclonal antibodies against soluble cytoplasmic (SCA) and insoluble cell wall antigens (ICWA) for monitoring modulation of mycelial antigens during growth cycle of T. indica. With SCA, continuous decrease in ELISA reactivity was observed in maturing fungus cultures, suggesting that SCA were expressed predominantly during early vegetative phase and their decreasing role was apparent as the fungus matures possibly towards sporogenous mycelium. In case of ICWA, the reaction profile showed an increase up to exponential phase of growth probably due to increase in the cell division and branching of mycelium. But later, ICWA antibody reactivity was decreased which may be due to conversion of mycelial phase to sporogenous phase, a quiescent stage of growth. Characterization of changes in antigenic configuration during developmental cycle of Tilletia indica by these antibodies could prove to be useful in identification of developmentally related and virulence marker(s).     

Electron-microscopic study of the serological affinity between the antigenic components of phages T4 and DDVI.

In an investigation of the antigenic fine structure of phages T4 and DDVI with the use of the neutralization reaction and electron-microscopic observation of the phage-antibody complexes, it has been possible to establish that the head of phage T4 consists of proteins which have antigenic determinants of two types: The first type is identical to the antigens of the head of phage DDVI, and the second type is apparently absent in phage DDVI. The phage DDVI head contains mostly determinants which are common to the phage T4 head, since it was not possible to detect antigenically specific components in the phage DDVI head. The tail sheaths of phage T4 and DDVI appear to be identical in the antigenic respect. A difference has been observed in the fibers and the base plates of the phages investigated. The presence of the following three types of antigens has been established: 1) common to phages T2, T4, and DDVI, 2) common to phages T4 and DDVI, and 3) specific for each phage investigated.     

Antigenic differences between corresponding DNA-dependent RNA polymerases from both phases of Histoplasma capsulatum

Reaction of antisera generated against DNA-dependent RNA polymerase III from the yeast phase of the dimorphic fungusHistoplasma capsulatum with the purified RNA polymerases of the organism indicated that there were major antigenic differences in the corresponding enzymes from the yeast and mycelial phases. These differences were most marked when the enzymes were reacted against antisera obtained early in the immunization and less after hyperimmunization. This extends our earlier demonstration of extreme differences in the corresponding RNA polymerases from two differentiated states of the same organism.     

Immunological studies of the activity of Aspergillus flavus Link. I. Antigen analysis and evaluation of its chemical composition and toxicity

The aim of this study was to get insight into chemical structure and toxigenicity of antigenic preparations obtained from A. flavus Link strain isolated from industrial environment. A microbiol multiplication and an antigenic fractions preparation scheme is presented. The method proposed allows to obtain antigens from culture filtrate (APP), mycelial extract (AEM), and two subfractions obtained from AEM: supernatant--AS and precipitated--API. The strain tested did not show aflatoxigenic activity and antigenic fractions obtained were free of aflatoxin B1, B2, G1, G2 in concentrations detectable by thin-layer chromatography. A chemical composition of the antigenic fractions was tested. A content, depending on fractions, of proteins ranged from 32.0 to 74.5 micrograms/ml, of sugars from 15.0-44.5 micrograms/ml, phosphorus 0.5-1.5 micrograms/ml, and nitrogen 2.5-4.9 micrograms/ml. Toxicity of APP and AEM antigens designated for laboratory animals immunisation was also determined. The values of LD50 for APP preparation was 2.00 mg/mouse and for AEM - 2.75 mg/mouse. These data give evidence of moderate toxicity of these preparations.     

Identification and purification of the specific antigen of Paracoccidioides brasiliensis responsible for immunoelectrophoretic band E.

A new purified antigen (E2) of Paracoccidioides brasiliensis mycelial growth phase was isolated by immunoadsorption from a crude metabolic soluble extract of the fungus. The antiserum prepared in a rabbit by inoculation of E2 antigen developed only one immunodiffusion line with the crude metabolic extract. Findings on immunological analysis showed that E2 antigen is the antigenic component of immunoelectrophoretic band E. The isolated antigens did not possess detectable alkaline phosphatase activity. It reacted in immunodiffusion tests with all the sera (14/14) from P. brasiliensis infected patients containing precipitating antibodies.     

Serological potential for fungal identification

POLONELLI, L. & MORACE, G., 1989. Serological potential for fungal identification. Specific antigens are valuable for the identification of fungal cultures. Early attempts to immunoidentify fungi were hampered by heterogeneity of antigens, antibody preparations and use of improper serological procedures. In recent years, the double diffusion exoantigcn technique has proved to be the most effective method for immunological identification of mycelial fungus cultures. Additional advances in perfecting methods occurred with the adoption of improved reference antisera obtained either through absorption or by immunizing animals with selected immunoelectrophoretic arcs or precipitin bands (reference antigens). Preliminary studies have shown that serodiagnostically important antigens may be used for accurately and rapidly identifying hyaline as well as dematiaceous fungi. Agglutination techniques consisting oflatex particles sensitized with rabbit anti- Cryptococcus neoformans globulin or Candida monospecific antisera permit the detection of specific yeast antigens in a few minutes. In spite of the great success obtained with the antigen test methods, some limitations in these procedures are apparent. The major problem derives from the occurrence of extensive cross reactions among congeneric species.
Hybridoma technology permits the production of uniform and standardized antisera (monoclonal antibodies) reacting with species-specific or strain-specific antigenic determinants (Western blotting technique) and the availability of functional pure epitopes (affinity chromatography). The current value and limitations as well as further avenues for the advance of the different procedures are reported.     

Trial of purification and serological characterization of human and murine transplantation antigens]

Human and murine antigens are purified by exclusion chromatography on Sephadex and preparative polyacrylamide gel electrophoresis and their purification stage checked by analytical methods. The study of antigenic preparations consists of electrofocusing, molecular weight estimation and chemical determinations. A cytotoxicity inhibition test with specific alloantisera reveals the antigenic potency. A microtest using 51Cr labelled tumoral cells has been achieved for the analysis of murine preparations.     

Antigenic composition of different forms of Sh. sonnei]

Data are presented on the study, with the aid of immunophoresis, of the antigenic composition of the I, II phases and R-forms of Sh. sonnei. From 5 to 7 antigens differing by thermoresistance, electrophoretic and diffuse mobility were found in the composition of microbial cells of various forms Sh. sonnei. Differences between the I phase on the one hand and cells of the II phase and R-form, on the other hand, consisted both in the quantitative composition of components of their thermostable O-antigens and in the structure of specific lipopolysaccharides of the forms.     

Analysis of Clonorchis sinensis antigens and diagnosis of clonorchiasis using monoclonal antibodies.

Clonorchis sinensis is a common parasite of man in Korea. Researches on the specific antigens of C. sinensis would be valuable not only because those elucidate the molecular characteristics of this fluke but also because it is applicable to immunodiagnosis. Although many monoclonal antibodies have been used in the field of parasite immunology, few articles on monoclonal antibodies against C. sinensis have been published so far. The aim of this study was to analyze C. sinensis antigens recognized by monoclonal antibodies, and to set up ELISA-inhibition test using C. sinensis specific monoclonal antibodies for improved specificity of immunodiagnostic tests. By fusion between spleen cells of the mice immunized with C. sinensis water-soluble crude adult worm antigens and plasmacytoma cells of mouse origin, 29 hybridoma clones secreting anti-C. sinensis monoclonal antibodies were made, and 8 clones among those were found specific. After cell cloning, isotypes of 6 selected specific monoclonal antibodies were determined to be IgG1, IgG2b and IgA. Four exposed antigenic determinants of natural infection were recognized by different specific monoclonal antibodies. By enzyme-immunoelectrotransfer blot, 10 KD, 34 KD antigenic determinants were found to be reacted with CsHyb 0714-20, CsHyb 0605-10 monoclonal antibodies, respectively. The antigenic determinant recognized by CsHyb 0714-20 monoclonal antibody was revealed to be located at the surface and parenchyme of a parasite by indirect immunofluorescent antibody technique, and those reacted with CsHyb 0605-10, CsHyb 0714-25 monoclonal antibodies were found at the parenchyme and intestine. The antigenic determinant reacted with CsHyb 0605-23 monoclonal antibody was found mainly around the uterine eggs. Four antigenic determinants recognized by specific monoclonal antibodies were all found to be present in the early eluted fractions of C. sinensis antigens separated by Sephadex G-200 gel filtration. By conventional ELISA, 75% of clonorchiasis cases were found positive, but 7.1% of normal controls and 37.5% of paragonimiasis cases showed false positives. However, by ELISA-inhibition test using C. sinensis specific monoclonal antibody (CsHyb 0605-23), 77.1% of clonorchiasis cases were found positive, and there were no false positives in normal controls or paragonimiasis cases, indicating 100% specificity.(ABSTRACT TRUNCATED AT 400 WORDS)     

The preparation and partial characterization of antigenic fractions obtained from the mycelial walls of several Aspergillus species

Extracts with immunological activity were prepared from Aspergillus fumigatus, A. flavus, A. terreus, A. niger and A. nidulans. In each case crude mycelial wall was extracted with an aqueous solution of Triton X-100 giving detergent-soluble material. Further fractionation was achieved by removing the detergent from this solution; the resultant precipitate was removed by centrifugation, and the aqueous supernatant was used as a source of soluble antigens. The sensitivity of these preparations was compared with that of water-soluble antigenic material, prepared from whole macerated mycelium, by double diffusion and counterimmunoelectrophoresis using homologous antisera and sera from patients suffering from aspergilloma and allergic bronchopulmonary aspergillosis. The selectivity of these antigenic preparations was monitored with heterologous antisera raised in rabbits. Batch variability was analysed for one strain of A. fumigatus using chemical and immunological methods. The nature of the antigenic sites involved in these reactions was investigated by studying the susceptibility of the preparations to proteolytic hydrolysis, periodate oxidation and concanavalin A treatment. The total protein and carbohydrate content of each fraction was determined and the constituent sugars analysed in an attempt to correlate chemical composition with antigenic activity.     

Analysis of the Crystal Antigens of Bacillus thuringiensis by Gel Diffusion

S ummary . The antigenic patterns of the crystal protein inclusions of Bacillus thuringiensis were determined. No specific antigenic patterns associated with previously described subgroups of this species were found. A larger number of categories of crystal antigen than of flagellar antigen or esterase type were found. In some cases isolates indistinguishable in classifications based on flagellar antigens or esterase types could be subdivided by their crystal antigenic pattern. The use of crystal antigens in classification is discussed.     

Identification of cell structure antigens of the causative agent of melioidosis by a 2-dimensional immunoelectrophoresis method

The antigenic composition of some cell structures of P. pseudomallei has been studied and the chemical nature of the antigens has been determined by the method of two-dimensional electrophoresis. In some cell components common antigens have been detected; at the same time these components have been found to possess their own characteristic antigenic complexes. The place of the cell structure antigens in the total antigenic structure of P. pseudomallei has been determined.     

Comparative studies of Trypanosoma vespertilionis Battaglia and Trypanosoma dionisii Bettencourt & França

SYNOPSIS. In diphasic blood agar media Trypanosoma vespertilionis developed spheroid clusters as compared to rather long, sausage-shaped (sometimes branched) clusters formed by Trypanosoma dionisii. The former species attained a greater population density (∼6 × 10⁷ organisms/ml) than the latter (∼ 2 × 10⁷ organisms/ml). Greater numbers of epimastigotes, some in active binary divisions, were observed during the logarithmic phase of growth, and morphologic changes occurred during cultivation which correlated with increased acidity and a depletion of glucose. Maximum numbers of trypomastigote forms were found during the stationary and early death phases. Most of the forms observed after 20 days were sphaeromastigotes. Glucose concentrations decreased to 0 M in T. vespertilionis and to 4.4 × 10⁻⁵ M in T. dionisii cultures during the stationary and death phases. By the 12th day of incubation cultures of T. vespertilionis were more acid (pH 5.5) than those of T. dionisii (pH 6.5). No antigenic changes during cultivation of each of the parasites were detected by immunodiffusion. Trypanosoma vespertilionis and T. dionisii contained common and specific antigens. At least 2–3 common antigens were detected in extracts reacted against heterologous antisera. Specific antigens were observed as nonidentical lines formed by extracts reacted against homologous and heterologous antisera and with antisera absorbed with heterologous antigens. At least 2 specific antigens were evident in extracts of T. vespertilionis and 1 in extracts of T. dionisii.     

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and nonendemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti -Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.     

Specific fluorescent antiglobulins for the detection and identification of blastomyces dermatitidis yeast-phase cells

Summary Two lots of rabbit anti-Blastomyces dermatitidis globulins were conjugated with fluorescein isothiocyanate. These reagents stained brightly elements of the yeast and mycelial phases of 10 strains ofB. dermatitidis. In addition, the labeled antibodies cross-reacted with elements of the yeast and mycelial phases of 7 strains ofHistoplasma capsulatum and cells of numerous other heterologous fungi. Adsorption of one lot of labeled antibodies twice with yeast cells ofH. capsulatum and once with elements ofGeotrichum candidum rendered the conjugate specific for the yeast phase ofB. dermatitidis. Three adsorptions with yeast cells ofH. capsulatum followed by a single adsorption with elements ofG. candidum rendered the second conjugate specific for yeast-phase cells ofB. dermatitidis. The specific reagents did not react with the mycelial phase of this fungus.