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.     

Biochemical and serological characteristics of soluble yeast phase antigens of Histoplasma Capsulatum

Soluble antigens of whole yeast-phase cells were extracted with a 0.1 M phosphate buffer containing 0.1 M sodium chloride and 0.02% iodoacetate. After being separated by differential filtration into fractions less than or greater than 50,000 daltons, these antigens were purified by molecular sieve and chromatographic separations on ionic exchange resins. Two high molecular weight fractions obtained from diethylaminoethyl-cellulose (DEAE) at pH 8.0 and 7.0 with tris (hydroxymethyl) aminomethane (Tris) buffer were M antigens; those obtained at pH 4.0 and 4.0 with salt were H antigens. The four fractions had protein to carbohydrate ratios of 7.3, 14.0, 8.4, and 6.5 respectively, and all had essentially the same amino acid composition with no methionine and tyrosine and little histidine, arginine, phenylalanine and lysine. They had high concentrations of glucose, less mannose and traces of galactose. The low molecular weight fractions had the new complex Y antigen, M antigen, and H antigen with protein to carbohydrate ratios of 1.4, 1.4 and 0.3 respectively. The amino acid and sugar composition of Y antigen strongly resembled the composition of the low molecular weight H and M antigens. Unlike the high molecular weight antigens, these low molecular weight antigens had methionine in relatively high concentrations; they had the same sugars as their respective high molecular weight counterparts. The yeast phase antigens differed from their respective mycelial counterparts in the following ways: glucose was the major sugar in the yeast phase with less amounts of mannose and traces of galactose, whereas in the mycelial antigens, mannose was the major sugar, with lesser amounts of galactose, glucose, and hexosamine. The H and M antigens of the yeast phase had high concentrations of glycine and alanine, whereas in the mycelial phase, these antigens had high concentrations of threonine and proline; the H and M antigens of the yeast phase had 5 to 16 times the protein to carbohydrate ratio observed for the same antigens of histoplasmin.     

Immunological studies with an m-deficient histoplasmin skin-test antigen

Previous studies have shown that a single skin test to histoplasmin may induce complement-fixing antibodies or M precipitins (or both) to histoplasmin in histoplasmin-sensitive, but serologically negative, individuals. Ideally a skin-test antigen should be one which detects hypersensitivity without stimulating humoral antibodies. Histoplasmin skin-test antigens presently used contain both H and M antigens. The present study was undertaken to evaluate an histoplasmin skin-test antigen deficient in the M component but containing the H antigen. Thirty histoplasmin-hypersensitive subjects were bled prior to administration of the experimental skin-test antigen and at various time intervals thereafter. Only six of the thirty hypersensitive subjects showed serological responses. The sera of the six, however, only showed weak precipitin reactions, five showed M bands, and only one showed an M and an H band. None showed complement-fixation titers with either the yeast or mycelial antigens of Histoplasma capsulatum. Our data suggest that the use of a skin-test antigen purified to contain only H component would detect histoplasmin hypersensitivity without inducing antibodies and would eliminate false-positive serological reactions caused by the M component.     

Development and Use of a Polyvalent Conjugate to Differentiate Histoplasma capsulatum and Histoplasma duboisii from Other Pathogens

Previous investigations have demonstrated the existence of five Histoplasma capsulatum serotypes. Available specific fluorescent-antibody reagents stain only four of the five serotypes. Antibodies produced against the most complete H. capsulatum serotype were labeled with fluorescein isothiocyanate to develop a reagent specific for H. capsulatum that was reactive with all the known serotypes. The unadsorbed reagent not only stained all the H. capsulatum serotypes, but it also stained cultures of Blastomyces dermatitidis, H. duboisii, several Candida species, and a variety of other fungi. Adsorption of the conjugate with antigens of C. albicans produced a reagent that intensely stained only H. capsulatum, H. duboisii, and B. dermatitidis. Differentiation of B. dermatitidis from the Histoplasma species was accomplished by application of a B. dermatitidis specific fluorescent antibody to antigens positive with the H. capsulatum reagent. At present, differentiation of H. capsulatum from H. duboisii may be accomplished only by animal inoculation. Our data substantiate the antigenic relationships hypothesized earlier, and they indicate that H. capsulatum shares at least two antigens with the other fungi that were studied.     

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

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.     

Factors Influencing the Production of H and M Antigens by Histoplasma capsulatum: Effect of Physical Factors and Composition of Medium

Stagnant culture methods have permitted only limited physiological studies of the production of H and M antigens by Histoplasma capsulatum because, with such methods, antigen production is uncontrolled. In this investigation, a shake culture method was used to convert yeast-phase inoculum to mycelialphase growth at 25 C. Results strongly suggest that the release of H and M antigens relates to autolysis of the cells. Among the factors influencing production of H and M antigens under shaking conditions, choice of strain was the most important. Alterations of carbon or nitrogen source or variations in amino acid to carbohydrate ratios had limited influence on antigen production. With a strain that produced both H and M antigens, however, proportions of titers of M to H antigens could be made to vary considerably by changes in the medium, the pH, and the temperature. Results suggest that the source of M antigen during autolysis is enzymatic dissolution of the cell wall. The source of H antigen is more obscure. Production of both antigens may be differentially controlled under conditions of good reproducibility by a correct choice of strain and manipulation of culture medium.     

Serological differences among the dermatophytes.

Antigens were prepared from young mycelial growth of 20 species of dermatophytes, and tested by double diffusion against homologous and heterologous antisera raised in rabbits. 48 distinct antigens were recognised by the procedures used. Although there were a considerable number of common reactions, there were significant differences between species and groups. Species of Microsporum, with the exception of M. gypseum and M. persicolor, form a coherent group distinct from Epidermophyton and Trichophyton. All Trichophyton species investigated with the exception of T. ajelloi, were serologically closely related. M. gypseum, T. ajelloi and M. persicolor showed affinities with both Microsporum and Trichophyton, and possibly form an intermediate group. The system used does not permit differentiation of species by serological means, but gives a new dimension to the inter-relationships of these fungi.     

Technique for the detection of Toxoplasma gondii antigens in mouse urine

A simple and rapid staphylococcal coagglutination test for the detection of Toxoplasma gondii antigens in mice urine is described. A suspension of protein-A containing Staphylococcus aureus coated with rabbit hyperimmune serum was used as reagent. The sensitivity of the antigen assay was found to be at least 118 ng of the antigen protein per ml. No coagglutination was observed when the reagent was challenged against antigenic solutions of other parasites. The suitability of the method for detecting antigens of T. gondii in urine samples was studied by experimental toxoplasma infection in mice. Before the staphylococcal test, the urine samples were double serially diluted in 0.1 M PBS. From the second day on all samples from infected mice were positive at 1/16 dilution. At this dilution, all samples from non infected mice were negative or did not produce coagglutination. This method might be used in the rapid etiological diagnosis also in human cases of acute toxoplasmosis.     

Factors Influencing the Production of H and M Antigens by Histoplasma capsulatum: Development and Evaluation of a Shake Culture Procedure

Studies were undertaken to improve the production of histoplasmin for use in complement-fixation tests and in the determination of H and M antibodies. A shake culture method performed at 25 C was developed with a yeast-phase inoculum. Eight strains of Histoplasma were tested in three synthetic media to evaluate the effects of strain and medium on H and M antigen production. Intrastrain variation was negligible, and antigen production was reproducible. All of the strains produced H antigen; six strains produced both H and M antigens, and two produced only H antigen. The time of H and M antigen appearance varied with the medium; M antigen appearance was dependent upon the strain and medium used. Titers of M antigen appeared to be greater in stagnant culture.     

Use of preparative isoelectric focusing in a Sephadex gel slab to separate immunizing and growth facilitating moieties in crude 3 M KCl extracts of a murine fibrosarcoma

Summary Crude 3 M KCl extracts of the methylcholanthrene-induced fibrosarcoma of C3H/HeJ mice, MCA-F, were demonstrated to contain two fractions, one inducing tumor resistance and the other facilitating the outgrowth of neoplastic cell challenge. In immunoprotection tests in syngeneic C3H/HeJ mice, optimal doses of crude solubilized tumor antigen afforded only a 28% reduction in growth compared with saline-treated controls. When crude extracts were fractionated by preparative isoelectric focusing (pIEF) in a slab of superfine Sephadex G-75, significant biologic activity was demonstrated in two fractions. Fraction (Fr) 1, pI 2.5–3.6, induced potent tumor facilitation, increasing the tumor size by more than 100%, while Fr 15, pI 5.8–6.0, engendered resistance that reduced their respective biological effects to MCA-F, but not the antigenically unrelated MCA-D tumor. Thus 3 M KCl extracts contain at least two biologically active components, one immunoprotective and one tumor-facilitating. Since the weak immunoprotective activity of crude materials may represent the vectorial effect of these antagonistic components, subsequent molecular characterization of both moieties may afford insight into the complex response of hosts toward tumors. Furthermore, TSTA purified by the rapid method of isoelectric focusing may be a more suitable reagent for immunotherapy than the parent crude 3 M KCl extracts by virtue of the absence of facilitating antigens.Abbreviations CE crude 3 M KCl extract - pIEF preparative isoelectric focusing - Fr fraction from pIEF - MCA-F and MCA-D antigenically different methylcholanthrene-induced fibrosarcomas of C3H/HeJ mice - TSTA tumor specific transplantation antigens     

Procedures for the production and separation of H and M antigens in histoplasmin: Chemical and serological properties of the isolated products

Two strains of Histoplasma capsulatum were required to prepare maximum yields of H and of M antigen from histoplasmin. The antigens were separated and partially purified by a series of procedures yielding an overall recovery of 70 to 90% of the individual antigens. Stable products suitable for use as reference products were obtained when the final purification step employed DEAE-cellulose with phosphate buffer elution at increasing molarity and decreasing pH. A final step of purification of each antigen with slab acrylamide gel electrophoresis gave products which were highly reactive and specific in a variety of serological tests with sera from persons with proven cases of histoplasmosis and with natural infections of heterologous deep mycoses. These antigens were maximally active at concentrations of 2 to 16 g protein in the complement fixation, capillary precipitin, microimmunodiffusion, or immunoelectrophoresis tests; 0.5 g gave a maximum delayed cutaneous hypersensitivity reaction in homologously infected animals and caused no appreciable reaction in control animals. Although these antigens appeared to be specific when tested with sera from persons with natural infections, the M and H antigens demonstrated the presence of an additional antigen reacting with sera of rabbits immunized with cell membrane and cell particulate fractions of Blastomyces dermatitidis. After purification by electrophoresis, both the H and M antigens of some preparations showed some decomposition and loss of reactivity after storage at 5 C for more than six months. The overall results suggest that the purified H and M antigens of Heiner (12) have multiple serological reactivity and may function in precipitin reactions, complementfixing reactions, hemagglutination of formalin-fixed goose red blood cells, and as antigens for delayed cutaneous tests.     

Analysis of lipopolysaccharides of Bacteroides fragilis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroblot transfer

Abstract Lipopolysaccharides (LPS) from three strains of Bacteroides fragilis were run on SDS-polyacrylamide gels and stained with silver. Each LPS produced a similar pattern, consisting of a series of regularly spaced discrete bands which decreased in intensity as they increased in M _r value. Electroblot transfer from duplicate SDS gels onto nitrocellulose membrane were reacted with antisera raised to whole cells of two of the strains and antigens were visualised with horse-radish peroxidase-antirabbit-IgG conjugate and colour reagent. Results revealed that the two lowest M _r bands of the LPS preparation (rough LPS) represented common antigens.     

Lateral organization of proteins in the chromatophore membrane ofRhodospirillum rubrum studied by chemical cross-linking

The organization of proteins in the chromatophore membrane, particularly of the reaction center and the light-harvesting polypeptide, was examined by the use of a hydrophobic and a hydrophilic cross-linking reagent, namely DSP (dithiobis-succinimidyl propionate) and glutaraldehyde. The linkage of proteins was studied by SDS polyacrylamide pore gradient electrophoresis. DSP was shown to link proteins within the core of the membrane. The subunit H of the reaction center is linked with DSP at a low concentration, either with itself or with other membrane proteins but not to the subunits M and L. In isolated reaction centers the subunits H are exclusively linked with each other. With increasing concentrations of DSP the bands of the subunits M, L, and the light-harvesting polypeptide disappear simultaneously from the gel, suggesting that these proteins are linked together. This hypothesis is supported by the finding that reaction centers isolated from chromatophores treated with DSP retain an appreciable amount of light-harvesting polypeptide. With increasing concentrations of the hydrophilic cross-linking reagent glutaraldehyde, the bands of all the three subunits of the reaction center, H, M, and L, progressively disappear from the gel, suggesting that they are linked together. The light-harvesting polypeptide remains free when this reagent is used.     

Use of laser-nephelometer to quantitate lipoprotein-bound immunoglobulins in human sera. Importance of the diluting reagent

If the method using Hyland LAS-RTM kit reagent recommended by the manufactorer yielded excellent results to measure immunoglobulins free in serum, the same is not true when one quantitates immunoglobulins bound with antigens, especially with lipidic antigens. Since the specificity of Hyland antisera is not the cause of this difference, we have studied therefore the effect of the diluting buffer on the antisera, on the blank, and its consequences on the assay. The abnormalities encountered in the results were due to precipitation of lipoproteins and immune complexes by the diluting buffer yielded in the kit. We avoided entirely these errors by preparing a blank, not in NaCl 0.15 M, but in the kit reagent used to dilute the antisera.     

Monoclonal antibodies to Mycoplasma pneumoniae antigens]

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

Soluble Antigens for Immunofluorescence Detection of Histoplasma capsulatum Antibodies

Soluble antigens from Histoplasma capsulatum in the mycelial and yeast phase were purified by gel filtration, fixed onto paper discs, and employed in an indirect immunofluorescence procedure to detect antibody in sera from individuals infected with H. capsulatum. The elution patterns of crude histoplasmin passed through Sephadex G-200 revealed two minor peaks of protein showing immunofluorescence, complement fixing, and precipitating-antigen activity. A large peak containing the pigment and other low molecular weight materials showed no serological activity. A polysaccharide antigen obtained from fragmented, deproteinized yeast-phase cells was reactive in the fluorescent-antibody test but showed no antigen activity in complement fixation or precipitin tests. Although certain sera from culturally proven cases of blastomycosis, coccidioidomycosis, and cryptococcosis reacted with the purified Histoplasma antigens, preliminary evaluation indicated that the immunofluorescence technique may be of value as a screening procedure for the serodiagnosis of histoplasmosis.     

The effect of hydrogen peroxide on the growth of microscopic mycelial fungi isolated from habitats with different levels of radioactive contamination

The effect of hydrogen peroxide ( 10(-9)-10(-1) M) on the mycelial growth of the fungi Alternaria alternata, Cladosporium cladosporioides, Mucor hiemalis, and Paecilomyces lilacinus has been studied. The growth of fungi isolated from habitats with a background level of radioactive contamination was stopped by H2O2 concentrations equal to 10(-3) and 10(-2) M, whereas the growth of fungi that were isolated from habitats with high levels of radioactive contamination was only arrested by 10(-1) M H2O2. The response of the different fungi to hydrogen peroxide was of three types: (1) a constant growth rate of fungal hyphae at H2O2 concentrations between 10(-9) and 10(-4) M and a decrease in this rate at 10(-3) M H2O2, (2) a gradual decrease in the growth rate as the H2O2 concentration was increased, and (3) an increase in the growth rate as the H2O2 concentration was increased from 10(-7) to 10(2)-5 M. The melanin-containing species A. alternata and C. cladosporioides exhibited all three types of growth response to hydrogen peroxide, whereas the light-pigmented species M. hiemalis and P. lilacinus showed only the first type of growth response. A concentration of hydrogen peroxide equal to 10(-1) M was found to be lethal to all of the fungi studied. The most resistant to hydrogen peroxide was found to be the strain A. alternata 56, isolated from the exclusion zone of the Chernobyl Nuclear Power Plant.     

Identification of a 75 kDa highly immunodominant antigen from Mycobacterium smegmatis and cross-reactivity with other species

Three monospecific antibodies MSAb 1, MSAb 2 and MSAb 3 were raised in BALB/C mice against respective antigens. M. smegmatis whole cell lysate was first separated on SDS-PAGE and randomly chosen bands were cut and then used for immunization. Antibodies were collected as ascites by injecting mice with myeloma cell line P3X63 Ag 658.4. All the three antibodies showed high reactivity with denatured antigens compared to native. Different extent of cross-reactivity was observed as evident from ELISA. MSAb1 recognized a 75 kDa immunodominant antigen from M. smegmatis and 66 kDa from M. tuberculosis (H37Ra), respectively. An apparently similar molecular weight antigen shown to be present in M. tuberculosis (H37Ra) an avirulent strain and BCG, but not recognized by MSAb1. The 75 kDa antigen has a stimulatory effect on T-cell proliferation.     

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.