Abstract
Salt gradient elution of crude histoplasmin on CM-Sepharose CL6B at pH 3.0 was used in essentially a one-step procedure to isolate the h, m, and non-m antigens ofHistoplasma capsulatum and free them of any c antigen common to other pathogenic fungi. The h antigen fraction was pure; the isolated m and non-m antigens contained less than 0.1% of the c antigen present in the original preparation. The residual c antigen in these fractions was removed by affinity chromatography on Affigel-10 coupled to aBlastomyces dermatitidis fungal antiserum. The final preparations of h, m, and non-m were pure when tested by immunodiffusion (ID), sodium-dodecyl-sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), or electrofocusing. The antigens were serologically stable at neutral pH for at least three months at 10°C. The relative molecular weights, isoelectric points, and amino acid composition of the antigens are described.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Literature Cited
Bradley G, Pine L, Reeves MW, Moss CW (1974) Purification composition, and serological characterization of histoplasmin-H and M antigens. Infect Immun 9:870–880
Department of Health, Education and Welfare, Food and Drug Administration (1977) Skin test antigens. Fed Register 40 (II):52674
Dickerson QH Jr, Busey JF (1968) Chromatographic separation of h and m antigens from histoplasmin. Proc Soc Exp Biol Med 128:654–658
Dubois M, Gillies KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356
Green JH, Harrell WK, Gray SB, Johnson JE, Bolin RC, Malcolm GB (1976) H and M antigens ofHistoplasma capsulatum: preparation of antisera and location of these antigens in yeast-phase cells. Infect Immun 14:826–831
Green JH, Harrell WK, Johnson JE, Benson R (1979) Preparation of reference antisera for laboratory diagnosis of blastomycosis. J Clin Microbiol 10:1–7
Green JH, Harrell WK, Johnson JE, Benson R (1980) Isolation of an antigen fromBlastomyces dermatitidis that is specific for diagnosis of blastomycosis. Curr Microbiol 4:293–296
Greene CH, De Lalla LS, Tompkins VN (1960) Separation of specific antigens ofHistoplasma capsulatum by ion-exchange chromatography. Proc Soc Exp Biol Med 105:140–141
Heiner DC (1958) Diagnosis of histoplasmosis using precipitin reactions in agar gel. Pediatrics 22:616–627
Kaufman L (1972) Micro agar-gel immunodiffusion tests. In: Manual of standardized serodiagnostic procedures for systemic mycoses. I. Agar immunodiffusion tests. Washington DC: Pan American Health Organization, Department of Research Development and Coordination, pp 9–14
Kaufman L (1981) Current methods for serodiagnosing systemic fungus infections and identifying their etiologic agents. Estratto Igiene Mod 76:427–442
Kaufman L, Standard P (1978) Improved version of the exoantigen test for identification ofCoccidioides immitis andHistoplasma capsulatum cultures. J Clin Microbiol 8:42–45
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:280–285
Lowry OH, Rosenbrough NA, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Mancini C, Carbonara AO, Heremans JF (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 1:235–254
Morrissey JH (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117:307–310
Pine L (1977) Histoplasma antigens: their production, purification and uses. Contrib Microbiol Immunol 3:138–168
Pine L, Gross H, Malcolm G, George JR, Gray SB, Moss CM (1977) Procedures for the production and separation of H and M antigens in histoplasmin: chemical and serological properties of the isolated products. Mycopathologia 61:131–141
Pine L, Gross H, Malcolm GB, Green JH, Barbaree JM, Harrell WK, Suggs MT, Blumer SO, Kaufman L, Ajello L, Smith SJ, May JC (1981) Evaluation of candidate international reference reagents and a microimmunodiffusion test for the identification of precipitins to the H and M antigens of histoplasmin. J Biol Stand 9:513–530
Pine L, Smith SJ, Gross H, Barbaree JM, Malcolm GB (1981) Studies on the thermal degradation of the H and M antigens of lyophilized histoplasmin. Sabouraudia 19:55–70
Pine L, Fears MB, Malcolm GB (1983) Heated histoplasmin as a control for non-specificity in the complement fixation test for histoplasmosis. Sabouraudia 21:233–237
Sparkman DH, Stein WH, and Moore S (1958) Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 30:1190–1206
Squire PG (1964) A relationship between the molecular weights of macromolecules and their elution volume based on a model for Sephadex gel filtration. Arch Biochem Biophys 107:471–478
Standard PG, Kaufman L (1976) Specific immunological test for the rapid identification of members of the genusHistoplasma. J Clin Microbiol 3:191–199
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Green, J.H., Pine, L. Preparation of h and m antigens ofHistoplasma capsulatum free of heterologous antigens. Current Microbiology 12, 209–215 (1985). https://doi.org/10.1007/BF01573333
Issue Date:
DOI: https://doi.org/10.1007/BF01573333