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. 1974 May;9(5):870–880. doi: 10.1128/iai.9.5.870-880.1974

Purification, Composition, and Serological Characterization of Histoplasmin—H and M Antigens

Georgia Bradley 1, Leo Pine 1, Michael W Reeves 1, C Wayne Moss 1
PMCID: PMC414900  PMID: 4137149

Abstract

To obtain purified H and M antigens suitable as primary standards in the serological diagnosis of histoplasmosis by agar gel double-diffusion tests, H and M reactive components of histoplasmin were fractionated by column chromatography by using Sephadex G-100, Sephadex G-200, and diethylaminoethyl cellulose. Six fractions from diethylaminoethyl cellulose were reactive in agar gel double-diffusion, complement fixation, and capillary precipitin tests. When examined by electrophoresis on acrylamide gel, one M antigen (fraction 2, molecular weight greater than 200,000) and two H antigens (fractions 5 and 6, molecular weight greater than 200,000) each gave essentially a single protein band. In agar gel double-diffusion and complement fixation tests with sera from patients with proven cases of histoplasmosis, blastomycosis, or coccidioidomycosis, these two fractions of H antigen and the one of M antigen reacted only with sera from proven or suspect cases of histoplasmosis and showed reactivity with those sera known to contain only the anti-H or anti-M antibody, respectively. Fraction 2 (M antigen) and fractions 5 and 6 (H antigens) had carbohydrate-to-protein ratios of 1.60, 0.77 and 0.78, respectively. Both antigens contained galactose, glucose, mannose, and hexosamine, with mannose being the predominant sugar. Fraction 2 was characterized by a high proline and glucose content, whereas fractions 5 and 6 contained higher concentrations of galactose, mannose, glycine, and alanine. Each of these products appeared to separate into two active fractions, one of a molecular weight greater than 200,000 and one of a molecular weight less than 35,000. The M antigen component of fraction 2 was still characterized by a high proline content, whereas the H antigen components of fractions 5 and 6 had a high content of glutamic acid, serine, glycine, and proline.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Besdine R. W., Pine L. Preparation and description of high-molecular-weight soluble surface antigens from a group A Streptococcus. J Bacteriol. 1968 Dec;96(6):1953–1960. doi: 10.1128/jb.96.6.1953-1960.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. CUMMINS C. S., HARRIS H. The chemical composition of the cell wall in some gram-positive bacteria and its possible value as a taxonomic character. J Gen Microbiol. 1956 Jul;14(3):583–600. doi: 10.1099/00221287-14-3-583. [DOI] [PubMed] [Google Scholar]
  3. Dickerson Q. H., Jr, Busey J. F. Chromatographic separation of h and m antigens from histoplasmin. Proc Soc Exp Biol Med. 1968 Jul;128(3):654–658. doi: 10.3181/00379727-128-33090. [DOI] [PubMed] [Google Scholar]
  4. Ehrhard H. B., Pine L. Factors influencing the production of H and M antigens by Histoplasma capsulatum: development and evaluation of a shake culture. Appl Microbiol. 1972 Feb;23(2):236–249. doi: 10.1128/am.23.2.236-249.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ehrhard H. B., Pine L. Factors influencing the production of H and M antigens by Histoplasma capsulatum: effect of physical factors and composition of medium. Appl Microbiol. 1972 Feb;23(2):250–261. doi: 10.1128/am.23.2.250-261.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. GREENE C. H., DELALLA L. S., TOMPKINS V. N. Separation of specific antigens of Histoplasma capsulatum by ion-exchange chromatography. Proc Soc Exp Biol Med. 1960 Oct;105:140–141. doi: 10.3181/00379727-105-26037. [DOI] [PubMed] [Google Scholar]
  7. Gehreke C. W., Nakamoto H., Zumwalt R. W. Gas-liquid chromatography of protein amino acid trimethylsilyl derivatives. J Chromatogr. 1969 Nov 25;45(1):24–51. doi: 10.1016/s0021-9673(01)86179-3. [DOI] [PubMed] [Google Scholar]
  8. HEINER D. C. Diagnosis of histoplasmosis using precipitin reactions in agargel. Pediatrics. 1958 Oct;22(4 Pt 1):616–627. [PubMed] [Google Scholar]
  9. Kaufman L. Value of immunodiffusion tests in the diagnosis of systemic mycotic diseases. Ann Clin Lab Sci. 1973 Mar-Apr;3(2):141–146. [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Moss C. W., Lambert M. A., Diaz F. J. Gas-liquid chromatography of twenty protein amino acids on a single column. J Chromatogr. 1971 Aug 5;60(1):134–136. [PubMed] [Google Scholar]
  12. PARK J. T., JOHNSON M. J. A submicrodetermination of glucose. J Biol Chem. 1949 Nov;181(1):149–151. [PubMed] [Google Scholar]
  13. Phillips L. S., Pine L. Evaluation of methods used to purify acid-extracted group A streptococcal M protein. Appl Microbiol. 1971 Dec;22(6):963–973. doi: 10.1128/am.22.6.963-973.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. SQUIRE P. G. A RELATIONSHIP BETWEEN THE MOLECULAR WEIGHTS OF MACROMOLECULES AND THEIR ELUTION VOLUMES BASED ON A MODEL FOR SEPHADEX GEL FILTRATION. Arch Biochem Biophys. 1964 Sep;107:471–478. doi: 10.1016/0003-9861(64)90303-0. [DOI] [PubMed] [Google Scholar]
  15. Schubert J. H., Wiggins G. L. Additional studies of histoplasmin formation. Mycopathol Mycol Appl. 1966 Nov 10;30(2):81–91. doi: 10.1007/BF02130354. [DOI] [PubMed] [Google Scholar]
  16. WIGGINS G. L., SCHUBERT J. H. RELATIONSHIP OF HISTOPLASMIN AGAR-GEL BANDS AND COMPLEMENT-FIXATION TITERS IN HISTOPLASMOSIS. J Bacteriol. 1965 Mar;89:589–596. doi: 10.1128/jb.89.3.589-596.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]

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