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Purification, composition, and serological characterization of histoplasmin-H and M antigens
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.
Insights
Purified H and M antigens from histoplasmin were isolated using chromatography for improved histoplasmosis diagnosis. These antigens specifically detected antibodies in patient sera, enhancing serological testing accuracy.
Area of Science:
- Immunology
- Medical Mycology
- Biochemistry
Background:
- Histoplasmosis diagnosis relies on serological tests using histoplasmin antigens.
- Current standards require purified H and M antigens for accurate serological diagnosis.
Purpose of the Study:
- To isolate and purify H and M antigens from histoplasmin.
- To establish these purified antigens as primary standards for histoplasmosis serological diagnosis.
Main Methods:
- Fractionation of histoplasmin using Sephadex G-100, Sephadex G-200, and diethylaminoethyl cellulose chromatography.
- Analysis of antigen reactivity using agar gel double-diffusion, complement fixation, and capillary precipitin tests.
- Examination of antigen purity and molecular weight via acrylamide gel electrophoresis.
Main Results:
- Six reactive fractions were obtained, with specific M (fraction 2) and H (fractions 5 and 6) antigens identified.
- Purified M and H antigens showed specific reactivity only with histoplasmosis patient sera.
- Characterization of carbohydrate-to-protein ratios and amino acid/sugar composition for M and H antigens.
Conclusions:
- Purified H and M antigens are suitable as primary standards for histoplasmosis diagnosis.
- These antigens enable specific detection of anti-H and anti-M antibodies.
- The study provides well-characterized antigens for improved serological testing of histoplasmosis.