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Purification and characterization of a new peptide antigen extracted from dermatophyte mycelia
Abstract:
A highly purified peptide antigen was produced from the cultured mycelia of Trichophyton mentagrophytes. Mycelia were extracted with 0.1 N-HCl at 37 degrees C repeatedly. The protein fraction was precipitated with picric acid and the final product was tentatively designated as Trichophyton mentagrophytes peptide (TMP). Total yield of TMP was about 3% of the original mycelia. When skin tests were done on sensitized guinea pigs, TMP showed a delayed-type reaction. The minimal dose which gave a positive reaction was 3 micrograms (0.1 ml of 30 micrograms/ml solution). Chemical analyses showed that TMP was almost exclusively of a peptide nature and digestion with bacterial peptidase (pronase) completely destroyed its skin test activity, thereby suggesting that protein fraction carried the entire antigenic activity. With chemical and/or physical processings, TMP showed firm stability; the skin test activity was unchanged after heating to 120 degrees C or after processing with HCl or NaOH. Fractionation of TMP by gel filtration demonstrated a considerable heterogeneity in its molecular size, while the antigenic activity was virtually the same in all the fractions. Disc electrophoresis also showed a wide-spread heterogeneous pattern. In the MIF test using peritoneal exudate cells from sensitized guinea pigs, 100 micrograms/ml of TMP had definite inhibitory effect on cell migration.
Insights
Researchers isolated a peptide antigen, Trichophyton mentagrophytes peptide (TMP), from fungal cultures. This peptide triggers delayed-type hypersensitivity reactions in guinea pigs, indicating its potential as a diagnostic tool.
Area of Science:
- Dermatology
- Immunology
- Mycology
Background:
- Trichophyton mentagrophytes is a common cause of fungal skin infections.
- Developing specific antigens is crucial for diagnosing and understanding fungal immune responses.
Purpose of the Study:
- To isolate and characterize a peptide antigen from Trichophyton mentagrophytes.
- To evaluate the antigenic activity and stability of the isolated peptide.
Main Methods:
- Extraction of fungal mycelia using 0.1 N-HCl.
- Purification of the peptide fraction using picric acid precipitation.
- Skin testing in sensitized guinea pigs to assess delayed-type hypersensitivity.
- Chemical analysis, peptidase digestion, and stability testing (heat, acid, alkali).
- Molecular size analysis using gel filtration and disc electrophoresis.
- Migration inhibition factor (MIF) assay using peritoneal exudate cells.
Main Results:
- A purified peptide, Trichophyton mentagrophytes peptide (TMP), was obtained with a yield of approximately 3%.
- TMP elicited a dose-dependent delayed-type hypersensitivity reaction in sensitized guinea pigs, with a minimal effective dose of 3 micrograms.
- Antigenic activity was attributed to the peptide nature, as pronase digestion abolished skin test reactivity.
- TMP demonstrated significant stability, retaining activity after heating to 120°C and exposure to HCl or NaOH.
- Fractionation revealed molecular heterogeneity, yet antigenic activity remained consistent across fractions.
- TMP inhibited cell migration in the MIF assay at concentrations of 100 micrograms/ml.
Conclusions:
- Trichophyton mentagrophytes peptide (TMP) is a stable, antigenic peptide capable of inducing delayed-type hypersensitivity.
- TMP shows potential as a specific diagnostic antigen for T. mentagrophytes infections.
- The peptide's stability and consistent antigenic activity across molecular sizes suggest its utility in immunological studies and diagnostics.