Related Experiment Videos
Characterization of allergen extracts by two-dimensional electrophoretic techniques: Micropolyspora faeni antigens
Abstract:
Thermophilic actinomycetes and saprobic fungi are important in the etiology of allergic occupational diseases such as "farmer's lung" disease. Each such organism produces several protein antigens. Inhaled, these antigens stimulate production of antibodies. Detection of precipitating antibodies has been useful in the diagnosis of diseases so induced. Characterization of allergen extracts from microorganisms associated with these diseases is important, to improve the sensitivity and precision of the precipitin analysis. For this purpose we submitted crude allergen extracts to electrophoresis and isoelectric focusing in agarose gels. Staining the gels revealed many protein components in each extract, especially after isoelectric focusing. After separation in one dimension, a lane of gel was cut out and the proteins were electrophoresed at right angles into another gel, which contained antibodies. Several arcs of immunoprecipitates, indicating different antigens, were seen. This technique ("crossed immunoelectrofocusing") has earlier been used with polyacrylamide in the first dimension, but it is improved by using instead agarose of a special quality. Further to improve the quantification, we isolated pieces of gel containing the proteins of interest and used them as samples in zone immunoelectrophoresis assay. This method is straightforward, easy to evaluate, and about 100-fold as sensitive as radial immunodiffusion. The amount of protein in each sample is usually proportional to the distance from the upper gel surface to the front of each immunoprecipitate. The increased sensitivity allows study of many hitherto unexamined antigens.
Insights
Characterizing microbial allergens is key for diagnosing farmer's lung disease. New methods like crossed immunoelectrofocusing improve antigen detection sensitivity, aiding in understanding allergic occupational diseases.
Area of Science:
- Immunology
- Microbiology
- Occupational Health
Background:
- Thermophilic actinomycetes and saprobic fungi cause allergic occupational diseases like farmer's lung.
- These microorganisms produce protein antigens that trigger antibody production upon inhalation.
- Detecting precipitating antibodies is crucial for diagnosing these diseases.
Purpose of the Study:
- To characterize allergen extracts from microorganisms linked to allergic occupational diseases.
- To enhance the sensitivity and precision of precipitin analysis for improved diagnosis.
- To explore novel methods for antigen identification and quantification.
Main Methods:
- Crude allergen extracts were analyzed using electrophoresis and isoelectric focusing in agarose gels.
- Crossed immunoelectrofocusing, utilizing special quality agarose, was employed for antigen separation and detection.
- Zone immunoelectrophoresis assay was used for quantifying specific antigens after isolation from gels.
Main Results:
- Isoelectric focusing revealed numerous protein components within the allergen extracts.
- Crossed immunoelectrofocusing successfully identified multiple antigens through immunoprecipitate arcs.
- Zone immunoelectrophoresis assay demonstrated a 100-fold increase in sensitivity compared to radial immunodiffusion.
Conclusions:
- The developed crossed immunoelectrofocusing technique offers improved characterization of microbial allergens.
- Enhanced sensitivity allows for the study of previously undetected antigens.
- These advancements can lead to more precise diagnostic tools for allergic occupational diseases.