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.