Identification of IgE-binding components of citrus red mite in sera of patients with citrus red mite-induced asthma.

Abstract

Our previous investigations demonstrated that citrus red mite (CRM) antigen could cause IgE-mediated bronchoconstriction in exposed farmers working on citrus farms. This study was performed to confirm IgE-binding components and major allergens within the CRM antigens. Ten subjects who had been diagnosed as having CRM-induced asthma were enrolled. Serum-specific IgE antibodies to CRM antigens were measured by using an ELISA. To identify IgE-binding components and major allergens, SDS-PAGE, 2-dimensional PAGE, IgE-immunoblot analysis, and amino acid sequencing of major allergens were performed. All the asthmatic subjects had high specific IgE antibodies to CRMs. Twelve percent SDS-PAGE analysis showed more than 10 protein bands ranging from 6 to 64 kd. SDS-PAGE and IgE-immunoblot analysis with each individual serum showed 5 IgE-binding components (11, 24, 35, 40, and 64 kd), with 2 (24 and 35 kd) of them bound in more than 50% of the study subjects. Two-dimensional PAGE and IgE-immunoblot analysis demonstrated that the major allergen at 24 kd had 2 bands with different isoelectric points of 4.75 and 5.1. Thirty-five kilodaltons had one band with an isoelectric point of 4.75. All amino acid sequencing of the 2 major allergens was performed, which was not homologous with any previously characterized allergens. Five IgE-binding components and 2 major allergens (24 and 35 kd) were identified within the CRM antigen. The N-terminal amino acid sequence of the 2 major allergens (24 and 35 kd) was determined.