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

Background: Our previous investigations demonstrated that citrus red mite ( CRM) antigen could cause IgE-mediated bronchoconstriction in exposed farmer s working on citrus farms. Objective: This study was performed to confirm IgE-binding components and m ajor allergens within the CRM antigens. Methods: Ten subjects who had been diagnosed as having CRM-induced asthma w ere enrolled. Serum-specific IgE antibodies to CRM antigens were measured b y using an ELISA. To identify IgE-binding components and major allergens, S DS-PAGE, 2-dimensional PAGE, IgE-immunoblot analysis, and amino acid sequen cing of major allergens were performed. Results: All the asthmatic subjects had high specific IgE antibodies to CRM s. Twelve percent SDS-PAGE analysis showed more than 10 protein bands rangi ng from 6 to 64 kd. SDS-PAGE and IgE-immunoblot analysis with each individu al 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 al lergen at 24 kd had 2 bands with different isoelectric points of 1.75 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 wa s not homologous with any previously characterized allergens. Conclusion: Five IgE-binding components and 2 major allergens (24 and 35 kd ) were identified within the CRM antigen. The N-terminal amino acid sequenc e of the 2 major allergens (24 and 35 kd) was determined.