[Development of an indirect ELISA for detection of specific IgG subclasses for saffron pollen allergens]

Allergic reactions to plant pollens are a common problem. There are some evidences that saffron pollen has allergenic properties. Although IgE-mediated hypersensitivity has been reported, other immunologic mechanisms may be involved based on a variety of clinical manifestations in allergic cases. One of the mechanisms that have been suggested is specific IgG and its subclasses. In this study, an Enzyme-linked Immunosorbent Assay [ELISA] was developed for detection of specific IgG subclasses against saffron pollen extract. Briefly, microtiter plates were coated with crude extract of saffron pollen and then blocked with bovine serum albumin [BSA], and the sera were added to the plates which followed by the addition of anti-human IgG. TMB was used as substrate. Finally, after stopping the reaction with addition of HCl, plates read at 450 nm. The method was verified by testing two groups of samples, first, sera from individuals living near saffron farms in Khorasan and second, sera from individuals living in areas not having any saffron farms. The results indicated significant differences between the two groups

Immunoblot analysis of saffron pollen proteins recognized by human IgE antibodies

Pollen is one of the major causes of allergy worldwide. Pollen allergens can cause an immediate hypersensitivity [type 1, IgE-mediated] response in susceptible individuals. Hypersensitivity to saffron pollen is a serious problem, especially among the people who working with Saffron flowers. The aim of this study was to identify protein with IgE binding activity [allergenic protein] in a saffron pollen extract. Proteins of the saffron pollen extract recognized by human IgE antibodies were investigated by SDS-PAGE and Western blotting. Proteins with IgE binding activity were identified with sera from 23 patients who suffered from continues sneezing, rhino rhea, conjunctivitis, throat irritation, shortness of breath during exposure with Saffron flowers. A chemiluminescent immuno-detection method was used to study IgE binding activity'of saffron pollen proteins with patients's sera.Six IgE immimoreactive proteins ranging in molecular weight from 13.5 to 85 KD were identified. From among of these proteins, four with apparent molecular weight of 13.5-14, 19.5-20, 42-43 and 85 KD could be identified with more than 35% of our allergic patient's sera. While two proteins with IgE binding activity [58 and 67 KD] could be demonstrated for only some of our saffron allergic patients.In conclusion, in this study we demonstrated the presence of several IgE binding proteins in the total extract of saffron pollen