Production and Characterization of Monoclonal Antibody against Saffron Pollen Profilin, Cro s 2

Background: Allergy to Saffron (Crocus sativus) pollen has been described in people involved in processing of saffron flower stamens. Profilins have been identified as a pan-allergen in different plant pollens and foods. This molecule is an actin-binding pro-tein with a molecular weight of 12-16 kDa found in eukaryotic species. Objective: The aim of this study was to generate monoclonal antibody against Cro s 2 in order to char-acterize this major allergen of saffron pollen. Methods: BALB/c mice were immunized to obtain adequate humoral response. Splenocytes were prepared from the immunized animals, mixed with the P3-X63-Ag8.653 myeloma cells and fused by means of PEG 1500. After two weeks of culturing in HAT-containing media, the supernatant from those wells growing hybridomas were screened by ELISA using plates coated with Cro s 2. Cells from positive wells were cloned at least 3 times by limiting dilution. Specific-ity and cross-reactivity of the mAbs were determined by Western blot analysis and sandwich ELISA. Results: Two stable hybridoma clones secreting mAbs against Cro s 2 were obtained and expanded. The anti-Cro s 2 mAbs were also found to cross-react with other plant profilins. Isotype of this mAb was identified as μ heavy chain and k light chain. Conclusion: The anti-Cro s 2 mAb could be a useful tool for characteriza-tion and standardization of many pollen and fruit-derived profilins.