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Production and characterization of monoclonal antibody against Saffron pollen profilin, Cro s 2
Mojtaba Sankian1, Forough Glosaz-Shirazi, Majid Arafi
1Immunobiochemistry lab, Immunology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
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 protein 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 characterize 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. Specificity 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 micro heavy chain and k light chain.
Conclusion:
The anti-Cro s 2 mAb could be a useful tool for characterization and standardization of many pollen and fruit-derived profilins.
