In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant eqTSLP protein and generate TSLP-specific antibodies.
EqTSLP was cloned from a skin biopsy sample from a horse with chronic urticaria and eqTSLP protein was expressed in E.coli and in mammalian cells. Recombinant proteins were designed to include C-terminal Histag, which allowed subsequent purification and detection by Histag-specific Ab. Polyclonal and monoclonal eqTSLP-specific Ab were generated after immunization of mice with E.coli-expressed TSLP. Their specificity was tested by western blotting and ELISA. In addition, a commercially available polyclonal human TSLP-specific antibody was tested for cross-reactivity with eqTSLP.
Expression of TSLP protein was confirmed by western blotting using Histag-specific Ab. E.coli-expressed TSLP appears as a band of 鈭?3 kDa, whereas mammalian cell-expressed TSLP showed several bands at 20-25 kDa, probably representing several glycosylation forms. Polyclonal and monoclonal antibodies generated in this study, as well as commercially available human TSLP-specific Ab reacted with both E.coli- and mammalian cell-expressed TSLP in western blotting and ELISA. A capture ELISA was established to quantitate TSLP in cell supernatants and validated using supernatants from primary equine keratinocytes and peripheral blood leukocytes (PBL). Increased TSLP concentrations were found after stimulation of keratinocytes with PMA + ionomycine and with Culicoides extract. Similarly, increased TSLP concentrations were detected in PBL after stimulation with ConA, Culicoides extract, or IgE cross-linking.
In conclusion, recombinant TSLP proteins and TSLP-specific antibodies produced in this study will allow further studies of the role of TSLP in equine allergic diseases.