A novel photoclick hydrogel electrophoretic and in situ immunoblotting method for EPO-receptor agonists screening

Code: 22B05PC

Erythropoietin receptor agonists (ERAs), e.g. darbepoetins (dEPO), erythropoietins (EPO), EPO-Fc were listed in WADA's Prohibited List under Class S2.1. Traditional polyacrylamide gel-electrophoretic analytic methods were applied by most of the WADA certified laboratories for ERAs detection for the last 20 years. In this project, a novel photoclick hydrogel electrophoretic and in situ immunoblotting method for the screening of ERAs in urine and blood samples of athletes will be developed with high sensitivity and high throughput. A tetrazole-functionalized photoclick hydrogel based chip will be designed first which can satisfy the processing of 15 samples simultaneously. Optimization of the parameters in SAR-PAGE and intensity immunoblotting assay will be carried out on this chip to realize the separation of ERAs, especially the distinguishment of endogenous EPO and recombinant human EPO )rhEPO). Along with the confocal microscope for image acquisition, the LOD of this method can be lower than 1 IU/L (plasma) and 0.1 IU/L (urine) with limited sample volumes. Meanwhile, the whole analysis time can be reduced within 6 hours for plasma samples. Finally, an intravenous administration of rhEPO drugs on healthy subjects will be carried out to verify the practicability of this new method.