Reliable assay for rapid detection of potentially unknown erythropoietin mimetics chemically unrelated to endogenous hormone in biological samples

Code: ISF17B03BS 

Recombinant erythropoietins (EPOs) have long been one of the most frequently abused drugs for increasing blood oxygen capacity. However, numerous compounds with EPO-like activity but completely different chemical structures (either short peptides or even nonpeptidic synthetic molecules) have been developed so far and due to such chemical diversity, detection of their potential abuse is notoriously difficult. To our knowledge, no universal and reliable analytical method is available to confirm the presence of all EPO mimetics in biological sample regardless of their chemical structure. Here, we describe development of a rapid and universal assay for detection of any ligand binding to the erythropoietin receptor (EpoR) which could be used as a low-cost screening test in doping control to identify individuals for further detailed examination. Our innovative platform comprises combination of immunoprecipitation steps and two modified ELISA tests where recombinant filamentous phage particles displaying EpoR are used instead of primary antibodies. As evaluated with spiked artificial urine, such assay can detect the presence of EpoR ligands chemically unrelated to EPO in specimens. Site-specific proteolytic cleavage and isolation of EpoR:ligand complex from phage particles enables analysis of the complex by mass spectrometry and potential identification of previously unknown EpoR ligand. The described platform may also be further developed into the lateral flow immunoassay format similar to common over-the-counter pregnancy test which would greatly increase its applicability out of the laboratory. To emphasize, such concept can also be applied to detection and identification of other growth factor or hormone mimetics by implementation of appropriate combination of receptor, ligands and antibodies. Moreover, based on our platform a multiplex assay capable of simultaneous detection of substances with distinct activity can be developed by utilizing Luminex® technology and appropriate combination of recombinant phage particles displaying different hormone and growth factor receptors. 

Main findings

During the work, we have identified technical issues in most steps of the method, and have addressed them appropriately. The myctag was re-located from the linker between the displayed EpoR and carrier capsid protein p3 to the N-terminus of the displayed EpoR. The folding of the EpoR and display of functional EpoR on recombinant phage particles was improved. We have also adopted the helper plasmid pTUM4 harboring genes for four periplasmic chaperones, which were used to co-transform the host bacteria for production of recombinant phage particles displaying EpoR. The novel recombinant phage M13-mer-Ser (myc-tag on the N-terminus of the EpoR, substitution of the Cys218 for Ser, produced at 16 °C) was further tested for the use in the platform. The platform was evaluated using real human sera. We established that novel recombinant phages M13-mER-Ser bind to all targets in presence of human serum, however, matrix effects were evident. We noted that it is important to deplete endogenous EPO from the serum prior subjecting it to the assay. Addition of protease inhibitors to the serum improves the binding of recombinant phages in ELISA assays but the addition is not critical. Binding of M13-mER-Ser phages to immobilized B-EPO is competitive with recombinant EPO and model EPO-mimetics (B-EMP1 monomer and [B-EMP1]4SA tetramer). Although the assay was improved, the resulting recombinant phages still gave high signals in ELISA2 after depletion using SA/B-EPO beads. Additional efforts are needed to validate the present platform using higher number of real human sera.