Ultra-sensitive immunochromatographic assay for quantitative determination of erythropoietin

Abstract

An ultra-sensitive quantitative EPO (erythropoietin) lateral flow immunochromatographic test with a detection limit of 1.2 fM (10−15 M), 0.035 ng EPO/L, which is 50–100 times more sensitive than a corresponding enzyme based immunoassay, is presented. In comparison with commercial lateral flow tests for other analytes, like cardiac troponins that also require high sensitivity, the detection limit achieved in the presented test is about three orders of magnitude lower. The thin zone for capture and concentration of the analyte, the carbon black nano-strings used as label and the use of a conventional image scanner for the quantitative determination are the key components that enable the high sensitivity obtained. The convective flow in the lateral flow monolith creates short diffusion distances between immobilised antibody, analyte and labelled antibody thus enhancing the binding efficiency. This rapid and sensitive EPO test procedure can be used both to process hundreds of samples in 1 h and be utilized as a 15-minute dipstick test for single determinations.The technique is demonstrated by measuring EPO in urine. EPO, like many of the other urine proteins, is often found in the urine precipitates and the specimens are therefore treated with a urine precipitate dissolvation buffer before analysis. It is shown that EPO in urine from normal individuals occurs in low concentration in a wide range between 1.7 and 51 ng/L. The concentration is however subjected to a wide variation during the day due to the EPO production variation and the urine concentration by the kidneys. It is also shown that the presented lateral flow device can be used as a miniaturized affinity column to distinguish an analyte (EPO) from its analogue (darbepoetin), directly by comparing the affinity profiles obtained after interaction with the immobilised antibody.The method for measuring the amount of EPO present in urine, the possibility to rapidly check the amount of EPO after a pre-treatment concentration step, and the potential to identify affinity differences between EPO and its analogues should make the presented method a valuable tool in the fight against EPO doping.