Abstract
BackgroundNeuropilin‐1 (NRP1) is a highly interactive molecule that exists as transmembrane and soluble isoforms. Measurement of circulating levels of soluble NRP1 (sNRP1) in human serum and plasma has proven to be difficult due to present matrix interferences and due to the lack of a reliable technique.MethodsWe developed a highly specific and sensitive sandwich ELISA assay for total sNRP1 quantification in peripheral blood, and we validated the test according to ICH guidelines. The linear epitopes of the employed polyclonal and monoclonal anti‐human NRP1 antibodies were mapped with microarray technology. We included a sample pre‐treatment step with guanidine hydrochloride (GuHCl) to release sNRP1 from existing interferants.ResultsThe ELISA assay which is calibrated with sNRP1 isoform 2 and covers a calibration range from 0.375 to 12 nmol/L detects sNRP1 in human serum and plasma (heparin, EDTA, and citrate). Multiple linear epitopes recognized by the polyclonal coating antibody are distributed over the whole sNRP1 sequence. The monoclonal detection antibody binds to a linear epitope which is in the N‐terminal region of the a1 domain of human sNRP1. Assay parameters like precision (intra‐assay: 6%), dilution linearity (95%‐115%), specificity (98%), and spike recovery (81%‐109%) meet the international standards of acceptance.ConclusionOur novel sandwich ELISA provides a reliable tool for the quantitative determination of total human sNRP1. The assay detects free and previous ligand‐bound total NRP1.
Highlights
Neuropilin‐1 (NRP1) is a single‐pass transmembrane glycoprotein of 923 amino acids first described in 1987 by Fujisawa et al.[1]
Interferences of semaphorin 3A (SEMA3A) or VEGF‐A165 with soluble NRP1 (sNRP1) binding of the monoclonal anti‐human NRP1 detection antibody employed in the sandwich ELISA were further tested in bio‐layer interferom‐ etry measurements
We describe the development and performance of a highly specific, accurate, and robust human sNRP1 sandwich ELISA that uses a polyclonal sheep anti‐human NRP1 capturing antibody and a labeled monoclonal mouse anti‐human NRP1 detection antibody
Summary
Neuropilin‐1 (NRP1) is a single‐pass transmembrane glycoprotein of 923 amino acids first described in 1987 by Fujisawa et al.[1]. Transmembrane NRP1 functions as co‐ receptor that mediates signaling in a variety of cell types by binding to proteins containing a PDZ domain.[3,4,12] In contrast, sNRP1 binds to the same ligands as transmembrane NRP1, but acts as decoy. In this context, sNRP1 was described as VEGF165 antagonist that has antitumor activities.[8]. We validated the assay based on the principles of bio‐ analytical validation defined by the ICH harmonized tripartite guide‐ line Q2 (R1)
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