Characterization of a sandwich ELISA for quantification of total human soluble neuropilin‐1

• Published in: Journal of clinical laboratory analysis Vol. 33; no. 7; pp. e22944 - n/a
• Main Authors: Gadermaier, Elisabeth, Tesarz, Manfred, Wallwitz, Jacqueline, Berg, Gabriela, Himmler, Gottfried
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01-09-2019; John Wiley and Sons Inc
• Subjects: Animals; Antibodies - metabolism; biomarker; Citric acid; Cross Reactions - immunology; ELISA; Enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Epitopes; Epitopes - metabolism; Guanidine - pharmacology; Guanidine hydrochloride; Heparin; Humans; Interferometry; International standards; Isoforms; Ligands; Mice; Models, Molecular; Monoclonal antibodies; Neuropilin; Neuropilin-1 - blood; Neuropilin-1 - chemistry; Peripheral blood; Protein Binding - drug effects; Protein Stability - drug effects; Recombinant Proteins - metabolism; sandwich ELISA; Sensitivity and Specificity; Solubility; soluble human neuropilin‐1; soluble neuropilin‐1; Vascular Endothelial Growth Factor A - metabolism; soluble human neuropilin-1; soluble neuropilin-1; biomarker; sandwich ELISA; ELISA
• ISSN: 0887-8013; 1098-2825
• DOI: 10.1002/jcla.22944

Background
Neuropilin‐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.
Methods
We 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.
Results
The 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.
Conclusion
Our 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.