total soluble Neuropilin-1 ELISA

Highly specific - epitope mapped antibody and characterized reagents. Fully validated. Proprietary product. Only 10 μl sample volume required.

Assay characteristics:

Protocols for urine, cell culture and non-human samples available.

Sensitivity:

LOD: 0.09 nmol/l (0 nmol/l + 3 SD); LLOQ: 0.09 nmol/l

Precision:
Intra-assay (n=6) ≤ 11%, Inter-assay (n=12) ≤ 10%

Spike/Recovery:

The mean recovery of recombinant NRP1 (1.5 nM and 6 nM) in human samples is:

serum (n=6): 90% / 92%
EDTA plasma (n=6): 91% / 93%
citrate plasma (n=1): 98% / 108%
heparin plasma (n=1): 115% / 97%

Dilution linearity:

The mean recovery of endogenous NRP1 in human samples for 1+1 and 1+3 dilution is:

serum (n=6): 95% / 104%  
EDTA plasma (n=6): 107% / 110%
citrate plasma (n=1): 101% / 102%
heparin plasma (n=1): 99% / 104%

Values from apparently healthy individuals:
Median (serum, n=24): 2.0 nmol/l  
Median (EDTA plasma, n=24): 1.7 nmol/l
Median (heparin plasma, n=24): 1.7 nmol/l  
Median (citrate plasma, n=24): 1.9 nmol/l    
It is recommended to establish the normal range for each laboratory. 

ADDITIONAL INFORMATION

Neuropilin-1 (NRP1) is a single-pass transmembrane glycoprotein of 923 amino acids, composed of a large extracellular region, a short transmembrane domain and a short cytoplasmic tail https://www.uniprot.org/uniprot/O14786. Due to alternative splicing or shedding, the extracellular region can be released into circulation as soluble Neuropilin. NRP1 is an essential cell surface receptor functioning in many key biological processes including the cardiovascular, neuronal, and immune systems (1,2). Multiple ligands bind to the extracellular region of NRP1, like class III semaphorins which have a key role in axonal guidance, or members of the VEGF family of angiogenic cytokines. Ligand-binding to transmembrane NRP1, which has co-receptor function, leads to signaling via receptor proteins containing a PDZ domain. In contrast, ligand-binding to soluble Neuropilin-1 (sNRP1) has antagonistic properties by acting as decoy (1,3). NRP1 is expressed by a variety of cells and tissues. For instance, the transmembrane protein is expressed by neuronal cells, endothelial cells, vascular smooth muscle cells, cardiomyocytes, multiple tumor cell lines and neoplasms, osteoblasts, naïve T cells or platelets. Expression of soluble Neuropilin-1 is further described in a variety of non-endothelial cells, e.g. in liver hepatocytes and kidney distal and proximal tubules. NRP1 is implicated in a multitude of physiological and pathological settings, e.g. in axon guidance, vascularization, tumor growth or regeneration and repair (4-9). Neuropilin-1 is described to stimulate osteoblast differentiation, to act as potential biomarker for the prediction of heart failure outcome or to play a role in renal fibrogenesis (6, 10,11). As a co-receptor for VEGF, NRP1 is a potential target for cancer therapies (12). The Neuropilin-1 enzyme immunoassay is a four hour ELISA to quantify human total soluble Neuropilin-1 (sNRP1). The assay is validated for human serum and plasma samples (EDTA, citrate, heparin) (13) (see validation data: www.bmgrp.com). To remove potentially bound ligands, samples are pre-treated with guanidine hydrochloride before testing. Recombinant human soluble Neuropilin-1, isoform 2 is used as calibrator.

1. Neuropilin Functions as an Essential Cell Surface Receptor. HF Guo and CW Vander Kooi, J Biol Chem, 2015; 290(49):29120-29126.

2. Neuropilin signalling in angiogenesis. Koch S, Biochem Soc Trans, 2012; 40(1):20-25. 

3. Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165. Gu C et al., J Biol Chem, 2002; 277(20): 18069-18076.

4. Multifaceted Role of Neuropilins in the Immune System: Potential Targets for Immunotherapy. Roy S et al., Front Immunol, 2017; 10 (8): 1228. 

5. Neuropilin 1 expression in human aortas, coronaries and the main bypass grafts. Alattar M et al., Eur J Cardiothorac Surg, 2014; 46(6): 967-973.

6. Role of Neuropilin-1 in Diabetic Nephropathy. Bondeva T et al., J Clin Med, 2015; 4(6): 1293-1311.

7. Neuropilins: role in signalling, angiogenesis and disease. Zachary I, Chem Immunol Allergy, 2014; 99: 37–70. 

8. Neuropilin-1 is upregulated in the adaptive response of prostate tumors to androgen-targeted therapies and is prognostic of metastatic progression and patient mortality. Tse BWC et al., Oncogene, 2017; 36(24): 3417-3427. 

9. Dual Function of NRP1 in Axon Guidance and Subcellular Target Recognition in Cerebellum. Telley L et al., Neuron, 2016; 21; 91(6): 1276-1291.

10. Nrp1, a Neuronal Regulator, Enhances DDR2-ERK-Runx2 Cascade in Osteoblast Differentiation via Suppression of DDR2 Degradation. Zhang Y et al., Cell Physiol Biochem, 2015; 36(1): 75-84.

11. Neuropilins: structure, function and role in disease. Pellet-Many C et al., Biochem J, 2008; 411(2): 211-226.

12. Neuropilin-1 as Therapeutic Target for Malignant Melanoma. G Graziani1 and PM Lacal, Front Oncol, 2015; 5: 125. 

13. CPMP/ICH/381/95 ICH Topic Q2 (R1) „Validation of Analytical Procedures: Text and Methodology” including: ICH Q2A “Text on Validation of Analytical Procedures” ICH Q2B “Validation of Analytical Procedures: Methodology”.

14. Neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex. Janssen BJC et al., Nat struct Mol Biol, 2012; 19:1293-1299.

REFERENCES, APPLICATIONS

Characterization of a sandwich ELISA for the quantification of total soluble human Neuropilin-1

ISN Frontiers poster #P-135, Tokyo February 2018