Total Soluble Neuropilin-1 ELISA Assay Kit

$720.00

The Eagle Biosciences Total Soluble Neuropilin-1 ELISA Assay Kit is intended for the quantitative determination of total soluble neuropilin-1 in serum, EDTA plasma, heparin plasma or citrate plasma. The Eagle Biosciences Total Soluble Neuropilin-1 ELISA Assay Kit is for research use only and should not be used for diagnostic procedures.

Total Soluble Neuropilin-1 ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: LOD: (0 pmol/l + 3 SD): 0.09 pmol/l; LLOQ: 0.09 pmol/l
Dynamic Range: 0.37 to 12 nmol/l
Incubation Time: 4 hours
Sample Type: EDTA plasma, heparin plasma, citrate plasma and cell culture
Sample Size: 10 µL

Controls Included

Product Developed and Manufactured in Austria by Biomedica

Additional Information

Assay Background

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. Due to alternative splicing or shedding, the extracellular region can be released into circulation as a 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 binf to the extracellular region of NPR1, 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).

Assay Principle

The Eagle Biosciences Total Soluble Neuropilin-1 ELISA Assay Kit is a sandwich enzyme immunoassay for the direct determination of total soluble Neuropilin-1 in humanserum and plasma samples. STD/CTRL/Sample require pre-treatment with an equal amount of guanidine hydrochloride (GuHCl) to remove potentially bound ligands, followed by pre-dilution with assay buffer. In a next step, pre-treated and diluted STD/CTRL/Sample and detection antibody (mouse monoclonal anti human Neuropilin-1 IgG) are pipetted into the wells of the microtiter strips, which are pre-coated with anti Neuropilin-1 antibody. Neuropilin-1 present in STD/CTRL/Sample binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody. In the washing step all non-specific unbound material is removed. Subsequently, the conjugate (Streptavidin-HRPO) is pipetted into the wells and reacts with the detection antibody. After another washing step, the substrate (TMB Tetramethylbenzidine) is pipetted into the wells. The enzyme catalysed colour change of the substrate is directly proportional to the amount of Neuropilin-1. This colour change is detectable with a standard microtiter plate ELISA reader. A dose response curve of the absorbance (optical density, OD at 450 nm) vs. standard concentration is generated, using the values obtained from the standard. The concentration of Neuropilin-1 in the sample is determined directly from the dose response curve.

Assay Procedure

In pre-dilution plate:

  1. Pipette 10 μl STD/CTRL/SAMPLE (Standard/Control/Sample) into respective wells.
  2. Add 10 μl GuHCl (Guanidin Hydrochloride, clear cap) into each well. Swirl gently.
  3. Cover tightly and incubate for 30 minutes at room temperature (18-26°C).
  4. Add 200 μl ASYBUF (Assay buffer, red cap) into each well. Swirl gently.

In pre-coated plate:

    1. Add 50 μl ASYBUF (Assay buffer, red cap) into each well.
    2. Transfer 50 μl pre-treated STD/CTRL/SAMPLE (Standard/Control/Sample) from pre-dilution plate intorespective wells. Swirl gently.
    3. For the transfer of pre-treated STD/SAMPLE/CTRL into the coated plate it is recommended to use a multichannel pipette. Transfer should be performed as soon as possible.
    4. Add 50 μl AB (biotinilated anti NRP-1 antibody, green cap) into each well. Swirl gently.
    5. Cover tightly and incubate for 2 hours at room temperature (18-26°C).
    6. Aspirate and wash wells 5x with 300 μl diluted WASHBUF (Wash buffer, natural cap). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel.
    7. Add 150 μl CONJ (Conjugate, amber cap) into each well. Swirl gently.
    8. Cover tightly and incubate for 1 hour at room temperature (18-26°C), in the dark.
    9. Aspirate and wash wells 5x with 300 μl diluted WASHBUF (Wash buffer, natural cap). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel.
    10. Add 150 μl SUB (Substrate, blue cap) into each well. Swirl gently.
    11. Incubate for 30 min at room temperature (18-26°C) in the dark.
    12. Add 50 μl STOP (Stop solution, white cap) into each well. Swirl gently.
    13. Measure absorbance immediately at 450 nm with reference 630 nm, if available.

Typical Standard Curve

Manual

Product Manual


Publications

Literature

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 viaSuppression 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.