ELISA Kit for the Quantitative Analysis of Human NEUROPILIN-1

 

 

 

 

 

The human Neuropilin-1 ELISA (enzyme-linked immunosorbent assay) kit is used for detection of human Neuropilin-1 in cell culture supernatants,human serum and plasma.THE ELISA KIT IS FOR RESEARCH USE ONLY. Please read this instruction manual

carefully and check out the material provided before use, and you can contact with our company if any questions. You can enter our website or call us for other aim.

Introduction

Neuropilin-1 (Npn-1 or Nrp1), also known as CD304, is an approximately 140 kDa transmembrane glycoprotein . Neuropilin-1 contains a large extracellular domain (ECD) with two N-terminal CUB domains (termed a1a2), two domains with homology to coagulation factors V and VIII (termed b1b2) and a MAM domain (termed c) that mediate the dimerization of Neuropilin-1. Neuropilin-1 is usually expressed as a homodimer, but it can also form heterodimers with Npn-2. Human Neuropilin-1 is a 923 amino acid (aa) protein that functions as a co-receptor for a number of extracellular ligands. Human Neuropilin-1 shares 93% aa sequence identity with the mouse and rat orthologs.

In addition to neurons, melanocytes, and keratinocytes, and on epithelial cells found in breast, uterus, kidney and lung, Neuropilin-1 has also been shown to be expressed on the surface of several immune cell types including plasmacytoid  dendritic cells, resting T cells, natural FoxP3 + regulatory T cells, and a subset of follicular helper T cells. Implicated in mediating the proliferation, survival, migration, and invasion of tumor cells, Neuropilin-1 is also expressed in multiple cancer types.It is additionally involved in a variety of physiological processes including angiogenesis, axon guidance, cell survival and migration, and tumor cell invasion. Apart from interaction with members of the FGF family, Neuropilin-1 also interacts with heparin-binding members of the VEGF family via its b1b2 domains.

Principles of the Test

The kits is a solid sandwich enzyme-linked immunosorbent assay for detection of human Neuropilin-1. An anti-human Neuropilin-1 monoclonal antibody has been absorbed onto the wells of the microtiter strips provided. Samples including specimens or standards were pipetted into wells. The human Neuropilin-1 in specimens or standards would be captured by the coated antibody and the free others were removed by washing. The human Neuropilin-1 biotin-conjugated antibody were added and binds to human Neuropilin-1 captured by the first antibody, which formed a sandwich. Streptavidin-HRP would be added and binds to the biotin conjugated antibody, then free Streptavidin-HRP would be removed during a wash step. After this, subtrate solution would be added and catalyzed by the HRP, and a coloured product is formed. The intensity of the colored product is used to calculate in proportion to the amount of human Neuropilin-1 in the original specimen.  

Materials provided with the kits:

Reagent	96/48Test Kit
Human Neuropilin-1 Antibody-Coated Wells	12 strips/6 strips
Standard Diluent	20 ml/10ml
Human Neuropilin-1 Standard	4/2vial(s)
Human Neuropilin-1 biotinylated Antibody	10ml/5ml
Streptavidin conjugated HRP	10ml/5ml
Wash Buffer Concentrate 20×	30ml/15ml
TMB	10ml/5 ml
Stop Solution	5ml/3 ml
Plate Covers	3/2
Complete Instruction Manual	1

Specimen Collection

1.Collecting specimen as following:

A.The particulate of the cell culture supernatants should be removed before use.

B.Serum was obtained from clot at room temperature.

C.Please collect plasma with EDTA.

D.Assay immediately or store samples at - 20℃. Avoid free-thaw cycles.

2.Antiseptic and anticoagulant should not appear in Serum samples.

3. Any particulate should be removed from samples before use.

4. Do not use grossly hemolyzed or lipemic samples.

Note: Strongly recommend that the serum and plasma samples should be diluented before use.

Precautions for use:

1. Please storage the Kit at 2～8℃。

2.Washing buffer concentrate may have crystal in low temperature, and you can melt its in water-bath before use.

3. Please discard the remains after use of the dissolved standard

4. Avoid contact of substrate solution with oxidizing agents and metal.

5. Usage of disposable pipette tips avoid microbial contamination or cross-contamination of reagents or specimens.

6. Do not mix or substitute reagents with those from other lots or other sources.

7. To ensure the adequate mixure of added reagents, please tap gently the plate after the wells were filled with liquid.

8. Incubation temperature should be 25～28℃.

9. Wash step was crucial for whole assay process.

10. Duplicate wells of the same sample were recommended in assay process.

11. Avoid the foam while pour the liquid into wells.

12.For serum or plasma samples ,the biotin-conjugated antibody should be incubate for at least 90 minutes.

Reagent Preparation

1.The reagents should be warmed up to room temperature before use. The remanent reagents must reseal and put into refrigeratory again as soon as possible.

2.Dilute 1ml of wash buffer Concentrate into 19ml deionized or distilled water to work.

3. If you have a 5x standard diluent, please dilute it with double steaming water or deionized water.

4. Add standard dilution solution to the bottle according to the volume of the label and wait 15 minutes for complete dissolution. Incubation temperature should be 25～28℃.  And in turn add the half concentration diluent by standard diluent .

Wash step:

Automated microplate washer or operating by pipette: Each well should be pour into300ul wash buffer and soak 15 or 30 seconds,then be aspirated, five times process were repeated. After the last wash, remove remaining wash buffer by aspirating. Invert the plate and blot it against clean paper towels.

Materials Required But Not Provided

1. pipettes and pipette tips

2. Microwell strip reader capable of reading at 450 nm (540 nm asoptional reference wave length)

3. automated microplate washer

4.Glass-distilled or deionized water

Assay procedure

1.The needed strips were putted into the frame, the remains were returned into foil pouch and resealed.

2.Blank well were recommended, which only color reagent and stop solution be added. It is suggested that each testing with gradient density of standard for standard curve.

3.Add 100ul of standard or sample. Then cover with the Plate Covers provided. Incubate for 2 hours at room temperature If assay the serum sample, If is plasma serum samples, different sample dilution ratio is different, generally range in 120 ~ 250times, if there is no definite scope, advice from 120 times dilution, if the sample concentration is too high, more than test scope, please increase after diluted times dilution test again。

4.Five times wash process were repeated.

5.Add 100ul of detetion antibody. Cover with the Plate Covers provided.Incubate for 1 hour at room temperature.

6.Five times wash process were repeated.

7.Add 100ul of Streptavidin-HRP. Cover with the Plate Covers provided. Lucifugal incubation for 20 minutes at room temperature.

8.Five times wash process were repeated.

9.Add 100ul of TMB，Lucifugal incubation for 20 minutes at room temperature.

10.Add 50ul of stop solution to each well, determine the optica density of each well within 10 minutes.  

Calculation of Results

1.Duplicates should be within 20 percent of the mean. Average absorbance values for each set of duplicate samples were used as detection results.

2.The blank absorbance values of subtract should be deducted.

3.Drawing a best fit curve through the points of graph. Draw the standard curve by plotting assayed OD valure (on the Y axis) vs. concentration (on the X axis). The sample concentration was obtained based on its OD value ​​founding in the standard concentration curve.

4.If the values obtained are not within the expected range of the standard, Samples should be dilute and assay again.

Typical Data and Standard Curve

concentration (pg/ml)	Typical data 1	Typical data 2	Average
0	0.116	0.127	0.1215
62.5	0.296	0.25	0.273
125	0.412	0.386	0.399
250	0.666	0.648	0.657
500	0.966	0.943	0.9545
1000	1.436	1.382	1.409
2000	2.308	2.189	2.2485

Human  NEUROPILIN-1 Standard Curve

 

Sensitivity, Specificity, Repeatability

Sensitivity: repeated assays were evaluated and the minimum detectable dose was 28.5pg/ml.

Specificity: No significant cross-reactivity or interference with human MFG-E8,Neuropilin-2/Fc Chimera,PlGF-2,Semaphorin 3A/Fc Chimera,Semaphorin 3E,VEGF,.

Repeatability: The coefficient of variation between wells or plates is less than 10 per cent.