Human Nephronectin ELISA Kit is a 90 minute sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of human Nephronectin in serum, plasma, and other biological fluids.

Assay Type

Sandwich (quantitative)

Principle of Assay

Human Nephronectin ELISA Kit (A314019) employs the sandwich enzyme immunoassay technique for the quantitative measurement of human Nephronectin in serum, plasma or other biological fluids. An antibody specific for Nephronectin has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the Nephronectin present in each sample is bound to the wells by the immobilized antibody. Biotinylated Anti-Nephronectin Antibody, which also binds the Nephronectin present in each sample, and Streptavidin-HRP, which binds the Biotinylated Anti-Nephronectin Antibody, are added and the microtiter plate is incubated. Following incubation, unbound Biotinylated Anti-Nephronectin Antibody and unbound Streptavidin-HRP are removed by washing, and two substrate solutions are added to the wells. Color develops in proportion to the amount of Nephronectin captured in each well. The color development is stopped by addition of stop solution which changes the color from blue to yellow and the intensity of the color is then measured. The concentration of Nephronectin in the samples can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.

Platform

Pre-coated Microplate (12 x 8 well strips)

Detection Type

Colorimetric

Instrument

Colorimetric Microplate Reader

Sample Type

Serum, plasma or other biological fluids.

Sensitivity

0.029 ng/ml

Product Range

0.75-12 ng/mL

Assay Time

1h 30m

Reactivity

Human

Recovery

Precision

Intra-assay CV: 3.8%, Inter-assay CV: < 10%

General Notes

Information online is representative. Always refer to the Product Manual inside the kit.

Storage

Store at +4°C. Do not use past expiration date!

Synonyms

EGFL6L, NPNT, NPNT_HUMAN, POEM, Preosteoblast EGF like repeat protein with MAM domain, Preosteoblast EGF-like repeat protein with MAM domain, Protein EGFL6 like, Protein EGFL6-like

Disclaimer

This product is for research use only. It is not intended for diagnostic or therapeutic use.

Kit Components

Item	Quantity	Storage
Pre-Coated 96 Well Microplate	12 x 8 Well Strips	+4°C
Standard Solution	500µl	+4°C
Standard Diluent	3ml	+4°C
Biotinylated Detection Antibody	1ml	+4°C
Streptavidin-HRP	6ml	+4°C
Wash Buffer (25X)	20ml	+4°C
Substrate Solution A	6ml	+4°C
Substrate Solution B	6ml	+4°C
Stop Solution	6ml	+4°C
Plate Sealers	5 Adhesive Strips	-
Foil Pouch	1 Zip-Sealed Pouch	-

Required Materials

The following materials are not included in the kit, but are required to run this assay:

Microplate reader capable of measuring absorbance at 450nm ± 10nm.
Squirt bottle, multichannel pipette reservoir, or automated microplate washer.
Graph paper or computer software capable of generating logarithmic functions.
Test tubes and Eppendorf tubes to prepare standards and samples.
Multi- and single-channel pipettes and sterile pipette tips.
Deionized or distilled water.
Absorbent paper towels.
37°C incubator.

Important Notes

Samples cannot be diluted for use with this kit. Owing to the material used to prepare the kit, sample matrix interference may falsely depress the specificity and accuracy of the assay.
Sample concentrations should be estimated before being used in the assay. If the sample concentration is not within the range of the standard curve, users must contact us to determine the optimal sample for their experiments.
Samples containing Sodium Azide (NaN₃) cannot be used in this assay. NaN₃ inhibits the activity of Horseradish Peroxidase (HRP) which is used in the detection step.

Bring all reagents and samples to room temperature before use.

We recommend that all standards, controls, and samples be assayed in duplicate.

Step 1

Prepare all reagents, samples, and working standards as directed in the Product Manual.

Step 2

Determine the number of microplate strips required for the assay and insert them into the plate frame. Unused strips should be stored in the foil pouch at +4°C.

Step 3

Add 50μl of standard to the standard wells.

Step 4

Add 40μl of samples to the sample wells.

Step 5

Add 10μl of Biotinylated Detection Antibody to sample wells (but not to the standard wells).

Step 6

Add 50μl of Streptavidin-HRP to sample wells and standard wells (but not to the blank control well). Mix well. Cover with the adhesive plate sealer provided. Incubate for 60 minutes at 37°C.

Step 7

Remove the plate sealer and wash the plate 5 times with Wash Buffer. Soak wells with 300μl Wash Buffer for 30 seconds to 1 minute for each wash. For automated washing, aspirate or decant each well and wash 5 times with Wash Buffer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Blot the plate onto paper towels or other absorbent material.

Step 8

Add 50μl of Substrate Solution A to each well and then add 50μl of Substrate Solution B to each well. Cover with a new adhesive plate sealer and incubate for 10 minutes at 37°C in the dark.

Step 9

Add 50μl of Stop Solution to each well. The color in the wells should change from blue to yellow immediately. Note: If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

Step 10

Determine the optical density (O.D. value) of each well within 10 minutes, using a microplate reader set to 450 nm.