Solid phase sandwich ELISA for the determination of ET-3 in serum, EDTA-plasma, supernatant of cell culture media and extract from tissue. For research use only, not for use in diagnostic procedures.
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Properties

Protocol PDF Click for Protocol
Catalog Number TK01279
Species Human
Design Solid phase sandwich ELISA using 2 kinds of high specific antibodies.
Standards 8 standards, serially diluted from 1 prepared lyophilized standard.
Controls None provided.
Sample Types Serum, EDTA-plasma, supernatent of cell culture media, and the extracted solution from tissue.
Sample Volume 100 μL of properly diluted unknown / determination.
Assay Desc. Overnight incubation (4˚C) + 30 min. (37˚C) + 30 min. (RT) = Overnight + 1 hour total incubation time.
Standard Range 0 / 0.78 - 50 pg/mL
Sensitivity 0.36 pg/mL
Radioactivity
Other Names
Storage 2 - 8°C
Postscript For research use only, not for use in diagnostic procedures.

Intended use

This ELISA kit applies to the in vitro quantitative determination of Endothelin-3 concentrations in serum, plasma and other biological fluids.

Test principle

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antigen specific to Endothelin-3. Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Endothelin-3 and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Endothelin-3, biotinylated detection antigen and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Endothelin-3. You can calculate the concentration of Endothelin-3 in the samples by comparing the OD of the samples to the standard curve.

Typical data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Precision

Intra-assay Precision (Precision within an assay):
3 samples with low, middle,high concentration of Endothelin-3. And the sample of Endothelin-3 were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of Endothelin-3. And the sample of Endothelin-3 were tested on 3 different plates, 30 replicates in each plate.

Recovery

Matrices listed below were spiked with certain level of Endothelin-3 and the recovery rates were calculated by comparing the measured value to the expected amount of Endothelin-3 in samples.

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 88-102 98
EDTA plasma (n=5) 87-97 94
heparin plasma (n=5) 91-96 93

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Endothelin-3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

    Serum (n=5) EDTA plasma (n=5) heparin plasma (n=5)
1:2 Range (%) 90-99 93-108 90-108
Average (%) 93 95 94
1:4 Range (%) 93-108 87-101 88-101
Average (%) 94 95 93
1:8 Range (%) 87-105 87-102 94-103
Average (%) 93 95 94
1:16 Range (%) 92-107 91-108 88-104
Average (%) 95 93 95

Stability

The stability of Endothelin-3 is determined by the loss rate of activity. The loss rate of Endothelin-3 is less than 5% within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Item Specifications Storage
Micro ELISA Plate(Dismountable) 8 wells ×12 strips -20℃, 6 months
Reference Standard 2 vials
Concentrated Biotinylated Detection Ab (100×) 1 vial, 120 μL
Concentrated HRP Conjugate (100×) 1 vial, 120 μL -20℃(shading light), 6 months
Reference Standard & Sample Diluent 1 vial, 20 mL 4℃, 6 months
Biotinylated Detection Ab Diluent 1 vial, 14 mL
HRP Conjugate Diluent 1 vial, 14 mL
Concentrated Wash Buffer (25×) 1 vial, 30 mL
Substrate Reagent 1 vial, 10 mL 4℃(shading light)
Stop Solution 1 vial, 10 mL 4℃
Plate Sealer 5 pieces
Product Description 1 copy
Certificate of Analysis 1 copy

Other supplies required

Microplate reader with 450 nm wavelength filter
High-precision transfer pipette, EP tubes and disposable pipette tips
Incubator capable of maintaining 37℃
Deionized or distilled water
Absorbent paper
Loading slot for Wash Buffer

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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