Solid phase sandwich ELISA for the determination of Human VEGFR-3/Flt-4 in human serum, EDTA-plasma and cell culture supernatant. For research use only, not for use in diagnostic procedures.
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Properties

Protocol PDF Click for Protocol
Catalog Number TK01754
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 Human serum, EDTA-plasma and cell culture supernatant.
Sample Volume 100 μL of properly diluted unknown / determination.
Assay Desc. 1 hour incubation (37°C) + 30 min. (4°C) + 30 min. (RT) = 2 hours total incubation time.
Standard Range 0 / 0.03 - 2.00 ng/mL
Sensitivity 0.004 ng/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 VEGFR-3/Flt-4 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 VEGFR-3/Flt-4. Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for VEGFR-3/Flt-4 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 VEGFR-3/Flt-4, 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 VEGFR-3/Flt-4. You can calculate the concentration of VEGFR-3/Flt-4 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 VEGFR-3/Flt-4. And the sample of VEGFR-3/Flt-4 were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of VEGFR-3/Flt-4. And the sample of VEGFR-3/Flt-4 were tested on 3 different plates, 30 replicates in each plate.

Recovery

Matrices listed below were spiked with certain level of VEGFR-3/Flt-4 and the recovery rates were calculated by comparing the measured value to the expected amount of VEGFR-3/Flt-4 in samples.

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 87-102 95
EDTA plasma (n=5) 91-103 96
heparin plasma (n=5) 86-99 94

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of VEGFR-3/Flt-4 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 (%) 90-103 94-102 93-100
Average (%) 93 93 94
1:4 Range (%) 94-101 90-101 88-107
Average (%) 94 95 94
1:8 Range (%) 87-105 92-102 92-108
Average (%) 94 94 95
1:16 Range (%) 90-100 89-107 90-106
Average (%) 95 95 94

Stability

The stability of VEGFR-3/Flt-4 is determined by the loss rate of activity. The loss rate of VEGFR-3/Flt-4 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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