Tissue-Type Plasminogen Activator

Enzyme-linked immunosorbent assay (ELISA) for the determination of human t-PA in cell culture supernatant and serum. For research use only, not for use in diagnostic procedures.
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Properties

Protocol PDF Click for Protocol
Catalog Number TK01706
Species Human
Design Enzyme-linked immunosorbent assay (ELISA) technique.
Standards 7 calibrators, serially diluted from 1 prepared lyophilized calibrator.
Controls None provided.
Sample Types Cell culture supernatant and serum.
Sample Volume 10 μL / determination.
Assay Desc. 2 hour incubation (RT) + 10 min. (RT) = 2 hours, 10 min. total incubation time.
Standard Range 16 - 1000 pg/mL
Sensitivity
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 Tissue-Type Plasminogen Activator 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 Tissue-Type Plasminogen Activator. Standards or samples are added to the micro ELISA plate wells and combined with the specific antigen. Then a biotinylated detection antigen specific for Tissue-Type Plasminogen Activator 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 Tissue-Type Plasminogen Activator, 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 Tissue-Type Plasminogen Activator. You can calculate the concentration of Tissue-Type Plasminogen Activator 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 Tissue-Type Plasminogen Activator. And the sample of Tissue-Type Plasminogen Activator were tested 30 times on one plate, respectively.
Inter-assay Precision (Precision between assays):
3 samples with low, middle,high concentration of Tissue-Type Plasminogen Activator. And the sample of Tissue-Type Plasminogen Activator were tested on 3 different plates, 30 replicates in each plate.

Recovery

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

Matrix Recovery range (%) Average Recovery (%)
Serum (n=5) 90-99 98
EDTA plasma (n=5) 89-97 92
heparin plasma (n=5) 91-96 94

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Tissue-Type Plasminogen Activator 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 (%) 94-108 91-106 89-108
Average (%) 94 93 93
1:4 Range (%) 91-100 90-103 88-106
Average (%) 93 93 94
1:8 Range (%) 89-103 93-105 94-107
Average (%) 94 94 95
1:16 Range (%) 91-102 94-99 89-108
Average (%) 94 93 93

Stability

The stability of Tissue-Type Plasminogen Activator is determined by the loss rate of activity. The loss rate of Tissue-Type Plasminogen Activator 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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