Human PAI-1 ELISA Kit

Catalog Number: abs551341 Application: ELISA Reactivity: Human Conjugation:

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$368 USD

Size: 96T

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Product Details

Product Specification

protein

PAI-1

Usage

Preparations before testing:

1. Please advance 10 Minutes remove the kit from the refrigerator and equilibrate to room temperature.
2. Standard gradient working solution preparation : join 1mL Universal diluent into lyophilized standard and let stand 15 Minutes until it is completely dissolved and then gently mix ( The concentration is 4000pg/mL) And then according to the following concentrations: 4000pg/mL 、 2000pg/mL 、 1000pg/mL 、 500pg/mL 、 250pg/mL 、 125pg/mL 、 62.5pg/mL 、 0pg/mL The dilution was performed.

Double dilution method: Take 7 branch EP Tubes, each tube is added 500μL Universal diluent ,4000pg/mL Pipette from the standard working solution 500μL To the first EP Mix evenly in a tube 2000pg/mL According to this step, suck and mix the standard working solution in sequence. The last tube is directly used as a blank hole, so there is no need to suck liquid from the penultimate tube, as shown in the figure below.

HRP Preparation of anti-resistance detection working fluid : Before use 15 Minutes will 100× concentrate HRP Detect resistance to 1000×g Centrifugation 1 Minutes, with a universal diluent 100× concentrate HRP Detection resistance dilution to 1× Working concentration ( Example :10μL Concentrate +990μL Universal diluent ) , now available for use.

1× Wash liquid preparation : Take 10mL 20× Wash liquid to 190mL In distilled water (the concentrated washing liquid taken out from the refrigerator may have crystals, which is a normal phenomenon. It can be placed at room temperature and prepared after the crystals are completely dissolved ) 。

Self-prepared test equipment required for the experiment:

1. Microplate reader ( 450nm ）
2. High-precision pipettes and tips: 5-10μL 、 5-50μL 、 20-200μL 、 200-1000μL
3.37℃ Incubator
4. Distilled or deionized water

Sample handling and requirements:

1. The detection range of the kit is not equivalent to the concentration range of the test substance in the sample It is recommended to estimate the concentration of the test substance in the sample through relevant literature before the experiment and determine the actual concentration of the sample through pre-experiment. If the concentration of the test substance in the sample is too high or too low, please dilute or concentrate the sample appropriately.

2. If the sample tested is not among the sample types listed in the instructions, it is recommended to conduct pre-experiments to verify its detection effectiveness.

3. Serum : The whole blood sample collected in the serum separation tube was placed at room temperature 2 Hour or 2-8℃ Overnight, then 1000×g Centrifugation 20 Minutes, take the supernatant, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided.

4. Plasma : with EDTA Or heparin as an anticoagulant to collect a sample, and collect the sample after collection 30 Within minutes 2-8℃1000×g Centrifugation 15 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided.

5. Tissue homogenate : with pre-cooled PBS (0.01M, pH=7.4) The tissue is flushed to remove residual blood (lysed red blood cells in the homogenate can affect the test results), and the tissue is weighed and cut into pieces. Combining the shredded tissue with the corresponding volume PBS (Generally according to 1:9 Weight to volume ratio, such as 1g The tissue samples correspond to 9mL Of PBS The specific volume can be appropriately adjusted according to the experimental needs and recorded. Recommended in PBS Add protease inhibitor) into a glass homogenizer and grind thoroughly on ice or grind in a homogenizer. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was mixed in 5000×g Centrifugation 5~10 Minutes, take the supernatant for detection.

6. Cell culture supernatant : Please 1000×g Centrifugation 20 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided.

7. Cell lysate Pre-cooling for adherent cells PBS Gently washed, followed by trypsinization, 1000×g Centrifugation 5 Cells were collected after minutes; The suspended cells can be collected directly by centrifugation. The collected cells were pre-cooled with PBS Washing 3 Times, every 1×10^6 Added to cells 150-200μL PBS Resuspension (recommended at PBS Adding a protease inhibitor; If the content is very low, it can be appropriately reduced PBS Volume) and the cells were disrupted by repeated freeze-thaw or sonication. The extracts were mixed in 2-8℃ ， 1500×g Centrifugation 10 Minutes, take the supernatant for detection.

8. Other biological samples ： 1000×g Centrifugation 20 Minutes, take the supernatant to detect.

9. Sample Appearance : The sample should be clear and transparent, and the suspended solids should be removed by centrifugation.

10. Sample preservation : After sample collection, if 1 Those tested within weeks can be stored in 4℃ , if it cannot be detected in time, please pack it according to the one-time usage amount and freeze it in -20℃ （ 1 Within months), or -80℃ （ 6 Test within months) to avoid repeated freezing and thawing. Hemolysis of the sample will affect the final test result, so hemolyzed samples are not suitable for this test.

Sample dilution protocol:

Please estimate the concentration range of the sample in advance. If your test sample needs to be diluted, refer to the following dilution scheme: Dilution 100 Times: One step dilution. Take 5μL Sample to 495μL Within universal diluent, do 100 Double dilution; Dilution 1000 Times: Two-step dilution. Take 5μL Sample to 95μL Within universal diluent, do 20 Dilute, and then take 5μL 20 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 1000 Times;

Dilution 100000 Times: Three-step dilution. Take 5μL Sample to 195μL Within universal diluent, do 40 Dilute, and then take 5μL 40 Double dilute sample to 245μL Within universal diluent, do 50 Time dilution, and finally take 5 μL 2000 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 100000 Times;

The amount of liquid taken during each dilution step is not less than 3μL , the dilution factor is not more than 100 Times 。 Each step of dilution should be mixed evenly to avoid foaming.

Operation steps:

1. Equilibration from room temperature 10 After minutes, remove the required slats from the aluminum foil bag, and seal the remaining slats with ziplock bag and put them back 4℃ 。

2. Loading samples: respectively add samples or different concentration standards according to 100μL Each well is added to the corresponding well, and the blank well is added 100μL Universal diluent. After covering the sealing film 37℃ Incubation 60 Min. ( Recommendation: Minimum dilution of sample to be tested with universal diluent 1 Times later, add the enzyme labeled plate for testing. So as to reduce the influence of matrix effect on the test results, and finally, the sample concentration needs to be multiplied by the corresponding dilution factor when calculating. It is recommended to set up double wells for all samples and standards to be tested during testing).

3. Plate washing: discard liquid and add per well 300μL 1x Wash liquid, stand 1 Minutes, throw off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 3 Times (the plate can also be washed with a plate washing machine).

4. Add HRP Anti-resistance test: After washing the plate, add directly to each well 100μL HRP Check the resistance to working fluid and cover the sealing film 37° Incubation 60 Minutes.

5. Plate wash: discard liquid as per step 3 Washing method, wash plate 5 Times.

6. Substrate addition: added per well 90μL Substrate (TMB) Covered with a sealing film, 37℃ Incubation protected from light 15 Minutes.

7. Add stop solution: take out the enzyme labeled plate and add it directly to each well 50μL Stop solution, immediately in 450nm Wavelength measurement of each well OD Value.

Calculation of experimental results:

Result judgment ：

1. Calculate the average of standards and sample replicates OD Value and subtract the blank hole's OD Values as correction values. Taking concentration as the abscissa, OD The value is the ordinate, and the standard curve of the four-parameter logic function is drawn on the double logarithmic coordinate paper.

2. If the sample OD If the value is higher than the upper limit of the standard curve, the test should be retested after appropriate dilution and multiplied by the corresponding dilution factor when calculating the sample concentration.

The following data and curves are for reference only. Experimenters need to establish standard curves according to their own experiments

Note: This figure is for reference only, and the sample content should be calculated from the standard curve drawn from each experimental data.

Kit Performance:

1. Repeatability: In-plate coefficient of variation is less than 10% , the interplate coefficient of variation is less than 10% 。

2. Recovery rate: added to selected healthy human plasma, tissue homogenate and cell lysate 3 People with different concentration levels PAI-1 , calculate the recovery.

Sample Type	Range ( % ）	Average recovery ( % ）
Plasma (n=8)	84-101	96
Tissue homogenate (n=8)	92-105	102
Cell lysate (n=8)	96-108	105

3. Linear dilution: in selected 4 A portion of healthy human plasma, tissue homogenate and cell lysate were added with high concentration human PAI-1 , linearity was assessed by dilution within the standard curve kinetic range.

Dilution ratio	Recovery ( % ）	Plasma	Tissue homogenate	Cell lysate
1 ： 2	scope	84-95	88-96	90-110
1 ： 2	Average recovery	91	93	96
1 ： 4	scope	89-103	87-108	105-115
1 ： 4	Average recovery	94	98	108

Problem analysis:

If the experimental results are not good, please take photos of the color development results in time, save the experimental data, keep the used slats and unused reagents, and then contact our company's technical support to solve the problem for you. At the same time, you can also refer to the following information:

Problem Description	Possible cause	Corresponding countermeasures
Poor scaling curve	Incorrect dilution of standard	Ensure that the standard is dissolved and diluted according to the recommended method
	Inaccurate pipetting	Calibrate the pipette periodically and check tip tightness
	Evaporation of the reaction solution	Enzyme-labeled plate is sealed with a sealing membrane
	Incomplete plate washing	Sufficient number of washes and addition of sufficient amount of washing liquid
	Foreign matter at the bottom of the hole	Clean bottom of plate before reading
Weak or colorless chromogenic	Incubation time is not enough	Ensure incubation time
	Incorrect incubation temperature	Incubate at recommended temperature
	Insufficient reagent volume addition	Inspect the pipette and follow the procedure exactly
	Incorrect dilution	Test Reagent Dilution Step
	Enzyme conjugate inactivation	Mixed enzyme conjugate and substrate, checked by color reaction

Theory

This kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA). To the microwells pre-coated with human plasminogen activator inhibitor 1 (PAI-1) capture antibody, sample, standard, HRP-labeled detection antibody were sequentially added, incubated and washed in the middle, and colored with substrate TMB. TMB turned blue under the catalysis of peroxidase (HRP), and turned final yellow under the action of acid. There was a positive correlation between the depth of color and human plasminogen activator inhibitor 1 (PAI-1) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450nm, and the sample concentration was calculated. Sensitivity: 24.93 pg/mL Specificity: Human PAI-1 can be detected in samples without significant cross-reaction with its analogs.

Source

Human

Synonym

Human Plasminogen Activator Inhibitor 1 ELISA Kit

Detection Type

Double antibody sandwich method

Composition

Name	96 Hole configuration	remark
Pre-coating 96 Well plate	8 Hole ×12 Strip	without
Standard	2 branch	Dilute as per instructions
Universal diluent	2×20mL	without
concentrate HRP Anti-detection 100×	120uL	Dilute as per instructions
20× Washing liquid	2×10mL	Dilute as per instructions
Bottom thing （ TMB ）	10mL	without
Stop liquid	6mL	without
Sealing film	4 Zhang	without
Instructions	1 Share	without

Background

PAI-1 is also known as endothelial prothrombin activator inhibitor or serum protein E1. It is a serine protease inhibitor (serpin). Its main function is to inhibit urokinase proplasmin activator (uPA). It is an enzyme responsible for cleaving proplasmin to form plasma protein. Plasma proteins mediate the degradation of the extracellular matrix either by themselves or together with matrix metalloproteinases. In this case, PAI-1 inhibits uPA through active site binding, preventing the formation of plasma proteins. Additional inhibition is through PAI-1 binding to the uPA/uPA receptor complex, resulting in degradation of the latter. Therefore, it can be said that PAI inhibits the serine proteases tPA and uPA/urokinase and is therefore an inhibitor of fibrinolysis, the physiological process that degrades blood clots. In addition, PAI-1 can also inhibit the activity of matrix metalloproteinases, which play a key role in the invasion of malignant cells through the basal layer.

General Notes

1. Carry out incubation in strict accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C prior to use. Store reagents in refrigeration immediately after use.
2. Incorrect plate washing may lead to inaccurate results. Make sure to drain the liquid from the wells as much as possible before adding the substrate. Do not allow the wells to dry out during incubation.
3. Eliminate the residual liquid and fingerprints at the bottom of the plate, otherwise it will affect the OD value.
4. The substrate color development solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used.
5. Avoid cross-contamination of reagents and specimens to avoid wrong results.
6. Avoid direct exposure to strong light during storage and incubation.
7. Any reaction reagent cannot come into contact with the bleaching solvent or the strong gas emitted by the bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagents in the kit.
8. Expired products cannot be used, and components with different item numbers and batch numbers cannot be mixed.
9. Recombinant proteins from sources other than the kit may not match the antibodies in this kit and are not recognized.
10. If the disease may be spread, all samples should be managed well, and the samples and testing devices should be handled according to the prescribed procedures.

Storage Temp.

Unopened kit, stored at 4 °C, shelf life 6 months.

Test Range

62.5-4000pg/mL

Applications

Plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids

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Human PAI-1 ELISA Kit

Human PAI-1 ELISA Kit