1. Intended Use
This Human Total PAI-1 ELISA kit is designed for quantification of Human total PAI-1 in serum and plasma. This assay kit is for in vitro research use only.
Features
• The total assay time is less than 2.5 hours.
• The kit measures human total PAI-1 in serum and plasma.
• Components of the kit are provided ready-to-use or concentrated.
2. Storage, Expiration
The kit should be stored at 2-8oC upon receipt, and should be equilibrated to room temperature before assay.
Refer to expiration dates on all reagents prior to use. Do not mix reagents from different kits unless they have the same lot numbers.
Remove any unused strips from the human adiponectin microplate, return them to the foil pouch and keep at 4 oC.
3. Introduction
Plasminogen activator inhibitor 1 (PAI-1) is an important endogenous inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator, and is the key determinant of fibronyltic activity.1 PAI-1 is a member of the seine protein inhibitor family.2 This glycoprotein consists of 379 amino acids residues with an apparent molecular weight of 48 kDa.3 The elevated circulating PAI-1 level is shown to be associated with obesity, cardiovascular diseases and type 2 diabetes (T2DM).4-9 Furthermore, PAI-1 play a key role in cell migration and is suggested to be involved tumor growth and angiogenesis.10
4. Test Principle
The PAI-1 ELISA is based on a solid phase enzyme-linked immunosorbent assay using monoclonal antibodies that against human PAI-1. The assay system is designed for the quantitative detection of human PAI-1 in plasma or serum in 2.5 hours. A mouse monoclonal antibody has been pre-coated onto 8-well strips which could be mould onto the strip holder provided. PAI-1 molecules in the samples are sandwiched by another rabbit polyclonal antibody labeled with biotin. The signal will be further amplified by the binding streptavidin-conjugated horseradish peroxidase (HRP-SPT). All unbound materials are washed away before the addition of HRP substrate. The color developed by the enzyme activity is determined after stopping the reaction by the addition of acidic solution. The color intensity represents the amount of PAI-1 presents in the samples.

5. Reagents Supplied
Each kit is sufficient for one 96-well plate and contains the following components:
1. Antibody Coated 8-well Strips (12 strips, total 96 wells)
8-well strips coated with mouse anti-human PAI-1 monoclonal antibody.
2. Human PAI-1 Standard (5 ng/ml, 1 ml)
After reconstituted, contains 5 ng/ml recombinant human PAI-1 in phosphate-buffered saline-BSA solution.
3. Assay Diluent (5×, 10 ml): A concentrated phosphate-buffered saline-BSA solution.
4. Wash Buffer (10×, 20 ml): Concentrated detergent-containing phosphate-buffered saline.
5. Detection Antibody Solution (100×, 100 ul): Rabbit anti-human PAI-1 polyclonal antibody labeled with biotin
6. HRP-SPT Solution (100×, 100 ul): Streptavidin conjugated HRP.
7. TMB Substrate Solutions A and B (6 ml each)
8. Stop Solution (6 ml)
9. Quality controls, high and low (1 vial each, 0.4 ml/vial): A diluted serum sample of known PAI-1 concentrations in phosphate-buffered saline-BSA solution.

6. Preparation of Reagents
Pre-warm all reagents to room temperature (18-25°C) before the assay. All reagents should be freshly diluted before use. Diluted reagents are not stable under the storage condition suggested in this manual.
Pre-warm all reagents to room temperature (18-25 oC) before the assay. All reagents should be freshly diluted before use. Diluted reagents are not stable under the storage condition suggested in this manual.
1) 1x Assay diluent. Dilute 5x concentrated Assay Diluent with distilled or deionized water to 1× working concentration.
2) 1x Wash buffer. Dilute 10x Washing Buffer with distilled or deionized water to 1× working concentration.
3) 1x Biotinylated Antibody Solution. Dilute desired amount of Biotinylated Antibody Solution, 100 ul of the 1x Biotinylated Antibody Solution is required per well. Prepare only as much 1x Detection antibody solution as needed. Return the 100x Biotinylated Antibody Solution to 2-8 oC immediately after the necessary volume is removed.
4) 1x HRP-SPT Solution. Dilute desired amount of HRP-SPT Solution, 100 ul of the 1x HRP-SPT Solution is required per well. Prepare only as much 1x HRP-SPT Solution as needed. Return the 100x HRP-SPT Solution to 2-8 oC immediately after the necessary volume is removed.
5) Substrate solution. Mix the desired amount of TMB Substrate Solutions A and B in a 1:1 ratio immediately before starting the reaction. Do not pre-mix the solutions during preparation of other reagents. Return Substrate A & B to 2-8 oC immediately after the necessary volume is removed.
6) Standard set.. Prepare the standards according to the following table. Thoroughly mix by vortexing.

7. Preparation of Samples
1. This kit is designed for quantitative detection of human total PAI-1 in plasma or serum samples. The intended use of this kit for detection of human total PAI-1 in other biological fluids or solutions was not tested.
2. Blood should be collected using standard venipuncture techniques. After blood clotted, centrifuge samples at 1,500 to 2,000g for 15 minutes and collect the supernatants.
3. Dilute the serum 100× with Assay Diluent by adding3 ul of serum to 297 ul of Assay Diluent in a 1.5 ml microtube and mix well by vortexing.
4. Specimens that cannot be assayed within 24 hours after collection should be frozen at -20 oC or below, and will be stable for up to 6 months.
8. Assay Procedure
1. Secure the desired number of coated strips into the holder and seal unused strips in the tin foil bag.
2. Dispense 100 ul of PAI-1 calibrators, diluted samples or controls into appropriate wells.
3. Incubate at room temperature (18-25 oC) for 45 minutes.
4. Discard the contents and strike the strips on dry paper towels to remove residual solution. (Note: Do not wash the wells after this step!)
5. Dispense 100 ul diluted biotinylated antibody into each well and incubate at room temperature for 30 minutes.
6. Discard the contents into a waste container and dispense 300 ul washing buffer into each well and soak for 30 seconds.
7. Discard the washing buffer and strike the strips on dry paper towels to remove residual buffer.
8. Repeat steps 6-7 for 4 times.
9. Dispense 100 ul diluted HRP-SPT into each well and incubate at room temperature for 20 minutes.
10. Repeat steps 6-7 for four times.
11. Dispense 100 ul TMB substrate mixture into each well and gently mix for 10 seconds.
12. Incubate at room temperature for 15 minutes, or till desired blue color intensity is developed.
13. Stop the reaction by adding 50 ul of 2 N H2SO4 into each well and gently mix for 10 seconds.
14. Read the absorbance at 450 nm with a micro-plate reader.
Note:
1. Pre-warmed all coated strips, standards, diluted samples and controls to room temperature for 15 minutes prior to assay.
2. It is recommended that all standards, samples and controls should be run in duplicate.
3. The absorbance should be read within 10 minutes after the addition of stop solution.

9. Calculations
1. Subtract the absorbance of the blank from that of standards, samples and controls.
2. Calculate the mean absorbance values of each standard. Generate a standard curve by plotting the absorbance obtained (y-axis) against PAI-1 concentrations (x-axis). The best fit line can be generated with any curve-fitting software by regression analysis. Any curve of 4-parameter or log-log curve fitting can be used for calculation.
3. Determine PAI-1 concentration of samples from standard curve and multiply the value by the dilution factor of 100.
Example of standard curve
Note: This standard curve is for the purpose of illustration only, and should not be used for calculating the unknown PAI-1 concentration. Users should construct their own standard curves for independent assays.

10. Performance Characteristics
1) Sensitivity. The minimum detectable PAI-1 level of this ELISA assay as determined by 2SD from the mean of 10 zero calibrator is estimated to be 0.04 ng/ml. The functional sensitivity was determined as 0.08 ng/ml.
2) Specificity. The assay was tested to have no cross-reactivity with mouse or rat PAI-1, or other cytokines or hormone molecules, such as adiponectin, IL-2, IL-6, resistin, leptin and TNF-alpha et al.
3) Precision.
a. Intra-assay variation: Within-run precision was determined by triplicate determination of PAI-1 concentrations of five serum samples in one assay.
b. Inter-assay variation: Between-run precision was determined by replicate determination of PAI-1 concentrations of five serum samples over a series of independent assays.

11. Troubleshooting and FAQs
1/ Weak signal in all wells
Possible explanations:
- Omission of a reagent or a step
- Improper preparation or storage of a reagent
- Assay performed before reagents were allowed to come to room temperature
2/ High signal and background in all wells
Possible explanations:
- Improper or inadequate washing
- Overdeveloping; incubation time should be decreased before addition of Stop Solution
3/ High coefficient of variation (CV)
Possible explanation:
- Improper or inadequate washing
1
2. References
1. Dellas C. and Loskutoff D.J. (2005) Thromb Haemost, 93, 631-640.
2. Silverman G.A. et al (2001) J Biol Chem, 276, 33293-33296.
3. van Mourik J.A., Lawrence D.A. and Loskutoff D.J. (1984) J Biol Chem, 259, 14914-14921.
4. Festa A, D’Agostino R. Jr., Tracy R.P. and Haffner S.M. (2002) Diabetes, 51, 1131-1137.
5. Leurs P.B. et al. (2002) Diabetes Care, 25, 1340-1345.
6. Pannacciulli N. et al. (2002) Obes Res, 10, 717-725.
7. Festa A. et al. (1999) Arterioscler Thromb Vasc Biol, 19, 562-568.
8. Meigs J.B. et al (2000) JAMA, 283, 221-228.
9. Aso Y., et al. (2002) Metabolism, 51, 471-476.
10. McMahon G.A., et al (2001) J Biol Chem, 276, 33964-33968.