Research Use Only

PAI-1 ELISA

Intended Use
The PAI-1 ELISA is an enzyme-linked immunoassay used to quantitate PAI-1 (plasminogen activator inhibitor, type 1) found in human plasma.

Background
PAI-1 is a 50-kDa glycoprotein member of the serine protease inhibitor (SERPIN) superfamily and is the principal physiological inhibitor of both forms of plasminogen activators (PAs), uPA(urokinase-type plasminogen activator), and tPA (tissue-type plasminogen activator). It is secreted in an active form, which spontaneously converts to a latent form [1], but it can be stabilized in the active form by binding to the plasma protein vitronectin [2,3]. Both tumor cells and capillary endothelial cells express higher levels of PAI-1 than other cell types [4]. It is hypothesized that high levels of PAI-1 serve to protect the tumor stroma from degradation by high amounts of secreted uPA [5,6]. High PAI-1 also may contribute to tumor-induced angiogenesis [7, 8].

Initial studies focused on PAI-1 levels in tumor lysates and cytosols. Since PAI-1 is an inhibitor, it was surprising that increased levels were detected in tumor tissue. For example, a study in 2002 determined PAI-1 to be a strong prognostic indicator in tumors of primary breast cancer patients [9]. Tissue findings were then extended to plasma studies and similar results were seen. Specifically, elevated plasma levels of PAI-1 have been documented in research studies of colon cancer patients [10,11], and reports suggest that PAI-1 may be a potential marker for monitoring disease progression. Another study showed increased levels of PAI-1 above the normal cutoff in 24% of breast cancer plasmas, 30% of colon cancer plasmas, 40% of lung cancer plasmas, and 12% of prostate cancer plasmas [12]. More recently, it was shown that metastatic breast cancer patients who had elevated pretreatment plasma PAI-1 had a shorter overall survival than metastatic breast cancer patients with normal levels of PAI-1 [13].

In order to understand the potential clinical utility of measuring PAI-1 in plasma, additional studies are necessary. Plasma samples can be analyzed for PAI-1 levels using the PAI-1 ELISA. This assay detects all forms of human PAI-1.

The availability of a quantitative, reproducible, standardized assay measuring PAI-1 in plasma allows researchers to examine the potential clinical value of measuring this circulating marker at any time during cancer. Although previous research on PAI-1 levels was conducted using tissue cytosols, plasma sample testing is more convenient and less invasive than tissue cytosol analysis and could provide investigators with the ability to measure this analyte in various cancer types.

Assay Principle
The PAI-1 ELISA measures all forms of human PAI-1. It is a sandwich-type immunoassay that utilizes a monoclonal antibody to human PAI-1 as the capture reagent. The Capture Antibody has been immobilized on the interior surface of microtiter plate wells. To perform the test, an appropriate volume of specimen is incubated in the coated well to allow binding of the antigen by the Capture Antibody. The immobilized antigen is then reacted with the PAI-1 detector rabbit antiserum. The amount of Detector Antibody bound to antigen is measured by binding it with a goat-anti-rabbit IgG-horseradish peroxidase (HRP)conjugate. Color development by incubation with OPD (o-phenylenediamine) Substrate enables quantitation of captured PAI-1. The colored reaction product is quantitated by spectrophotometry and reflects the amount of PAI-1 protein in the sample.

Patents
Two issued patents have been licensed covering the PAI-1 ELISA reagent system, U.S. patent 5,422,245 and European patent 0229116.

Selected References

1. Deng G, Royle G, Seiffert D, et al. The PAI-1/vitronectin interaction: Two cats in a bag? Thrombosis and Haemostasis 1995;74:66–70.
2. Lawrence DA, Berkenpas MB, Palaniappan S, et al. Localization of vitronectin binding domain in plasminogen activator inhibitor-1. J Biol Chem 1994 May 27;269(21):15223–15228.
3. Salonen E-M, Vaheri A, Pollanen J, et al. Interaction of plasminogen activator inhibitor (PAI-1) with vitronectin. J Biol Chem 1989 Apr 15;264(11):6339–6343.
4. Bajou K, Noel A, Gerard RD, et al. Absence of host plasminogen activator inhibitor-1 prevents cancer invasion and vascularization. Nat Med 1998 Aug;4(8):923–928.
5. Kristensen P, Pyke C, Lund LR, et al. Plasminogen activator type 1 in Lewis lung carcinoma. Histochemistry 1990;93(6):559–566.
6. Bacharach E, Itin A, and Keshet E. In vivo patterns of expression of urokinase and its inhibitor PAI-1 suggest a concerted role in regulating physiological angiogenesis. Proceedings of the National Academy of Sciences, USA, 1992;89:10686–10690.
7. Pyke C, Kristensen P, Ralfkiaer E, et al. The plasminogen activation system in human colon cancer: Messenger RNA for the inhibitor PAI-1 is located in endothelial cells in the tumor stroma. Cancer Res 1991 Aug 1;51(15):4067–4071.
8. Bajou K, Masson V, Gerard RD, et al. The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies. J Cell Biol 2001 Feb 19;152(4):777–784.
9. Look MP, van Putten WL, Duffy MJ, et al. Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J Natl Cancer Inst 2002 Jan 16;94(2):116–128.
10. Nielsen HJ, Pappot H, Christensen J, et al. Association between plasma concentrations of plasminogen activator inhibitor-1 and survival in patients with colorectal cancer. BMJ 1998 Mar 14;316(7134):829–830.
11. Nielsen HJ, Christensen IJ, Sorensen S, et al. Preoperative plasma plasminogen activator inhibitor type-1 and serum C-reactive protein levels in patients with colorectal cancer. Ann Surg Oncol 2000 Sep;7(8):617–623.
12. Hamer PJ, Morris LD, Jarosz DE, et al. PAI-1 and uPA:PAI-1 complex levels in normal plasma and elevations in cancer plasma. AACR. 2002; Abstract #4496.
13. Hamer PJ, Goshgarian N, Smith J, et al. Plasma PAI-1 and uPA:PAI-1 complex levels a predictive and prognostic factors in patients with metastatic breast cancer. AACR. 2003; Abstract #6356.