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 tPA CASSIA (ELISA)

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Haematologic Technologies, Inc.

57 River Road

Essex Junction, VT USA

Tel: 802.878.1777

Fax: 802.878.1776

Email: hti@haemtech.com

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tPA CASSIA (Colorimetric Active Site Specific Immunoassay)

The underlying biochemical mechanism governing coagulation and fibrinolysis involves a controlled cascade of proteolytic reactions characterized by the conversion of serine protease zymogens to enzymes. Depending upon the initiating stimulus, the physiological outcome of these reactions is blood clot formation/dissolution (1-4). The relative concentrations of serine protease zymogen, enzyme, or enzyme-inhibitor complex in the circulation will vary in accord with the state of vascular damage. Many conventional antigen-based clotting factor-specific immunoassays do not distinguish between zymogen and enzyme. Colorimetric Active Site Specific Immunoassay or CASSIA technology utilizes a rapid and irreversible biotinylated peptide chloromethylketone inhibitor to specifically active-site label the enzyme component within a complex milieu of zymogen and enzyme (5,6). Circulating serine proteases which are free from endogenous inhibitors at the time of blood collection incorporate the biotinylated-inhibitor and allow the investigator to measure levels of serine proteases (6,7). Tissue-type plasminogen activator (tPA) circulating in plasma exists in free forms as well as in tPA-inhibitor complexes. The tPA CASSIA kit employs a monoclonal antibody for capture which specifically recognizes both single- and two-chain tPA (7). The avidin-peroxidase conjugate then detects the biotinylated active-site modified tPA bound to the microtiter wells. The addition of a peroxidase substrate results in a colorimetric signal which is proportional to the amount of bound antigen. The total incubation time for the assay is less than two hours.

 
Extensive analyses have been performed on this assay in order to eliminate the potential for interferences. Linearity of the assay extends from 20 ng/ml to 0.3 ng/ml, with the lower limit of detection in plasma being 3 ng/ml. The intra-assay coefficient of variation (CV) is less than or equal to 7.7%, while the inter-assay CV is less than or equal to 4.5%. Recovery from normal control plasma is 85-110%. The effects of bilirubin, lipid, and lysed red blood cells on recovery from normal control plasma have been studied as well, and were found to have no significant effects on recovery. 

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References 
1. Davie, E.W. and Fugikawa, K. Ann. Rev. Biochem. 44, 799 (1975). 
2. Mann, K.G. et al., Ann. Rev. Biochem. 57, 915 (1988). 
3. Collen, D. and Lignen, H.R. CRC Crit. Rev. Oncol. Hematol. 4, 249 (1986). 
4. Collen, D. J. Cell. Biochem. 33, 77 (1987). 
5. Williams, E.B. et al., J. Biol. Chem. 264, 7536 (1989). 
6. Mann, K.G. et al., Blood 76, 755 (1990). 
7. Hartshorn, J.N. et al., Blood 78, 833 (1991).

 Please inquire about assays not listed.  We will work with you to custom develop and manufacture assays.