DiaPharma Group, Inc.

The physiologically most important plasmin inhibitor is the serpin alpha2-antiplasmin. It is a single chain 70 KD glycoprotein synthesised in the liver with a half-life of 3.3 days. The rapid reaction between plasmin and alpha2-antiplasmin results in the formation of an inactive complex composed of one molecule of each component. Two steps are involved in this process. First a reversible complex is formed between the lysine binding sites on plasmin and complementary sites on the COOH-terminal region of the alpha2-antiplasmin molecule. In a second step, an irreversible complex is formed in association with the cleavage of a peptide bond in the inhibitor.The plasma concentration of alpha2-antiplasmin is approximately 1μM or 70μg/mL. Approximately 20% of alpha2-antiplasmin present in plasma is crosslinked when blood is clotted. During thrombolytic therapy alpha2-antiplasmin is consumed. Measurement of plasmin-alpha2-antiplasmin complexes (PAP complexes) provides information concerning the extent of plasminemia and, thus, the possibility of hemorrhagic phenomena. For this application the high level version of the kit should be used.In all diseases which are accompanied by increased fibrin formation and, thus, by an increased reactive plasminemia, one encounters increased PAP complex formation and, in certain situations, there exists a correlation between the occurrence of fibrin split products and the occurrence of PAP complexes. Thus, the determination of these complexes serves as a good indicator of ongoing thrombotic-thrombolytic events. For this application the low level version of the kit should be used.

Application

The PAP complex test can be used to detect elevated levels of this complex which may occur during thrombotic events, in cases of endogenous hyperfibrinolysis and during thrombolytic therapy.

Test principle

The TC PAP-test is a solid phase enzyme immunoassay

Specificity

The TC PAP Elisa measures plasmin-alpha2-antiplasmin complexes only. It is not affected by free plasminogen, alpha2-antiplasmin or other plasmin complexes.

In the assay 15 -150ng/mL PAP complexes are measured. Normal plasma values will range from 0-514 ng/mL PAP complexes. The inter- and intraassay variations are less than 10% and 5%, respectively.

Test samples

Use "inhibitor cocktail" plasma samples (citrate plasma containing 2000 KIU/mL aprotinin and 20mM benzamidine). For this purpose collect the blood of patients to be tested in pre coated plastic or siliconized tubes already containing 3.2% buffered citrate in 1:10 ratio to blood as anticoagulant as well as aprotinin and benzamidine in a ratio yielding the final concentrations of 2000 KIU/mL and 20mM, respectively. After filling, samples should be gently mixed by inverting the tube 5 times, and, then are placed in a crushed ice-water mixture. Centrifuge the blood within 30. min. after the puncture at 2000xg for 30min. at 4°C (preferably in a cold centrifuge with swing out rotor).Immediately after centrifugation, plasma should be carefully pipetted off and collected in a small plastic tube. After carefully mixing of the pooled plasma, precooled and prelabelled plastic tubes for storage are filled. The freezing operation is very important: snapfreezing, that means almost instantaneous freezing, is highly recommended. Storage temperature should be kept constantly below a temperature of -30°C, and preferably at -70°C. The total time between blood collection and plasma freezing should not exceed 90 min. Thawing and refreezing of plasma aliquots is not recommended. Thawing for assay is achieved rapidly using a waterbath at 37°C. After thawing, plasma samples are placed in a crushed ice-water mixture until analysis. Haemolytic and lipaemic plasmas may be used if no other samples are available.

Commercially available standard control plasmas may not be used as normal controls for this assay.

Kit components

1. Plate + plate cover: 12 x 8 well microtitre strips precoated with a monoclonal directed against the neoantigen in the PAP complex and blocked with 1% bovine serum albumin (BSA), lyophilized (TC-Code GY)
2. Standard: 1x lyophilized PAP Standard (TC-Code BM)
3. POX-antibody: 1 x conjugated anti Plasminogen antibodies (concentrated). (TC-Code BK)
4. Dilution buffer: (white cap) 2 x 20mL 2.5x concentrated (PBS, 1% bovine serum albumine BSA, 2000 KIU/mL aprotinin, and 20mM benzamidine) (TC-Code AF).
5. Substrate: (green cap) 1 x 12 mL TMB (Tetramethylbenzidine) in substrate buffer containing H2O2. Ready to use. (TC-Code KN)
6. Stop solution: (red cap) 1x 12 mL 0.5 mol/l Sulphuric Acid (TC-Code KK)
7. Wash buffer: (blue cap) 2 x 20mL 12.5x concentrated (PBS 0.5% Tween 20) (TC-Code BE)