DiaPharma Group, Inc.

For Research Use Only. Not For Use In Diagnostic Procedures

Matched Pair Aintibodies for ELISA for Thrombin-Heparin Cofactor II (T-HCII) Complexes

1. Capture Antibody (Catalog No. T:HCII-EIA-C): Yellow capped tube containing 0.50 ml of affinity purified sheep antibody to Thrombin for coating plates.
2. Detecting Antibody (Catalog No. T:HCII-EIA-D): Red capped tube containing 0.50 ml of peroxidase conjugated, affinity purified goat antibody to Heparin Cofactor II for detection of HCII complexed to Thrombin.

These reagents are supplied in 50% glycerol and should be stored at -20°C.

Assay Procedure

Preparation of Thrombin-HCII Reference Standard: Prepare a solution of purified HCII (330 micrograms/milliliters = 5 micrometers) in TBS containing 1 mM EDTA an 0.05 micro/milliliters heparin. Add purified thrombin to a final concentration of 1 micrometers (37 micrograms/milliliters) and incubate at 37°C for 30 minutes. Check that no thrombin activity remains by clot-time or chromogenic assay.This solution contains 1 micrometers T:HCII complex. By diluting this complex into Factor II Deficient Plasma, a series of standards can be constructed. For example a dilution of 1/1,667 into plasma gives a 600 pM standard, a dilution of 1/5,000 for a 200 pM standard, a 1/16,667 for a 60 pM standard and a 1/50,000 for a 20 pM standard.

Coating of plates: Dilute the capture antibody 1/100 (100 microliters IgG in 10 ml coating buffer preferably a polypropylene tube) and immediately add 100 microliters to every well in the microplate. Incubate 2 hrs @ 22°C or overnight @ 4°C.

Blocking: Empty contents of plate and add 150 microliters of blocking buffer to every well and incubate for a minimum of 90 minutes @ 22°C. Wash plate X4 with PBS-Tween.

Samples: Standard and test plasmas are diluted 1/4 in diluent. Apply 100 microliters/well and incubate plate @ 22°C for 2 hours. Wash X 4 with PBS-Tween.

Detecting Antibody: Dilute the detecting antibody 1/100 in diluent and apply 100 microliters to every well. Incubate plate @ 22°C for 1 hour. Wash X 4 with PBS-Tween, then once with water.

OPD Substrate: Apply 100 microliters of freshly prepared OPD substrate to every well. Allow color to develop for 10-15 minutes at ambient temperature, then quench reaction with the addition of 50 microliters/well of 2.5 M H2SO4.

The acidified plate can be read at a wavelength of 490 nm. Results can be calculated by plotting concentration of standards versus absorbance at 490 nm on log-log graph paper, and reading unknowns from this line.