For Research Use Only. Not For Use In Diagnostic Procedures.

Reagents -- Composition, Packaging and Storage

1. Capture Antibody (Catalog No. APCAT-EIA-C): yellow capped tube containing 0.50 ml of affinity purified sheep anti-Protein C (PC) IgG for coating plates.
2. Detecting Antibody (Catalog No. APCAT-EIA-D): red capped tube containing 0.50 ml or peroxidase conjugated, affinity purified sheep anti-alpha1-antitrypsin (alpha1-AT) for detection of alpha1-antitrypsin complexed to activated Protein C (APC).

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

Procedure:

Preparation of APC-alpha1-Antitrypsin (APCAT) Reference Standard: Purified activated Protein C (2 micrograms/milliliters = 33 nMolar) is incubated with purified alpha1-antitrypsin (1.5 mg/ml = 27.3 micromolar) in 0.05 M Hepes, 0.15 M NaCl, pH 7.4. Complex formation is monitored by measuring residual activity by anticoagulant or chromogenic assay. When approximately half of the APC activity has been inhibited the reaction is quenched by inactivating residual APC with the addition of PPACK (Calbiochem) to 50 micromolar final concentration. The concentration of complex is calculated from the amount of APC activity inhibited by alpha1-antitrypsin before the addition of the PPACK. The APC-alpha1-AT complex (APCAT) is then diluted to 5 nMolar in PC deficient plasma.

Coating of plates: Dilute the capture antibody 1/100 in coating buffer in 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 plasma is diluted 1/2 (2.5 nM) down to 1/32 (0.078 nM) preferably into PC deficient plasma). The standards and samples are further diluted 1/100 in HBS-Tween-BSA 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 HBS-Tween-BSA and apply 100 microliters to every well. Incubate plate @ 22°C for 60 minutes. Wash X 4 with PBS-Tween.

OPD Substrate: Apply 100 microliters of freshly prepared OPD substrate to every well. Allow color to develop for 10-15 minutes, then stop color development with the addition of 50 microliters/well of 2.5 M H2SO4. The acidified plate can be read at a wavelength of 490 nm.