DiaPharma Group, Inc.

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

Reagents -- Composition, Packaging and Storage

1. Capture Antibody (Catalog No. APCPCI-EIA-C): Yellow capped tube containing 0.5 ml of affinity purified sheep anti-Protein C (PC) IgG for coating plates.
2. Detecting Antibody (Catalog No. APCPCI-EIA-D): Red capped tube containing 0.5 ml of peroxidase labelled anti-Protein C Inhibitor (PCI) for detection of PCI complexes.

Both reagents are supplied in 50% glycerol and should be stored at -10 to -20°C.

Assay Procedure:

Preparation of APC-PCI Reference Standard: Purified activated Protein C (final concentration of 2 micrograms/milliliters = 33 nMolar) is incubated with purified PCI (final concentration 50 micrograms/milliliters = 0.88 uMolar) in 0.05 M Hepes, 0.15 M NaCl, pH 7.4 and 0.25 micro/milliliters heparin. 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 to 50 uMolar final concentration. The concentration of complex is calculated from the amount of APC activity inhibited by PCI before the addition of the PPACK. The APC-PCI complex is then diluted to 5 nMolar in Protein C deficient plasma. Standards are not stable and if stored frozen in aliquots should be re-assayed against fresh standard.

Coating of plates: Dilute the capture antibody 1/100 in coating buffer 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 (0.156 nM) preferably into PC-deficient plasma). The standards and samples are further diluted 1/40 in HBS-Tween-BSA. 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 1 hour. 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 reaction with the addition of 50 microliters/well of 2.5 M H2SO4. Read plate at a wavelength of 490 nm.