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

Reagents -- Composition, Packaging Storage

1. Capture Antibody (PS-EIA-C): One yellow-capped vial containing 0.5 ml of affinity-purified polyclonal anti-Protein S antibody for coating plates.
2. Detecting Antibody (PS-EIA-D): One red-capped vial containing 0.5 ml of peroxidase conjugated polyclonal anti-Protein S antibody for detection of captured Protein S.

Note: Antibodies are supplied in a 50% (v/v) glycerol solution for storage at -10 to -20°C. Keep vials tightly capped. Do not store in frost-free freezers.

Principle of Sandwich-style ELISA

Affinity-purified polyclonal antibody to PS is coated onto the wells of a microtitre plate. Any remaining binding sites on the plastic wells are blocked with bovine serum albumin. The plates are washed and plasma or other fluids containing PS are applied. The coated antibody will capture the PS in the sample. After washing the plate to remove unbound material, a peroxidase conjugated second antibody to PS is added to the plate to bind to the captured PS. After washing the plate to remove unbound conjugated antibody, the peroxidase activity is expressed by incubation with ophenylenediamine (OPD). After a fixed development time the reaction is quenched with the addition of H2SO4 and the color produced is quantified using a microplate reader. The color generated is proportional to the concentration of PS in the sample.

Assay Procedure:

1. Coating of plates: Dilute the capture antibody 1/100 in coating buffer (preferably in a polypropylene tube) and immediately add 100 milliliters to every well in the plate. Incubate overnight at 2-8°C.
2. Blocking: Empty contents of plate and add 150 milliliters of blocking buffer to every well and incubate for 90 minutes @ 22°C. Wash plate X 3 with wash buffer.
3. Samples:
   Total Protein S: Reference plasma is diluted 1/400 (100%) then serial 1/2's down to 1/12,800 (3.13%). Sample plasmas are diluted 1/800, 1/1,600 & 1/3,200.
   Free Protein S: Reference plasma and samples are PEG precipitated before use. Reference plasma supernatant is diluted 1/200 (100%) then serial 1/2's down to 1/6,400 (3.13%). Sample plasma supernatants are diluted 1/400, 1/800 & 1/1600. All dilutions are made in HBS-BSA-T20 sample diluent. Apply 100 milliliters/well and incubate plate @ 22°C for 120 minutes. Wash plate X 3 with wash buffer.
4. Detecting Antibody: Dilute the detecting antibody 1/100 in HBS-BSA-T20 sample diluent and apply 100 milliliters to each well. Incubate plate @ 22°C for 90 minutes. Wash plate X 3 with wash buffer.
5. OPD Substrate: Apply 100 milliliters of freshly prepared OPD substrate to every well. Allow color to develop for 5-10 minutes then stop color reaction with the addition of 50 milliliters/well of 2.5 M H2SO4. The plate can be read at a wavelength of 490 nm.

Related Products:

• GAPS-IG - Goat anti-human Protein S, whole IgG from antiserum
• GAPS-AP - Goat anti-human Protein S, affinity purified IgG
• GAPS-HRP - Goat anti-human Protein S, peroxidase labelled IgG
• SAPS-IG - Sheep anti-human Protein S, IgG from antiserum
• SAPS-AP - Sheep anti-human Protein S, affinity purified IgG
• SAPS-HRP - Sheep anti-human Protein S, peroxidase labelled IgG
• PS-DP - Human plasma deficient in PS, immune depleted