DiaPharma Group, Inc.

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

Reagents -- Composition, Packaging and Storage

1. Capture Antibody (HCII-EIA-C): One yellow-capped vial containing 0.4 ml of polyclonal affinity purified anti-HCII antibody for coating plates.
2. Detecting Antibody (HCII-EIA-D): Four neutral-capped tubes each containing 10 ml of pre-diluted peroxidase conjugated polyclonal anti-HCII antibody for detection of captured HCII.

Store reagents at 2-8°C

Principle of Sandwich-style ELISA

Affinity-purified antibody to HCII is coated onto the wells of a microtitre plate. Any remaining binding sites on the plastic wells are blocked with an excess of bovine serum albumin. The plates are washed and plasma or other fluids containing HCII are applied. The coated antibody will capture the HCII in the sample. After washing the plate to remove unbound material, a peroxidase conjugated second antibody to HCII is added to the plate to bind to the captured HCII. 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 HCII present 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 microliters to every well in the plate. Incubate 2 hrs @ 22°C or overnight @ 4°C.
2. 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 X 3 with wash buffer.
3. Samples: Reference plasma is diluted 1/500 (100%) then serial 1/2's down to 1/16000 (3.13%). Sample plasmas are diluted 1/1000, 1/2000 & 1/4000. All dilutions are made in HBS-BSA-T20 sample diluent. Apply 100 microliters/well and incubate plate @ 22°C for 60 minutes. Wash plate X 3 with wash buffer.
4. Detecting Antibody: Apply the pre-diluted detecting antibody, 100 microliters to each well. Incubate plate @ 22°C for 60 minutes. Wash plate X 3 with wash buffer.
5. 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. The plate can be read at wavelength of 490 nm.

Related Products:

• GAHC2-IG - Goat anti-HCII, whole IgG from antiserum
• GAHC2-AP - Goat anti-HCII, affinity purified IgG (APIgG)
• GAHC2-APHRP - Goat anti-HCII, APIgG-peroxidase labelled
• SAHC2-IG - Sheep anti-HCII, whole IgG from antiserum
• THCII-EIA - Antibody set for ELISA of thrombin-HCII complexes
• HCII-DP - Human HCII-deficient plasma, immune depleted
• ATHC-DP - Human ATIII & HCII def plasma, immune depleted