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

Reagents -- Composition, Packaging Storage

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

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 antibody to tPA 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 tPA are applied. The coated antibody will capture the tPA in the sample. After washing the plate to remove unbound material, a peroxidase conjugated second antibody to tPA is added to the plate to bind to the captured tPA. 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 tPA present in the sample.

Assay Procedure:

1. Coating of plates: Dilute the capture antibody 1/100 in coating buffer (in a polypropylene tube) and immediately add 100 microliters per well in the plate. Incubate for 2 hours at ambient temperature or overnight at 2-8°C.
2. Blocking: Empty contents of plate and add 150 microliters of blocking buffer to every well and incubate for 60 minutes @ 22°C. Wash plate X 3 with wash buffer.
3. Preparation of tPA Reference Standards: Reconstitute vials of tPA standard and tPA/PAI-1 deficient plasma according to manufacturers instructions. After reconstitution, dilute the tPA standard into tPA/PAI-1 deficient plasma to achieve six reference standard plasmas with final tPA concentrations of 50, 25, 1 2.5, 6.25, 3.13 and 1.56 nanograms/microliters respectively.
4. Samples: Reference plasmas prepared in step 3 and test plasmas are diluted ¼ in HBS-BSA-T20 sample diluent. Apply 100 microliters/well and incubate plate @ 22°C for 90 minutes. Wash plate X 3 with wash buffer.
5. Detecting Antibody: Dilute the detecting antibody 1/100 in HBS-BSA-T20 sample diluent and apply 100 microliters to each well. Incubate plate @ 22°C for 90 minutes. Wash plate X 3 with wash buffer.
6. OPD Substrate: Apply 100 microliters 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 microliters/well of 2.5 M H2SO4. The plate can be read at a wavelength of 490 nm.

Related Products:

• SATPA-IG - Sheep anti-human tPA, whole IgG from antiserum
• SATPA-AP - Sheep anti-human tPA, affinity purified IgG
• SATPA-HRP - Sheep anti-human tPA, peroxidase conjugated IgG