Product Specification

                11dhTxB₂ ELISA Test Kit

INTENDED USE

Complete ELISA kit-ready-to-use reagents

96-well microplate format

Single wash step

Assay time-2.5 hours

BACKGROUND

1.Aspirin,the foundation of antiplatelet therapy in cardiovascular medicine,is widely prescribed by physicians to prevent heart attack and stroke.It is estimated that more than 150,000 heart attacks each year could be prevented by the appropriate use of aspirin therapy.

2.Recently,physicians discovered that a significant number of individuals taking dosages of aspirin

considered therapeutic were experiencing vascular thrombotic events including acute coronary

syndromes,transient ischemic attacks,strokes and peripheral vascular events.In addition,clinical researchers,utilizing a variety of laboratory tests, discovered that some patients have a reduced or minimal response to aspirin administration.

3.Test Kit is a quantitative enzyme-linked immunoassay (ELISA)to determine levels of 11-dehydro thromboxane B2 (11dhTxB2) in human urine,which aids in the determination of platelet response to aspirin ingestion.

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PRINCIPLE AND PROCEDURE

Test Kit measures urinary 11-dehydrothromboxane B2(11dhTxB2)and is performed as acompetitive ELISA.Diluted samples,purified 11dhTxB2 conjugated to alkaline phosphatase(AP),purified mouse monoclonal antibody directed to 11dhTxB2 are combined and incubated in microwells coated with a polyclonal anti-mouse antibody. Incubation allows the endogenous 11dhTxB2 present in the samples to compete with the purified AP-conjugated 11dhTxB2 for binding to the mouse monoclonal anti-11dhTxB2 antibody.The monoclonal antibody then binds to the polyclonal anti-mouse antibody coated on the microtiter plate.The complex formed on the plate is composed of monoclonal antibody and endogenous or AP-conjugated 11dhTxB2.After the removal of unbound complexes by washing,the bound AP-11dhTxB2 conjugate is assayed by the addition of paranitrophenylphosphate (pNPP)chromogenic substrate.Color develops in the wells at an intensity inversely proportional to the sample urine concentration of 11dhTxB2,and is read on a spectrophotometer at 405nm.Results (pg/mL)are calculated against a reference curve prepared from the Reference Solution provided in the kit.Final results are reported as pg 11dhTxB2 per mg creatinine to normalize results for urine concentration.

CLINICAL PERFORMANCE

Results are presented as positive or negative,based on a cutoff of 1500 pg 11-dehydro thromboxane B2 per mg urinary creatinine.The detection range for 11-dehydro thromboxane B2 in the test is 300-4000 pg/mL urine.For greatest accuracy,samples that generate values greater than 4000 pg/mL should be retested at an appropriate dilution.The analyte concentration reported should be normalized by dividing the measured 11 dhTxB2 by the concentration of urine creatinine as measured by a separate assay.The test kit was evaluated for intra-assay(72 replicates per sample within 1 kit lot)and inter- assay(216 replicates per sample over 3 kit lots) precision using the kit urine controls.Results are summarized in the following table:

FEATURE

· Random Urine Sample

· Results reported as:

≤1500 pg 11dhTxB₂/mg of creatinine=ASPIRIN RESPONDER

>1500 pg 11dhTxB₂/mg of creatinine=ASPIRIN NON-RESPONDER

· Urinary 11-dehydro Thromboxane B₂ measurement

· Quantitative measurement of 11-dehydro Thromboxane B₂in human urine which aids in the qualitative detection of acetylsalicylic acid (ASA)effect

· NEW CAP(College of American Pathologists) proficiency sample available beginning 2012. Three (3)samples twice per year-listed under CAP:COAGULATION Code TBX

· Assay formats available for both manual and select automated platforms

PREPARATIONS

1.  Allow all specimens and reagents to reach room temperature (~25°C) and mix thoroughly by gentle inversion before use.

2.Prepare Wash Buffer by diluting Wash Concentrate 20-fold with deionized water. The diluted wash solution is stable in room temperature for at least one week.

PRECAUTION FOR USERS

1. Handling should preclude any pipetting by mouth. Use only pipettes with disposable tips for each specimen.

2.Do not mix materials from different master lots. Do not use kit components beyond the expiration date.

3.All materials should be brought to room temperature before use.