Etanercept (Enbrel) Pharmacokinetic ELISA

Etanercept (trade name Enbrel®) is a protein drug used to treat autoimmune diseases by adsorbing tumor necrosis factor (TNF; a soluble inflammatory cytokine) . Etanercept is a fusion protein produced through expression of recombinant DNA by linking the extracellular ligand-binding portion of TNFRSF1B to the Fc component of human immunoglobulin G1 (IgG1) . It reduces the effect of naturally present TNF, functioning as a decoy receptor that binds to TNF. Etanercept is indicated for the treatment of moderate to severe rheumatoid arthritis (RA), psoriatic arthritis, ankylosing spondylitis, and moderately to severely active polyarticular juvenile idiopathic arthritis. Serum concentration of Enbrel® may predict some clinical outcome during maintenance therapy.

Product Specifications

CAS Number

7732-18-5

Gene ID

7124

Reactivity

Human, mouse, rat

Reaction Volume

15 µL

Target Antigen

Etanercept (Enbrel)

Detection Method

Peroxidase / OD450

Assay Type

ELISA

Assay Principle

Quantification of Etanercept in biological matrices

Assay Protocol

The Etanercept ELISA kit is designed to measure free Etanercept with high specificity and sensitivity . This assay employs the sandwich enzyme immunoassay technique. A precoated anti-Etanercept 96 well plate is provided. Calibrator, quality control samples and test samples are pipetted into the appropriate wells. Etanercept present in biological matrices is bound by the immobilized capture antibody. After washing away any unbound substances, enzyme linked detection antibody is added to the wells. The plate is washed to remove any unbound antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of Etanercept present in test samples and the concentration is calculated from the standard series.

Assay Performance Time

2.5 hours

Sample Type

Serum Plasma

Sample Volume

15ul

Detection Range

50ng/ml - 1.56ng/ml

Precision

<10%, <10%

Sensitivity

Etanercept

Limit Of Detection

1.5ng/ml

Components

Coated microtiter plate, 96 wells Calibrator diluent. - 1.8ml Calibrator 12ul 10X wash buffer - 25ml Assay buffer - 50ml 1000X detection reagent - 17ul TMB - 12ml TMB stop solution - 12ml Plate sealers - 3

Shipping Conditions

Blue Ice

Storage Conditions

-20C, 1 year

Shelf Life

12 months

Specificity

Etanercept

Method

Direct sandwich ELISA

Preservative

None

Preparation

Prepare only the appropriate amount of required reagent on the day of use. Store all reagents as per instructions stated on the label. 1. Wash Buffer (1X) Preparation: Dilute wash buffer concentrate with ultra-pure water 1/10 before use (for example add 20mL concentrate to 180mL ultra-pure water) . Mix well. 2. Detection Reagent (1X) Preparation: Dilute detection reagent with assay buffer 1/1000 before use (for example add 11μl concentrate to 11ml of assay buffer) . Mix well. 3. Preparation of Calibrators: Prepare calibrators with concentrations ranging from 2500 ng/mL to 78 ng/mL. The following is an example calibrator curve.

Operating Procedure

This assay employs the sandwich enzyme immunoassay technique. Capture antibody is coated onto a 96 well microplate. Calibrator and test samples are pipetted into the appropriate wells. Etanercept present in biological matrices is bound by the immobilized anti-Etanercept antibody. After washing away any unbound substances, enzyme linked anti-Etanercept antibody is added to the wells. The plate is washed to remove any unbound antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of Etanercept present in test samples. The color development is stopped and the intensity of the color is measured.

Results Calculation

1. Construct a standard curve by plotting the absorbance obtained from each standard against concentration. Use a 4 or 5 parameter curve fit. Alternatively a log-log curve fit may be used. 2. The concentration of the unknowns can be read directly from this standard curve using the absorbance value for each sample. 3. Any sample undiluted or diluted still reading greater than the highest standard should be diluted appropriately with assay buffer and retested. If the samples have been diluted, the concentration determined from the standard curve must be multiplied by the dilution factor.

Sample Collection

This kit is compatible with EDTA-plasma, heparinplasma and serum samples. Samples can be stored at or below -20°C for up to 1 year.

Sample Preparation

Dilute calibrators and test samples 1/50 with assay buffer (for example add 5µL of prepared calibrator or sample to 245µL of assay buffer) . Mix well. Do not store diluted samples.