Tocilizumab (Actemra) Pharmacokinetic ELISA
Catalogue Number: EL-1611-202
Size: 1X96 wells
Antigen: Tocilizumab (Actemra)
Storage: -20°C, 1 year
Reactivity: Human, Mouse, Rat
Detection Range: 50ng/ml - 1.56ng/ml
Detection Minimum: 1.5ng/ml
Protocol: The Tocilizumab ELISA kit is designed to measure free Tocilizumab with high specificity and sensitivity. This assay employs the sandwich enzyme immunoassay technique. A precoated anti-Tocilizumab 96 well plate is provided. Calibrator, quality control samples and test samples are pipetted into the appropriate wells. Tocilizumab 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 Trastuzumab present in test samples and the concentration is calculated from the standard series.
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
Method Type: Direct sandwich ELISA
Detection Method: Peroxidase / OD450
Principle: Quantification of Tocilizumab in biological matrices
Plate: Strip
Sample Type: Serum Plasma
Sample Volume: 15ul
Assay Time: 2.5 hours
Specificity: Tocilizumab
Assay Precision: <10%, <10%
Preservative: None
Description: Tocilizumab (Actemra®) is an immunosuppressive humanized monoclonal antibody drug, mainly for the treatment of rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis, a severe form of arthritis in children. Tocilizumab binds the interleukin-6 receptor (IL-6R) thus blocking the signaling caused by interleukin 6 (IL-6). IL-6 is a cytokine that plays an important role in immune response and is implicated in the pathogenesis of many diseases.
Gene ID: 3570
Assay Procedure: This assay employs the sandwich enzyme immunoassay technique. Anti- Tocilizumab is coated onto a 96 well microplate. Calibrator and test samples are pipetted into the appropriate wells. Tocilizumab present in biological matrices is bound by the immobilized anti- Tocilizumab antibody. After washing away any unbound substances, enzyme linked antiTocilizumab antibody is added to the wells. This antibody is developed and purified specifically against Actemra® (domain residing in Fc portion of the Actemra® molecule). 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 Tocilizumab present in test samples. The color development is stopped and the intensity of the color is measured
Reagent 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 2,500 ng/mL to 78 ng/mL. The following is an example calibrator curve.
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 calibrator diluent 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.