Natalizumab (Tysabri ) is a humanized monoclonal antibody against the cell adhesion molecule alpha4-integrin used in the treatment of multiple sclerosis and Crohn's disease. Tysabri and was previously named Antegren . Natalizumab is administered by intravenous infusion every 28 days and reduces the ability of inflammatory immune cells to attach to and pass through the cell layers lining the intestines and blood brain barrier.
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 11ul 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.
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 preparation Dilute calibrators and test samples 1/50 with assay buffer (for example add 5uL of prepared calibrator or sample to 245uL of assay buffer). Mix well. Do not store diluted samples.
Intra-assay coefficient of variation (CV) <10%. Inter-assay CV <10%.
Notes
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.
Usage Notes
Quantification of Natalizumab in biological matrices
Protocol
The Natalizumab ELISA kit is designed to measure free Natalizumab with high specificity and sensitivity . This assay employs the sandwich enzyme immunoassay technique. A precoated anti-Natalizumab 96 well plate is provided. Calibrator, quality control samples and test samples are pipetted into the appropriate wells. Natalizumab 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 Natalizumab present in test samples and the concentration is calculated from the standard series.
Gene ID
1956
Applications
ELISA
Storage
-20C, 1 year
Reviews of Natalizumab (Tysabri) Pharmacokinetic ELISA