Infliximab (Remicade) Pharmacokinetic ELISA
Catalogue Number: EL-1611-091
Size: 1X96 wells
Antigen: Infliximab (Remicade)
Storage: -20°C, 1 year
Reactivity: Human, Mouse, Rat
Detection Range: 25ng/ml - 0.78ng/ml
Detection Minimum: 0.78ng/ml
Protocol: The Infliximab ELISA kit is designed to measure free Infliximab with high specificity and sensitivity. This assay employs the sandwich enzyme immunoassay technique. A precoated anti-Infliximab 96 well plate is provided. Calibrator, quality control samples and test samples are pipetted into the appropriate wells. Infliximab 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 Infliximab 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 Infliximab in biological matrices
Plate: Strip
Sample Type: Serum Plasma
Sample Volume: 15ul
Assay Time: 2.5 hours
Specificity: Infliximab
Assay Precision: <10%, <10%
Preservative: None
Description: Infliximab (Remicade®) is a chimeric monoclonal antibody against tumour necrosis factor alpha (TNF- α). Infliximab neutralizes the biological activity of TNF- α by binding with high affinity to the soluble (free floating in the blood) and transmembrane (located on the outer membranes of T cells and similar immune cells) forms of TNF-α, and inhibits or prevents the effective binding of TNF-α with its receptors.
Gene ID: 7124
Assay Procedure: This assay employs the sandwich enzyme immunoassay technique. Anti- Infliximab is coated onto a 96 well microplate. Calibrator, quality control samples (if desired) and test samples are pipetted into the appropriate wells. Infliximab present in biological matrices is bound by the immobilized anti- Infliximab antibody. After washing away any unbound substances, enzyme linked anti- Infliximab antibody is added to the wells. This antibody is developed and purified specifically against truncated Remicade® (domain residing in Fc portion of the Remicade® 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 Infliximab present in test samples. The color development is stopped and the intensity of the color is measured.
Reagent Preparation: Prepare 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 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 1/100 with assay buffer (for example add 5µL of prepared calibrator or sample to 495µL of assay buffer). Mix well. *Note that test samples may require further dilution when peak values are predicted to exceed 2.5ug/ml. Do not store diluted samples.