Natalizumab ELISA Kit
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- Target
- Natalizumab
- Reactivity
- Human, Mouse, Rat
- Detection Method
- Colorimetric
- Method Type
- Sandwich ELISA
- Detection Range
- 1.56-50 ng/mL
- Minimum Detection Limit
- 1.56 ng/mL
- Application
- ELISA
- Purpose
- Quantification of Natalizumab in biological matrices
- Sample Type
- Plasma, Serum
- Analytical Method
- Quantitative
- Specificity
- Natalizumab (Tysabri)
- Cross-Reactivity (Details)
- Human IgG1 and rituximab prepared at 250 ng/mL were assayed
- Sensitivity
- 1.5 ng/mL
- Components
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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 - Material not included
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Precision pipettes calibrated to deliver 5-1000μL
Multi-channel pipette calibrated to deliver 50-200μL
Plate shaker
Disposable tips
Vortex-Mixer
Distilled or de-ionized water
Microplate reader capable of reading 450nm with background subtrac
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- Application Notes
- Optimal working dilution should be determined by the investigator.
- Sample Volume
- 15 μL
- Assay Time
- 2.5 h
- Plate
- Pre-coated
- 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.
- Reagent Preparation
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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 20 mL concentrate to 180 mL ultra-pure water). Mix well. 2. Detection Reagent (1X) Preparation: Dilute detection reagent with assay buffer 1/1000 before use (for example add 11 μL concentrate to 11 mL 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.
- 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
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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.
- Assay Procedure
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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. Natalizumab present in biological matrices is bound by the immobilized anti-Natalizumab antibody. After washing away any unbound substances, enzyme linked anti-Natalizumab 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. The color development is stopped and the intensity of the color is measured.
- Calculation of Results
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- 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.
- Assay Precision
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Precision: The precision was determined by analyzing samples prepared at 625 ng/mL in 6 replicates on 6 different occasions. Intra-assay coefficient of variation (CV) < 10%. Inter-assay CV < 10%.
Recovery: 250 ng/mL of Natalizumab was spiked in 10 lots of human serum. Recovery ranges are from 90-108% with an average recovery of 96%. - Restrictions
- For Research Use only
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- Preservative
- Without preservative
- Precaution of Use
- Read manual completely before beginning
- Storage
- -20 °C
- Storage Comment
- Store kit components at -20°C unless specified otherwise. DO NOT USE past kit expiration date. Some vials contain a small amount of reagents. Spin tubes on pulse setting prior to opening.
- Expiry Date
- 12 months
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- Target
- Natalizumab
- Target Type
- Antibody
- Background
- Natalizumab (Tysabri®) is a humanized monoclonal antibody against the cell adhesion molecule α4-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.
- Gene ID
- 1956
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