Product Details
| abID | ab237650 |
|---|---|
| Cat # +Size | E4381-100 |
| Size | 100 assays |
| Detection Method | Absorbance (450 nm) |
| Species Reactivity | Human |
| Applications | This ELISA kit is used for quantitative measurement of Omalizumab in human serum and plasma |
| Features & Benefits | • Easy, convenient, sensitive and time-saving method to measure the level of Omalizumab in human serum and plasma. • Detection Range: 10 - 1000 ng/ml • Sensitivity: 10 ng/ml • Assay Precision: Intra-Assay: CV < 15%; Inter-Assay: CV < 15% (CV (%) = SD/mean X 100) • Recovery rate: 85 – 115% with normal human serum samples with known concentrations • Cross Reactivity: There is no cross reaction with native serum immunoglobulins and tested monoclonal antibodies such as infliximab, adalimumab, etanercept, bevacizumab, trastuzumab |
| Kit Components | • Micro ELISA Plate • Omalizumab Standards (S1 – S7) • Assay Buffer • HRP-conjugate Probe • TMB substrate (Avoid light) • Stop Solution • Wash buffer (20X) • Plate sealers |
| Storage Conditions | 4°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Omalizumab is a recombinant DNA-derived humanized IgG1k monoclonal antibody that selectively binds to human immunoglobulin E (IgE). Omalizumab inhibits the binding of IgE to the high-affinity IgE receptor (FceRI) on the surface of mast cells and basophils, blocking the IgE-mediated secretion of inflammatory mediators from these cells. Mast cell activation and the release of mediators, in response to allergen exposure and IgE, results in a cascade of events. This cascade culminates in the activation of B-lymphocytes, T-lymphocytes, eosinophils, fibroblasts, smooth muscle cells, and the endothelium. BioSimTM Omalizumab (Human) ELISA Kit has been developed for specific quantification of Omalizumab concentration in human serum or plasma with high sensitivity and reproducibility. It is based on the principle of sandwich ELISA. Standards and samples (serum or plasma) are incubated in the microtiter plate coated with the omalizumab. The wells are washed after incubation. HRP-Streptavidin is added and unbound conjugates are washed away with wash buffer. The HRP enzymatic reaction is detected using TMB-substrate. Finally, an acidic stop solution terminates the enzymatic reaction. The color developed is directly proportional to the amount of omalizumab antibodies in the sample. Results of the sample can be determined using the standard curve.
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What is the reagent used to coat the plate, IgE or anti-Omalizumab antibody? And what is the detection reagent, labeled IgE or another anti-Omalizumab antibody with labeling?
This is a sandwich ELISA. The plate is coated with IgE antibody and the detection reagent is an Fc specific anti-human IgG with HRP labeling.
What is a sandwich ELISA?
The analyte of interest in a sample is "sandwiched" by an immobilized capture antibody coated on the plate and a labeled antibody used for detection. Sandwich ELISA is very sensitive. The density of color is proportional to the amount of analyte captured from the samples and can be quantified when compared with standard curve.
What is a competitive ELISA?
The endogenous unlabled analyte of interest in a sample is "competed" by the exogenous labeled antigen coated on the plate for a limited amount of antibody binding sites. Therefore, the lower signal indicate higher concentration of the analyte.
What dilution range should I use for the samples?
A preliminary experiment is always recommended when working with new samples and ELISA Kits to ensure all results fall within the detection range.
Can I extend the standard curve (in either direction)?
Extended standard curve is not recommended by BioVision.
What type of software is needed to graph a 4-parameter or 5-parameter curve?
SoftMax Pro by Molecular Devices, SigmaPlot® by Systat Software Inc., or others can be used for this purpose.
Low absorbance; No signal
Target present below detection limits of assay
Reduce dilution factor to increase sample concentration (Preliminary experiment is recommended for optimal dilution range)
Incompatible sample types
Use samples recommended by the kit
Standard dilutions are unstable
Use fresh standard dilutions
Buffers/substrates are contaminated
Use new buffers and substrates
Insufficient incubation time
Extend incubation time
Cover or seal plates during all incubation
Plate washings too vigorous
Pipette wash buffer gently or make sure the automatic wash system has correct pressure
Wells scratched with pipette or washing tips
Use caution when dispensing and aspirating into and out of wells
Incubation temperature too low
Ensure incubations are carried out at temperatures recommended by the manual
Reagents are cold
Bring all reagents to room temperature before each assay
Plate read at incorrect wavelength
Ensure the plate reader is set at the correct wavelength recommended on the protocol
High Background
Insufficient washing
Increase number of washes; Add a 30 second soaking step in between washes; at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Residual buffer/substrates remain in wells
Remove all residual buffer and substrates at the end of each wash
Too much HRP-Streptavidin
Centrifuge and mix the substrate vial before use to avoid percipitation and inconsistent HRP concentration
Excessive incubation time
Reduce incubation time
Substrate incubation carry out in light or wait too long to read plate after adding stop solution
Avoid light during incubation and read signals immediately after adding stop solution
Standards improperly reconstituted or diluted
Briefly spin vials before opening; inspect for undissolved material after reconstitution
Poor standard curve
Standard improperly diluted
Confirm dilutions are made correctly
Standard dilutions are unstable
Use freshly diluted standards
Pipetting error
use calibrated pipette and stay consistent between each pipette
Curve doesn't fit scale
Try plotting using different scales (log-log, 4 parameter logistic, 5 parameter etc.)
Large intra-coefficient of variation (CV)
Bubbles in wells
Avoid bubbles during experiment, eliminate all bubbles prior to reading
Insufficient washing
Increase number of washes; add a 30 second soak step in between washes; at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Wells not washed equally/thoroughly
Use calibrated (automated) multi-channel pipette for consistent handling, make sure all parts of the automated washer are unobstructed
Edge effects
Seal the plate completely during incubation
Incomplete reagent mixing
Make sure all reagents are mixed thoroughly during each step
Inconsistent sample preparation or storage
Consistent sample preparation. Optimize sample storage conditions and minimize freeze and thaw cycles
Stacked plates
Avoid stacking plates during incubation
Plate sealers not used or reused
Cover assay plate with plate sealer, use a fresh sealer each time to prevent contamination
Large inter-coefficient of variation (CV)
Insufficient washing
Increase number of washes; add a 30 second soak step in between washes; at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Inconsistent incubation temperature
Make sure to follow recommended incubation temperatures. Avoid fluctuations in temperature due to environmental conditions.
Plate sealers not used or reused
Cover assay plate with plate sealer, use a fresh sealer each time to prevent contamination
