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Product Details
| Cat # +Size | K4696-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (450 nm) |
| Species Reactivity | human |
| Applications | This ELISA kit is used for quantitative measurement of human Apolipoprotein E (ApoE) |
| Features & Benefits | • Easy, convenient and time-saving method to assay for human ApoE. • Sensitivity: 25 ng/ml. Detection range: 25 ng to 1.6 µg/ml. • Cross Reactivity: No significant cross-reactivity or interference between this analyte and its analogues was observed. |
| Kit Components | • Plate Coated with ApoE Ab• Assay Diluent• Wash Buffer A (10x)• Wash Buffer B (10x)• rh ApoE Standard• Detection Antibody (100x)• HRP Conjugate (100x)• TMB Substrate • Stop Solution • Plate Sealer |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
BioVision’s Human Apolipoprotein E (ApoE) Enzyme-Linked Immunosorbent Assay (ELISA) Kit is an in vitro assay for the quantitative measurement of human ApoE. ApoE transports lipoproteins, fat-soluble vitamins, and cholesterol via the lymph system to the blood. It is synthesized mainly in liver, while it has also been identified in the brain, kidneys, and spleen. In the nervous system, ApoE is synthesized in non-neuronal cell types, most notably astroglia and microglia, while neurons preferentially express its receptors. Defects in ApoE lead to familial dysbetalipoproteinemia (increased plasma cholesterol and triglycerides). More recently, ApoE has been implicated in several biological processes not directly related to lipoprotein transport, including Alzheimer's disease, immunoregulation, and cognition. The assay employs an antibody specific for human ApoE coated on a 96-well plate. Standard and samples are pipetted into wells and ApoE present in the sample is bound to the wells by immobilized antibody. Wells are washed and human ApoE specific detection antibody is added. After washing away unbound detection antibody, HRP-conjugate is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells, and color develops in proportion to the amount of bound ApoE. Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. This ELISA kit recognizes ApoE-2, ApoE-3, and ApoE-4 isoforms. Sensitivity of the kit is 25 ng/ml and detection range is from 25 ng to 1.6 µg/ml. Recovery inside this range is between 86 and 111% (average recovery is 102%). The intra-assay reproducibility as measured by the coefficient of variation (CV) is < 8 % & inter-assay has CV < 12 %.
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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
