Vaspin (human) Serum ELISA Kit

WARNING: This product can expose you to chemicals including TMB, which is [are] known to the State of California to cause cancer.  For more information go to www.P65Warnings.ca.gov.
Catalog #: K4917 | abID:

Product Details

Cat # +Size K4917-100
Size 100 assays
Detection Method Absorbance (450 nm)
Species Reactivity Mammalian
Applications This ELISA kit is used for this assay detects human Vaspin in the range of 0.016 – 1 ng/ml Vaspin/ml. The lowest level of vaspin that can be detected by this assay is 12 pg/ml.
Features & Benefits • Simple procedure
• Fast and convenient
• High-throughput adaptable
• Cross Reactivity: No significant cross-reactivity or interference between this analyte and its analogues was observed.
Kit Components • 1 plate coated with human Vaspin Antibody
• 1 bottle Wash Buffer 10X
• 1 bottle Diluent 5X
• 1 bottle Detection Antibody
• 1 vial Detector 100X (HRP Conjugated anti-rabbit IgG)
• 1 vial human Vaspin Standard (lyophilized)
• 1 vial human Vaspin QC sample (lyophilized)
• 1 bottle TMB Substrate Solution
• 1 bottle Stop Solution
• 3 plate sealers (plastic film)
Storage Conditions 4°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

Vaspin, designated as visceral adipose tissue-derived serpin, is a serpin whose expression is restricted to visceral adipose tissue. Vaspin seems to retain the serpin signature conformation fold consisting of three β-sheets and nine α-helices as well as a reactive loop site that interacts with its cognate serine protease unidentified hitherto. Vaspin is remarkably upregulated at high-fat high-sucrose (HFHS) diet and at multiple metabolic dysfunctions like obesity or insulin resistance. Administration of thiazolidinedione (TZD) caused a significant induction of serum vaspin in mice. These facts imply that upregulation of vaspin may be a compensatory response to antagonize the action of other unknown proteases upregulated in obesity or insulin resistance. Indeed, administration of recombinant vaspin to obese mice fed with HFHS chow improved glucose tolerance and insulin sensitivity. Vaspin seems to directly act on white adipose tissue. This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human vaspin in biological fluids. A monoclonal antibody specific for vaspin has been precoated onto the 96-well microtiter plate. Standards and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, vaspin is recognized by the addition of a purified polyclonal antibody specific for vaspin (Detection Antibody). After removal of excess polyclonal antibody, HRP conjugated anti-rabbit IgG (Detector) is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of vaspin in the samples. The assay range is 0.016 – 1 ng/ml Vaspin/ml. The lowest level of vaspin that can be detected by this assay is 12 pg/ml.


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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