ANGPTL3 ELISA Kit (Angiopoietin-Like 3)

Details for Product ANGPTL3 ELISA Kit No. ABIN956437
Antigen
  • fb60e11
  • fb66h02
  • wu:fb60e11
  • wu:fb66h02
  • zgc:111943
  • ANGPTL3
  • LOC100230785
  • ANG-5
  • ANGPT5
  • ANL3
  • FHBL2
  • hypl
  • angiopoietin-like 3
  • angiopoietin like 3
  • angptl3
  • ANGPTL3
  • Angptl3
Reactivity
Human
Alternatives
Kits with alternative reactivity to:
7
5
4
3
2
1
1
1
1
1
1
1
Detection Method
Colorimetric
Method Type
Sandwich ELISA
Detection Range
0.156-10 ng/mL
Minimum Detection Limit
0.156 ng/mL
Application
ELISA
Options
Purpose The ANGPTL3 ELISA Kit is to be used for the in vitro quantitative determination of human ANGPTL3 in biological fluids. This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human ANGPTL3 in biological fluids. A monoclonal antibody specific for ANGPTL3 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, ANGPTL3 is recognized by the addition of a purified polyclonal antibody specific for ANGPTL3 (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 ANGPTL3 in the samples.
Sample Type Cell Culture Supernatant, Plasma, Serum, Tissue Samples
Analytical Method Quantitative
Detection Method Colorimetric
Specificity The ANGPTL3 ELISA Kit is to be used for the in vitro quantitative determination of human ANGPTL3 in biological fluids. This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human ANGPTL3 in biological fluids. A monoclonal antibody specific for ANGPTL3 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, ANGPTL3 is recognized by the addition of a purified polyclonal antibody specific for ANGPTL3 (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 ANGPTL3 in the samples.
Characteristics The angiopoietins are a family of growth factors that are specific for vascular endothelium. The full-length cDNA encoding angiopoietin-like protein 3 (ANGPTL3) from a human fetal liver/spleen cDNA library has 460-amino acid and the characteristic structure of angiopoietins: a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils, a short linker peptide, and a globular fibrinogen-like domain (FLD). Human ANGPTL3 shares 76 % amino acid sequence identity with mouse Angptl3. Northern blot analysis of human tissues showed a preferential expression of 4 ANGPTL3 transcripts being 4.5, 3.0, 2.8, and 1.7 kb in liver. ANGPTL3 can induce angiogenesis in the rat corneal assay. The FLD alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Microarray analysis showed that mouse hematopoietic stem cell (HSC)-supportive fetal liver CD3-positive cells expressed Angptl2 and Angptl3. The ANGPTL3 ELISA Kit is to be used for the in vitro quantitative determination of human ANGPTL3 in biological fluids. This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human ANGPTL3 in biological fluids. A monoclonal antibody specific for ANGPTL3 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, ANGPTL3 is recognized by the addition of a purified polyclonal antibody specific for ANGPTL3 (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 ANGPTL3 in the samples.

Features and Benefits:
  • Simple procedure
  • Fast and convenient
  • High-throughput adaptable
Components
  • 1 plate coated with human ANGPTL3 Antibody
  • 1 bottle Wash Buffer 10X
  • 1 bottle Diluent 5X
  • 1 bottle Detection Antibody
  • 1 vial Detector 100X (HRP Labeled Streptavidin)
  • 1 vial human ANGPTL3 Standard (lyophilized)
  • 1 vial human ANGPTL3 QC sample (lyophilized)
  • 1 bottle TMB Substrate Solution
  • 1 bottle Stop Solution
  • 3 plate sealers (plastic film)
Plasmids, Primers & others Plasmids, Primers & others ANGPTL3 products on genomics-online (e.g. as negative or positive controls)
Antigen
Alternative Name ANGPTL3 (ANGPTL3 ELISA Kit Abstract)
Background The angiopoietins are a family of growth factors that are specific for vascular endothelium. The full-length cDNA encoding angiopoietin-like protein 3 (ANGPTL3) from a human fetal liver/spleen cDNA library has 460-amino acid and the characteristic structure of angiopoietins: a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils, a short linker peptide, and a globular fibrinogen-like domain (FLD). Human ANGPTL3 shares 76 % amino acid sequence identity with mouse Angptl3. Northern blot analysis of human tissues showed a preferential expression of 4 ANGPTL3 transcripts being 4.5, 3.0, 2.8, and 1.7 kb in liver. ANGPTL3 can induce angiogenesis in the rat corneal assay. The FLD alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Microarray analysis showed that mouse hematopoietic stem cell (HSC)-supportive fetal liver CD3-positive cells expressed Angptl2 and Angptl3.
Application Notes This assay detects human ANGPTL3 in the range of 0.156-10 ng/mL ANGPTL3/mL. The lowest level of ANGPTL3 that can be detected by this assay is 75 pg/mL.
Comment

Absorbance (450 nm)
Simple procedure
Fast and convenient
High-throughput adaptable

Plate Pre-coated
Protocol a) Determine the number of 8-well strips needed for assay and insert them into the frame for current use. The extra strips should be resealed in the foil pouch and can be stored at 4 °C for up to 1 month.
b) Add 100 l of the Standards, Samples and QC Sample into the appropriate wells in duplicate.
c) Cover plate with plate sealer and incubate for 1 hr at 37 °C.
d) Aspirate and wash x 3 with 300 l of 1X Wash Buffer.
e) Warm Detection Antibody to room temperature. Add 100 l to each well and tap gently on the side of the plate to mix.
f) Cover plate with plate sealer and incubate for 1 hr at 37 °C.
g) Aspirate and wash x 3 with 300 l of 1X Wash Buffer.
h) Add 100 l of the 1X Detector to each well.
i) Cover plate with plate sealer and incubate for 1 hr at 37 °C.
j) Remove plate from 37 °C, aspirate and wash x 5 with 300 l of 1X Wash Buffer.
k) After last wash, tap inverted plate on a stack of paper towels. Complete removal of liquid is essential for good performance.
l) Add 100 µL to each well of TMB Substrate Solution.
m) Allow the color to develop at room temperature in the dark for 10 min.
n) Stop the reaction by adding 100 l of Stop Solution to each well.
o) Tap the plate gently to ensure thorough mixing. The substrate reaction yields a blue solution that turns yellow when Stop Solution is added. Caution: Stop Solution is a Corrosive Solution p) Measure the OD at 450 nm in an ELISA plate reader within 30 min.
Reagent Preparation

Prepare just the appropriate amounts for the assay.
a) 1X Wash Buffer: Dilute 10X Wash Buffer 1: 9 with dH2O to obtain 1X Wash Buffer.
b) 1X Diluent: Dilute 5X Wash Buffer 1: 4 with dH2O to obtain 1X Diluent.
c) 1X Detector 100X (HRP Labeled Streptavidin): Dilute 100X Detector 1: 99 with 1X Diluent to obtain 1X Detector. Note: The diluted Detector must be used within 1 hr of preparation.

Assay Procedure

Test Samples/Standards/QC Sample: (We recommend these be run in duplicate)
a) Serum: Use a serum separator tube. Let samples clot at room temperature for 30 min before centrifugation for 20 min at 1000 x g. Assay freshly prepared serum or store serum in aliquots at - 20 °C for future use. Avoid repeated freeze/thaw cycles.
b) Plasma: Collect using heparin, EDTA or citrate as an anticoagulant. Centrifugation for 15 min at 1000 x g within 30 min of collection. Assay freshly prepared plasma or store in aliquots at -20 °C for future use. Avoid repeated freeze/thaw cycles Note: Serum, Plasma, Urine or Cell Culture Supernatant has to be diluted in Diluent 1X. Samples containing visible precipitates must be clarified before use. As starting point 1/50 dilution of serum or plasma are recommended.
c) QC Sample: Reconstitute Human ANGPTL3 QC sample with 1 mL of dH2O. Mix the QC Sample to ensure complete reconstitution. Allow to sit for a minimum of 15 min. The QC Sample is ready to use- do not dilute it (refer to the C of A for current QC Sample concentration).
d) Standards: Reconstitute human ANGPTL3 Standard with 1 mL of dH2O to produce a stock solution (20 ng/mL). Mix the Stock solution to ensure complete reconstitution. Allow to sit for a minimum of 15 min. The reconstituted standard should be aliquoted and stored at -20 °C.
e) Prepare 1X Diluent: Dilute 5X Diluent 1:4 with dH2O.
f) Prepare Standard Curve using 2-fold serial dilutions with 1X Diluent.

Calculation of Results

a) Average the duplicate readings for each Standard, QC Sample and Test Sample and subtract the average blank value (obtained with the 0 ng/mL point).
b) Generate a Standard Curve by plotting the average absorbance on the horizontal (X) axis vs. the corresponding concentration (µg /mL) on the vertical (Y) axis.
c) Calculate the Test Sample ANGPTL3 concentrations by interpolation of the Standard Curve regression curve in the form of a quadratic equation.
d) If the Test Samples were diluted, multiply the interpolated values by the dilution factor 5. to calculate the corrected human ANGPTL3 concentrations.

Assay Precision 1. Intra-assay precision: Six samples of known concentrations of human ANGPTL3 were assayed in replicates 6 times to test precision within an assay.
2. Inter-assay precision: Six samples of known concentrations of human ANGPTL3 were assayed in 6 separate assays to test precision between assays.
3. Recovery: When samples (serum) are spiked with known concentrations of human ANGPTL3, the recovery averages 89 % (range from 80 to 105 %).
4. Expected values: ANGPTL3 levels range in plasma and serum from 20 to 150 ng/mL (from healthy donors).
Restrictions For Research Use only
Storage 4 °C
Storage Comment Reagents must be stored at 2 - 8 °C when not in use. Bring reagents to room temperature before use. Do not expose reagents to temperatures greater than 25 °C.
Expiry Date 12 months
Supplier Images
Image no. 1 for Angiopoietin-Like 3 (ANGPTL3) ELISA Kit (ABIN956437) Angiopoietin-Like 3 (ANGPTL3) ELISA Kit
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