Human COMP / TSP5 ELISA Pair Set

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Antigen
  • COMP
  • EDM1
  • EPD1
  • MED
  • PSACH
  • THBS5
  • TSP5
  • cartilage oligomeric matrix protein
  • COMP
  • sce3551
  • CJA_1292
  • Comp
Reactivity
Human
Host
Mouse
Conjugate
Biotin
Method Type
Sandwich ELISA
Application
ELISA
Options
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Purpose The human COMP / TSP5 ELISA Pair Set is for the quantitative determination of human COMP.
This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.
Detection Method Colorimetric
Sensitivity 39.1 pg/mL
Characteristics Capture Antibody: 0.5 mg/mL of mouse anti-COMP monoclonal antibody. Dilute to a working concentration of 2 μg/mL in CBS before coating.

Detection Antibody: Each vial contains 120 μg biotinylated rabbit anti-COMP polyclonal antibody. Reconstitute with sterile 1 mL distilled water. Dilute to a working concentration of 2 μg/mL in detection antibody dilution buffer before use.

Standard: Each vial contains 60 ng of recombinant COMP. Reconstitute with 1 mL detection antibody dilution buffer. A seven-point standard curve usi ng 2-fold serial dilutions in sample dilution buffer, and a high standard of 2500 pg/mL is recommended.

Sensitivity: The minimum detectable dose of human COMP / TSP5 was determined to be approximately 39.1 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
Components
  • Capture Ab
  • Detection Ab
  • Standard
Material not included CBS - 0.05 M
Na2CO3 - 0.05 M
NaHCO3, pH 9.6, 0.2 μm filtered
TBS - 25 mM
Tris, adjust pH to 7.4 by HCl
Wash Buffer - 0.05 % Tween20 in TBS, pH 7.2 - 7.4
Blocking Buffer - 5 % BSA in Wash Buffer
Sample dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Detection antibody dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Substrate Solution: To achieve best assay results, fresh substrate solution is recommended Substrate stock solution - 10 mg/mL TMB (Tetramethylbenzidine ) in DMSO Substrate dilution buffer - 0.05 M Na2HPO4 and 0.025 M citric acid , adjust pH to 5.5
Substrate working solution - For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilutionbuffer and then add 80 μL 0.75 % H2O2 , mix it well
Stop Solution - 2 N H2SO4
Alternative Name COMP
Background Cartilage Oligomeric Matrix Protein (COMP) , also referred to as thrombospondin-5, is a secretednoncollagenous extracellular matrix (ECM) protein and belongs to the subgroup B of the thrombospondinprotein family. The native glycoprotein incorporates five identical subunits, each with the molecular weight of87 KDa consists of four epidermal growth factor (EGF) -like domains, eight so-called thrombospondin type3 (T3) repeats (calcium-binding domain) and a carboxyl-terminal globular domain. A coiled-coil domain atthe amino terminus mediates the pentamerization resulting in a bouquet-shaped subunit arrangement viadisulfide-bonds. COMP is a component of cartilage, synovium, ligament, and tendon, and binds to otherECM proteins such as collagen I, II and IX with high affinities depending on the divalent cations Zn2+ orNi2+ . COMP is suggested to play a role in cell growth and development, and recent studies have revealedthe possible mechanism that it protects cells against death by elevating members of the IAP (inhibitor ofapoptosis protein) family of survival proteins. COMP mutations cause dominantly inheritedchondrodysplasias Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) characterizedby short stature and early-onset osteoarthrosi, and up-regulated expression of COMP are observed inrheumatoid arthritis and certain carcinomas
Research Area Stem Cells, Extracellular Matrix
Application Notes 50 μL of streptavidin conjugated to horseradish-peroxidase. 1:2000 Dilution in detection antibody dilution buffer before use.
Protocol This ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utilizes a monoclonal antibody specific for COMP /TSP5 coated on a 96-well plate. Standards and samples are added to the wells,and any COMP / TSP5 present binds to the immobilized antibody. The wells arewashed and a biotinylated rabbit anti-COMP polyclonal antibody is then added,producing an antibody-antigen-antibody "sandwich". To produces color inproportion to the amount of COMP / TSP5 present in the sample strepavidin-HRPand TMB substrate solution are loaded. The absorbances of the microwell areread at 450 nm.
Reagent Preparation
  1. Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4 °C.
    2. Aspirate each well and wash with at least 300 μL wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
    3. Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
    4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
  1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at roomtemperature.
    2. Repeat the aspiration/wash as in step 2 of plate preparation.
    3. Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1hour at room temperature.
    4. Repeat the aspiration/wash as in step 2 of plate preparation.
    5. Add 100 μL of Streptavidin-HRP to each well. Incubate for 1 hour at room temperature.
    6. Repeat the aspiration/wash as in step 2 of plate preparation.
    7. Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature (if substrate solutionis not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
    8. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each. Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.

Restrictions For Research Use only
Format Liquid
Precaution of Use The Stop Solution suggested for use with this Pair Set is an acid solution. Wear eye, hand, face, andclothing protection when using this material.
Handling Advice Avoid repeated freeze-thaw cycles.
Storage 4 °C,-20 °C,-80 °C
Storage Comment Capture Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Avoid repeated freeze-thaw cycles.

Detection Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Avoid repeated freeze-thaw cycles.

Standard: Store lyophilized standard at -20°C to -80°C for up to 6 months from date of receipt. Aliquot and store the reconstituted Standard at -80°C for up to 1 month. Avoid repeated freeze-thaw cycles.

Streptavidin-HRP: Store at 4°C and protect it from prolonged exposure to light. DO NOT FREEZE! It is stable for up to 6 months from date of receipt.
Expiry Date 6 months
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