COL18A1 ELISA Kit

Catalog No. ABIN1979625

Product Details COL18A1 ELISA Kit

Target: COL18A1 (Collagen, Type XVIII, alpha 1 (COL18A1))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 10-10000 pg/mL

Minimum Detection Limit: 10 pg/mL

Application: ELISA

Purpose: Human Endostatin ELISA Kit for cell culture supernatants, plasma, and serum samples.

Sample Type: Plasma, Cell Culture Supernatant, Serum

Analytical Method: Quantitative

Specificity: This ELISA kit shows no cross-reactivity with any of the cytokines tested: Human Angiogenin, BDNF, BLC, ENA-78, FGF-4, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, I-309, IP-10, G-CSF, GM-CSF, IFN-gamma, Leptin, MCP-1, MCP-2, MCP-3, MDC, MIP-1 alpha, MIP-1 beta, MIP-1 delta, PARC, PDGF, RANTES, SCF, TARC, TGF-beta, TIMP-1, TIMP-2, TNF-alpha, TNF-beta, TPO, VEGF.

Sensitivity: < 10 pg/mL

Characteristics:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Components:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Material not included:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis

Application Details

Application Notes: Recommended Dilution for serum and plasma samples100 fold

Sample Volume: 100 μL

Plate: Pre-coated

Protocol:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Reagent Preparation:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: If your samples need to be diluted, Assay Diluent A (Item D) should be used for dilution of serum/plasma samples. 1x Assay Diluent B (Item E) should be used for dilution of culture supernatants and urine. Suggested dilution for normal serum/plasma: 30-300 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
   3. Assay Diluent B should be diluted 5-fold with deionized or distilled water.
   4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture medium and urine, Assay Diluent B should be diluted 5-fold with deionized or distilled water) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 100 µL Endostatin standard from the vial of Item C, into a tube with 400 µL Assay Diluent A or 1x Assay Diluent B to prepare a 10,000 pg/mL stock standard solution. Pipette 400 µL Assay Diluent A or 1x Assay Diluent B into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 200 µL 100 µL standard + 400 µL 200myl 200 µL 200 µL 200 µL 200 µL 10,000 3333.3 1111.1 370.4 123.5 41.2 13.7 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP- Streptavidin concentrate should be diluted 400-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 35 µL of HRP-Streptavidin concentrate into a tube with 14 ml 1x Assay Diluent B to prepare a final 400 fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Assay Procedure:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Calculation of Results:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent A Human Endostatin concentration (pg/mL) O D =4 50 (n m ) 0.1 1 10 10 100 1,000 10,000 100,000 Assay Diluent B Human Endostatin concentration (pg/mL) O D =4 50 (n m ) 0.01 0.1 1 10 10 100 1,000 10,000 100,000
Sensitivity: The minimum detectable dose of Endostatin is typically less than 10 pg/mL.
Recovery: Recovery was determined by spiking various levels of human Endostatin into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 88.47 78-102 Plasma 92.31 80-104 Cell culture media 95.32 84-108
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 103 105 102 Range ( %) 94-115 95-117 93-116 1:4 Average % of Expected 98 110 105 Range ( %) 90-112 96-120 94-116
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Assay Precision: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Restrictions: For Research Use only

Handling

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

Storage Comment: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Expiry Date: 6 months

References for COL18A1 ELISA Kit (ABIN1979625)

Etulain, Mena, Negrotto, Schattner: "Stimulation of PAR-1 or PAR-4 promotes similar pattern of VEGF and endostatin release and pro-angiogenic responses mediated by human platelets." in: Platelets, Vol. 26, Issue 8, pp. 799-804, (2015) (PubMed).

Zhang, Yu, Lu, Xia, Xie, Hu, Hao, Xu, Qian: "Prognostic values of VEGF and endostatin with malignant pleural effusions in patients with lung cancer." in: Asian Pacific journal of cancer prevention : APJCP, Vol. 15, Issue 19, pp. 8435-40, (2014) (PubMed).

Biswal, Prentice, Dorey, Blanks: "A hypoxia-responsive glial cell-specific gene therapy vector for targeting retinal neovascularization." in: Investigative ophthalmology & visual science, Vol. 55, Issue 12, pp. 8044-53, (2014) (PubMed).

Etulain, Negrotto, Tribulatti, Croci, Carabelli, Campetella, Rabinovich, Schattner: "Control of angiogenesis by galectins involves the release of platelet-derived proangiogenic factors." in: PLoS ONE, Vol. 9, Issue 4, pp. e96402, (2014) (PubMed).

Etulain, Fondevila, Negrotto, Schattner: "Platelet-mediated angiogenesis is independent of VEGF and fully inhibited by aspirin." in: British journal of pharmacology, Vol. 170, Issue 2, pp. 255-65, (2013) (PubMed).

Fujii, Furumatsu, Yokoyama, Kanazawa, Kajiki, Abe, Ozaki: "Chondromodulin-I derived from the inner meniscus prevents endothelial cell proliferation." in: Journal of orthopaedic research : official publication of the Orthopaedic Research Society, Vol. 31, Issue 4, pp. 538-43, (2013) (PubMed).

Kerr, Whyte, Watson, Tolson, McFadyen: "A mixed-methods evaluation of the effectiveness of tailored smoking cessation training for healthcare practitioners who work with older people." in: Worldviews on evidence-based nursing, Vol. 8, Issue 3, pp. 177-86, (2012) (PubMed).

Etulain, Lapponi, Patrucchi, Romaniuk, Benzadón, Klement, Negrotto, Schattner: "Hyperthermia inhibits platelet hemostatic functions and selectively regulates the release of alpha-granule proteins." in: Journal of thrombosis and haemostasis : JTH, Vol. 9, Issue 8, pp. 1562-71, (2012) (PubMed).

Jing, Lu, Wu, Subramanian, Ramakrishnan: "Inhibition of ovarian cancer by RGD-P125A-endostatin-Fc fusion proteins." in: International journal of cancer, Vol. 129, Issue 3, pp. 751-61, (2011) (PubMed).

Ständker, Schrader, Kanse, Jürgens, Forssmann, Preissner: "Isolation and characterization of the circulating form of human endostatin." in: FEBS letters, Vol. 420, Issue 2-3, pp. 129-33, (1998) (PubMed).

Target Details for COL18A1

Target: COL18A1 (Collagen, Type XVIII, alpha 1 (COL18A1))

Alternative Name: Endostatin (COL18A1 Products)

Synonyms: KNO ELISA Kit, KNO1 ELISA Kit, KS ELISA Kit, cb371 ELISA Kit, cb529 ELISA Kit, fc96d06 ELISA Kit, sb:cb529 ELISA Kit, wu:fb47h10 ELISA Kit, wu:fb77g12 ELISA Kit, wu:fc50f04 ELISA Kit, wu:fc96d06 ELISA Kit, wu:fk02h12 ELISA Kit, kno ELISA Kit, kno1 ELISA Kit, collagen type XVIII alpha 1 chain ELISA Kit, collagen type XVIII alpha 1 chain a ELISA Kit, collagen, type XVIII, alpha 1 ELISA Kit, collagen type XVIII alpha 1 ELISA Kit, COL18A1 ELISA Kit, col18a1a ELISA Kit, Col18a1 ELISA Kit, col18a1 ELISA Kit

Target Type: Chemical

Background: Endostatin is found in the conditioned medium of the hemangioendothelioma cell line EOMA. It has been identified as a fragment of the nontriplehelical C-terminal NC1 domain of collagen type 18 and has been referred to as endostatin 18. Endostatin specifically inhibits proliferation of endothelial cells although it does not affect the proliferation of EOMA cells. It also potently inhibits angiogenesis and tumor growth. The Human Endostatin ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human Endostatin in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human Endostatin coated on a 96-well plate. Standards and samples are pipetted into the wells and Endostatin present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human Endostatin antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin 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 Endostatin bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.

Gene ID: 80781

UniProt: P39060