PDGF-BB Homodimer ELISA Kit

Catalog No. ABIN625073

This Colorimetric ELISA kit is designed for the quantitative measurement of Human PDGF-BB Homodimer. There are 19 publications available.

Quick Overview for PDGF-BB Homodimer ELISA Kit (ABIN625073)

Target: PDGF-BB Homodimer

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1 pg/mL - 400 pg/mL

Application: ELISA

Sample Type: Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Lysate

Product Details

Minimum Detection Limit: 1 pg/mL

Purpose: Human PDGF-BB ELISA Kit for cell culture supernatants, plasma, serum, and lysate samples.

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, RANTES, SCF, TARC, TGF-beta, TIMP-1, TIMP-2, TNF-alpha, TNF-beta, TPO, VEGF.

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

,

• 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: Suggested Plasma Dilution: 5 - 50 fold

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: 5-50 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 280 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture medium and urine) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 4 µL PDGF-BB standard from the vial of Item C, into a tube with 496 µL Assay Diluent A or 1x Assay Diluent B to prepare a 400 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 200myl 200 µL 200 µL 200 µL 200 µL 4 µL standard + 496 µL 400 133.3 44.44 14.81 4.94 1.65 0.549 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 800-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 20 µL of HRP-Streptavidin concentrate into a tube with 16 ml 1x Assay Diluen Bt to prepare a 800-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 PDGF-BB concentration (pg/mL) 0.1 1 10 100 1000 O D =4 50 n m 0.01 0.1 1 10 Assay Diluent B Human PDGF-BB concentration (pg/mL) 0.1 1 10 100 1000 O D =4 50 n m 0.1 1 10
Sensitivity: The minimum detectable dose of PDGF-BB is typically less than 1 pg/mL.
Recovery: Recovery was determined by spiking various levels of human PDGF-BB into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 93.57 82-103 Plasma 91.99 83-104 Cell culture media 95.43 84-104
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 93 93 94 Range ( %) 83-103 84-105 82-104 1:4 Average % of Expected 94 95 93 Range ( %) 84-104 82-103 85-103
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

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Gamal, Abdel-Ghaffar, Iacono et al.: "Gingival crevicular fluid vascular endothelial cell growth factor and platelet-derived growth factor-BB release profile following the use of perforated barrier membranes during treatment of intrabony ..." in: Journal of periodontal research, Vol. 51, Issue 3, pp. 407-16, (2016) (PubMed).

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Lima, Mano, Concheiro, Alvarez-Lorenzo: "Fast and mild strategy, using superhydrophobic surfaces, to produce collagen/platelet lysate gel beads for skin regeneration." in: Stem cell reviews, Vol. 11, Issue 1, pp. 161-79, (2015) (PubMed).

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Winsz-Szczotka, Komosińska-Vassev, Kuźnik-Trocha, Siwiec, Żegleń, Olczyk et al.: "Circulating keratan sulfate as a marker of metabolic changes of cartilage proteoglycan in juvenile idiopathic arthritis; influence of growth factors as well as proteolytic and prooxidative agents on ..." in: Clinical chemistry and laboratory medicine, Vol. 53, Issue 2, pp. 291-7, (2015) (PubMed).

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Stojek, Adrych, Rojek, Smoczynski, Sledzinski, Szrok, Swierczynski: "Decreased serum platelet derived growth factor BB levels in acute and increased in chronic pancreatitis." in: World journal of gastroenterology : WJG, Vol. 20, Issue 36, pp. 13127-32, (2014) (PubMed).

Cadamuro, Nardo, Indraccolo, Dallolmo, Sambado, Moserle, Franceschet, Colledan, Massani, Stecca, Bassi, Morton, Spirli, Fiorotto, Fabris, Strazzabosco: "Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma." in: Hepatology (Baltimore, Md.), Vol. 58, Issue 3, pp. 1042-53, (2014) (PubMed).

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Target Details

Target: PDGF-BB Homodimer

Alternative Name: PDGF-BB

Background:

Gene Symbols: PDGFB, SIS, PDGF2

Protein Name (Synonyms): Platelet-derived growth factor subunit B (PDGF subunit B) (PDGF-2) (Platelet-derived growth factor B chain) (Platelet-derived growth factor beta polypeptide) (Proto-oncogene c-Sis) (Becaplermin)

Gene ID: 5155

UniProt: P01127