IL-10 ELISA Kit

Catalog No. ABIN625196

Product Details IL-10 ELISA Kit

Target: IL-10 (IL10) (Interleukin 10 (IL10))

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 10-6000 pg/mL

Minimum Detection Limit: 10 pg/mL

Application: ELISA

Purpose: Rat IL-10 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: rat CINC-2, CINC-3, CNTF, IL-1 alpha, IL-1 beta, IL-4, IL-6, GM-CSF, IFN-gamma, Leptin, Lix, MCP-1, MIP-3 alpha, beta-NGF, TIMP-1, TNF-alpha, 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 samples2 - 10 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: 2-10 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) into Item C vial to prepare a 100 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 60 µL IL-10 standard from the vial of Item C, into a tube with 940 µL Assay Diluent A or 1x Assay Diluent B to prepare a 6,000 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 200myl 200 µL 200 µL 200 µL 200 µL 60 µL standard + 940 µL 6000 2000 666.7 222.2 74.07 24.67 8.23 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 140-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 100 µL of HRP-Streptavidin concentrate into a tube with 14 ml 1x Assay Diluent B to prepare a final 140 fold diluted HRP-Streptavidin solution. 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. Rat IL-10 concentration (pg/mL) O D =4 50 (n m ) 0.01 0.1 1 10 0 10 100 1,000 10,000 Assay Diluent A Rat IL-10 concentration (pg/mL) O D =4 50 (n m ) 0.01 0.1 1 10 0 10 100 1,000 10,000 Assay Diluent B
Sensitivity: The minimum detectable dose of IL-10 is typically less than 10 pg/mL.
Recovery: Recovery was determined by spiking various levels of Rat IL-10 into Rat serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 96.75 84-104 Plasma 95.97 82-102 Cell culture media 98.69 90-109
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 89 94 96 Range ( %) 80-99 87-106 85-104 1:4 Average % of Expected 93 96 99 Range ( %) 84-103 85-104 89-107
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 IL-10 ELISA Kit (ABIN625196)

Abdelkader, Safar, Salem: "Ursodeoxycholic Acid Ameliorates Apoptotic Cascade in the Rotenone Model of Parkinson's Disease: Modulation of Mitochondrial Perturbations." in: Molecular neurobiology, (2015) (PubMed).

Chen, Min, Wang, Leung, Shi, Zhou, Yu, Wang, An, Sha, Chen: "Pre-activation of mesenchymal stem cells with TNF-?, IL-1? and nitric oxide enhances its paracrine effects on radiation-induced intestinal injury." in: Scientific reports, Vol. 5, pp. 8718, (2015) (PubMed).

Wang, Zhao, Han, Tang: "Protective effects of Semen Crotonis Pulveratum on trinitrobenzene sulphonic acid-induced colitis in rats and H?O?-induced intestinal cell apoptosis in vitro." in: International journal of molecular medicine, Vol. 35, Issue 6, pp. 1699-707, (2015) (PubMed).

El-Gowilly, Helmy, El-Gowelli: "Pioglitazone ameliorates methotrexate-induced renal endothelial dysfunction via amending detrimental changes in some antioxidant parameters, systemic cytokines and Fas production." in: Vascular pharmacology, Vol. 74, pp. 139-50, (2015) (PubMed).

Gu, Zhang, Bi, Liu, Tan, Gong, Li, Chen: "Mesenchymal stem cells suppress neuronal apoptosis and decrease IL-10 release via the TLR2/NFκB pathway in rats with hypoxic-ischemic brain damage." in: Molecular brain, Vol. 8, Issue 1, pp. 65, (2015) (PubMed).

Ram, Singh, Kumawat, Kumar, Lingaraju, Uttam Singh, Rahal, Tandan, Kumar: "Deferoxamine modulates cytokines and growth factors to accelerate cutaneous wound healing in diabetic rats." in: European journal of pharmacology, Vol. 764, pp. 9-21, (2015) (PubMed).

Ram, Singh, Kumawat, Kant, Tandan, Kumar: "Bilirubin modulated cytokines, growth factors and angiogenesis to improve cutaneous wound healing process in diabetic rats." in: International immunopharmacology, Vol. 30, pp. 137-49, (2015) (PubMed).

Liu, Pu, Zheng, Cai, Ye: "Therapeutic efficacies of chitosan against Pneumocystis pneumonia of immunosuppressed rat." in: Parasite immunology, Vol. 36, Issue 7, pp. 292-302, (2014) (PubMed).

Ahmed, El Morsy, Ahmed: "Pomegranate extract protects against cerebral ischemia/reperfusion injury and preserves brain DNA integrity in rats." in: Life sciences, Vol. 110, Issue 2, pp. 61-9, (2014) (PubMed).

Alhusban, Fouda, Bindu Pillai, Ishrat, Soliman, Fagan: "Compound 21 is pro-angiogenic in the brain and results in sustained recovery after ischemic stroke." in: Journal of hypertension, Vol. 33, Issue 1, pp. 170-80, (2014) (PubMed).

Vashistha, Sharma, Jain: "Ameliorative potential of ferulic acid in vincristine-induced painful neuropathy in rats: An evidence of behavioral and biochemical examination." in: Nutritional neuroscience, (2014) (PubMed).

Amdekar, Singh, Kumar, Sharma, Singh: "Lactobacillus casei and Lactobacillus acidophilus regulate inflammatory pathway and improve antioxidant status in collagen-induced arthritic rats." in: Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, Vol. 33, Issue 1, pp. 1-8, (2013) (PubMed).

Shi, Liu, Bai, Cui, Li, Li, Hu, Wei: "In vitro effects of pirfenidone on cardiac fibroblasts: proliferation, myofibroblast differentiation, migration and cytokine secretion." in: PLoS ONE, Vol. 6, Issue 11, pp. e28134, (2012) (PubMed).

Agrawal, Gullaiya, Dubey, Singh, Kumar, Nagar, Tiwari: "Neurodegenerative Shielding by Curcumin and Its Derivatives on Brain Lesions Induced by 6-OHDA Model of Parkinson's Disease in Albino Wistar Rats." in: Cardiovascular psychiatry and neurology, Vol. 2012, pp. 942981, (2012) (PubMed).

Schaal, Garg, Ghosh, Lovera, Lopez, Patel, Louro, Patel, Tuesta, Chan, Pearse: "The therapeutic profile of rolipram, PDE target and mechanism of action as a neuroprotectant following spinal cord injury." in: PLoS ONE, Vol. 7, Issue 9, pp. e43634, (2012) (PubMed).

Salga, Ali, Abdulla, Abdelwahab: "Gastroprotective activity and mechanism of novel dichlorido-zinc(II)-4-(2-(5-methoxybenzylideneamino)ethyl)piperazin-1-iumphenolate complex on ethanol-induced gastric ulceration." in: Chemico-biological interactions, Vol. 195, Issue 2, pp. 144-53, (2012) (PubMed).

Taha, Salga, Ali, Abdulla, Abdelwahab, Hadi: "Gastroprotective activities of Turnera diffusa Willd. ex Schult. revisited: Role of arbutin." in: Journal of ethnopharmacology, Vol. 141, Issue 1, pp. 273-81, (2012) (PubMed).

Helmy, El-Gowelli: "Montelukast abrogates rhabdomyolysis-induced acute renal failure via rectifying detrimental changes in antioxidant profile and systemic cytokines and apoptotic factors production." in: European journal of pharmacology, Vol. 683, Issue 1-3, pp. 294-300, (2012) (PubMed).

Sidhu, Pandhi, Malhotra, Vaiphei, Khanduja: "Beneficial effects of Emblica officinalis in L-arginine-induced acute pancreatitis in rats." in: Journal of medicinal food, Vol. 14, Issue 1-2, pp. 147-55, (2011) (PubMed).

Mukherjee, Katki, Arisi, Foresti, Shapiro: "Early TBI-Induced Cytokine Alterations are Similarly Detected by Two Distinct Methods of Multiplex Assay." in: Frontiers in molecular neuroscience, Vol. 4, pp. 21, (2011) (PubMed).

Target Details for IL-10

Target: IL-10 (IL10) (Interleukin 10 (IL10))

Alternative Name: IL-10 (IL10 Products)

Synonyms: CSIF ELISA Kit, GVHDS ELISA Kit, IL-10 ELISA Kit, IL10A ELISA Kit, TGIF ELISA Kit, Il-10 ELISA Kit, IL10X ELISA Kit, interleukin 10 ELISA Kit, IL10 ELISA Kit, Il10 ELISA Kit

Background: Interleukin-10 (IL-10) (Cytokine synthesis inhibitory factor) (CSIF)

Gene ID: 25325

UniProt: P29456

Pathways: Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Production of Molecular Mediator of Immune Response, Maintenance of Protein Location, Cancer Immune Checkpoints