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|Antigen||Interleukin 12 (IL12) ELISA Kits|
Kits with alternative reactivity to:
|Method Type||Sandwich ELISA|
|Detection Range||1-600 pg/mL|
|Minimum Detection Limit||1 pg/mL|
|4 references available|
|Supplier||Log in to see|
Product Details IL12 ELISA KitTarget details Application Details Handling ProductDetails: References for IL12 Kit (ABIN625010) Images
|Purpose||Human IL-12 p70 ELISA Kit for cell culture supernatants, plasma, and serum samples.|
|Sample Type||Plasma, Cell Culture Supernatant, Serum|
|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 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||< 1 pg/mL|
|Material not included||
Target detailsProduct Details IL12 ELISA Kit Application Details Handling ProductDetails: References for IL12 Kit (ABIN625010) Images back to top
|Alternative Name||IL-12 p70 (IL12 ELISA Kit Abstract)|
|Background||IL-12 is a heterodimeric 70 kDa glycoprotein (IL-12-p70) consisting of a 40 kDa subunit. It is secreted by peripheral lymphocytes after induction and is produced mainly by B-cells and to a lesser extent by T-cells. The most powerful inducers of IL-12 are bacteria, bacterial products, and parasites. IL-12 is produced after stimulation with Phorbol esters or Calcium ionophore by human B-lymphoblastoid cells. IL-12 activates NK-cells positive for CD56, and this activity is blocked by antibodies specific for TNF-alpha. IL-12 promotes specific allogenic CTL reactions. IL-12 synergizes also with anti-CD3 antibodies and with allogeneic stimulation in mixed lymphocyte cultures in inducing T-cell proliferation. The Human IL-12(P70) ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human IL-12(P70) in serum, plasma, cell culture supernatants and urine. This assay employs an antibody specific for human IL-12(P70) coated on a 96-well plate. Standards and samples are pipetted into the wells and IL-12(P70) present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human IL-12 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 IL-12(P70) 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%.|
|Pathways||JAK-STAT Signaling, Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Activated T Cell Proliferation|
Application DetailsProduct Details IL12 ELISA Kit Target details Handling ProductDetails: References for IL12 Kit (ABIN625010) Images back to top
|Application Notes||Recommended Dilution for serum and plasma samples2 fold|
|Sample Volume||100 μL|
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) is 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 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 10 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 40 µL IL-12 (P70) standard from the vial of Item C, into a tube with 626.7 µL Assay Diluent A or 1x Assay Diluent B to prepare a 600 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 40 µL standard + 626.7 µL 600 200 66.7 22.2 7.4 2.5 0.8 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) before use. HRP-Streptavidin concentrate should be diluted 300-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 50 µL of HRP-Streptavidin concentrate into a tube with 15 ml 1x Assay Diluent B to prepare a final 300 fold diluted HRP-Streptavidin solution (don't store the diluted solution for next day use). Mix well.
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. Human IL-12(P70) 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 IL-12(P70) concentration (pg/mL) 0.1 1 10 100 1000 O D =4 50 n m 0.01 0.1 1 10 Assay Diluent A
Sensitivity: The minimum detectable dose of IL-12(P70) is typically less than 1 pg/mL.
Recovery: Recovery was determined by spiking various levels of human IL-12 (P70) into human serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 83.34 78-102 Plasma 85.63 79-101 Cell culture media 95.52 88-109
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 86 87 92 Range ( %) 77-101 78-102 81-104 1:4 Average % of Expected 92 90 91 Range ( %) 80-103 81-102 79-102 1:8 Average % of Expected 87 93 89 Range ( %) 81-102 82-104 80-103
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %
|Assay Precision||Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %|
|Restrictions||For Research Use only|
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|Handling Advice||Avoid repeated freeze-thaw cycles.|
|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|
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|Product cited in:||
Abdallah, Attia, Saad, El-Khateeb, Lotfi, Abdallah, El-Shennawy: "Serum Th1/Th2 and macrophage lineage cytokines in leprosy; correlation with circulating CD4(+) CD25(high) FoxP3(+) T-regs cells." in: Experimental dermatology, Vol. 23, Issue 10, pp. 742-7, 2014
Hashad, Hamed, El Gharabawy, El Metwally, Morsi: "Interleukins 12 and 13 levels among beta-thalassaemia major patients." in: Eastern Mediterranean health journal = La revue de santé de la Méditerranée orientale = al-Majallah al-ṣiḥḥīyah li-sharq al-mutawassiṭ, Vol. 19, Issue 2, pp. 181-5, 2013
Hashad, Hamed, El Gharabawy, El Metwally, Morsi: "Interleukins 12 and 13 levels among beta-thalassaemia major patients." in: Eastern Mediterranean health journal = La revue de santé de la Méditerranée orientale = al-Majallah al-?i???yah li-sharq al-mutawassi?, Vol. 19, Issue 2, pp. 181-5, 2013
Germann, Gately, Schoenhaut, Lohoff, Mattner, Fischer, Jin, Schmitt, Rüde: "Interleukin-12/T cell stimulating factor, a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells." in: European journal of immunology, Vol. 23, Issue 8, pp. 1762-70, 1993
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