IL12B ELISA Kit (Interleukin 12b)

Details for Product IL12B ELISA Kit No. ABIN1446078, Supplier: Log in to see
  • CLMF
  • CLMF2
  • IL-12B
  • NKSF
  • NKSF2
  • p40
  • Il-12b
  • Il12p40
  • Il-12p40
  • Il12
  • IL-12p40
  • IL-12
  • IL-12 p40
  • il12b
  • il12p40.b
  • zgc:152789
  • IL-12.p40
  • IL12p40
  • IL12B
  • CLMF p40
  • interleukin 12B
  • interleukin 12b
  • interleukin 12Ba
  • IL12B
  • Il12b
  • il12ba
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Method Type
Sandwich ELISA
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Purpose The IL-12p40 ELISA kit is a solid phase sandwich ELISA for the in-vitro qualitative and quantitative determination of IL-12p40 in supernatants, buffered solutions or serum and plasma samples. This assay will recognise both natural and recombinant human IL-12p40 monomer but also when in heterodimer with the p35 protein (IL-12p70) or with the p19 protein (IL-23).
Sample Type Cell Culture Supernatant, Plasma, Serum
Analytical Method Qualitative and Quantitative
Detection Method Colorimetric
Specificity The assay recognizes both natural and recombinant human IL-12p40. To define the specificity of this ELISA several proteins were tested for cross reactivity. There was no cross reactivity observed for any protein tested (IL-1α, IL-1β, IL-10, IL-2, IFNγ, IL-4, IL-6, TNFα, IL-8 and IL-13). The assay will also recognize the p40 when in heterodimer with the p35 protein (IL-12p70) or with the p19 protein (IL-23).
Sensitivity The sensitivity, minimum detectable dose of IL-12p40 using this IL-12p40 ELISA kit was found to be 20pg/mL. This was determined by adding 3 standard deviations to the mean OD obtained when the zero standard was assayed 40 times.
Components Pre-coated 12 strip plates, biotinylated secondary antibody, standards, controls (when available), buffers, Streptavidin-HRP, TMB, Stop Reagent.
Material not included Microtitre plate reader fitted with appropriate filters (450nm required with optional 620nm reference filter)
Microplate washer or wash bottle
10, 50, 100, 200 and 1,000 μL adjustable single channel micropipettes with disposable tips
50-300 μL multi-channel micropipette with disposable tips
Multichannel micropipette reagent reservoirs
Distilled water
Vortex mixer
Miscellaneous laboratory plastic and/or glass, if possible
Plasmids, Primers & others Plasmids, Primers & others IL12B products on genomics-online (e.g. as negative or positive controls)
Alternative Name IL-12p40 (IL12B ELISA Kit Abstract)
Background IL-12 is a potent regulator of cell mediated immune response produced by activated monocytes/macrophages cells, B lymphocytes and connective tissue type mast cells. The biologically active form of IL-12 is a 70 kDa heterodimeric glycoprotein consisting of disulfide-linked 35 kDa (p35) light chain and 40 kDa (p40) heavy chain subunits. The two subunits are genetically unrelated. The p70 form is the only biologically active form of IL-12. The p35 subunit has homology to IL-6, while p40 has homology with IL-23. The p40 subunit has been found to be expressed in a higher excess over p70. IL-12 has been found to bind to IL-12R. The p40 subunit can also form a homodimer which has been shown to bind IL-12R and thus acts as an IL-12 antagonist IL-12R has been reported to be present on IL-2 activated CD4+, CD8+ and CD56+ cells. IL-12 exerts a variety of biological effects on human T and NK cells. IL-12 induces an IFNγ production and other cytokines from peripheral blood T and NK cells. Its role is directing development and proliferation of Th1 cells. Thus IL-12 is linked with autoimmunity, high level have also been reported for chronic inflammatory reactions, bacterial and viral infection.
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 Notes Optimal working dilution should be determined by the investigator.

Spike recovery: The spike recovery was evaluated by spiking 3 concentrations of IL-12p40 in human serum in 2 separate experiments. Recoveries ranged from 99 to 110% with an overall mean recovery of 103%. 950.075 Human IL-12p40 ELISA kit insert version 7-30/04/2014 14 12.6. Stability Storage Stability Aliquots of spiked serum samples were stored at -20°C, 2-8°C, room temperature (RT) and at 37°C and the IL-12p40 level determined after 24h. We observed no significant loss of IL-12p40 immunoreactivity during storage. Freeze-thaw Stability Aliquots of spiked serum were stored frozen at -20°C and thawed up to 5 times and the IL-12p40 level was determined. There was no decrease in activity of IL-12p40 after cycles of freezing and thawing. 12.7. Expected serum values A panel of 24 human sera was tested for IL-12p40. 3 are below the detection level, 21 are ranged from 65 to 483 pg/mL with a mean at 187 pg/mL and a SD at 126 pg/mL.

Assay Time 1 - 2 h
Plate Pre-coated
Protocol Add 100 μL of sample and diluted standard/controls and 50 μL Biotinylated anti-IL-12p40
Incubate 1 hours at room temperature
Wash three times
Add 100 μL of Streptavidin-HRP
Incubate 30 min at room temperature
Wash three times
Add 100 μL of ready-to-use TMB Protect from light. Let the color develop for 12-15 mn.
Add 100 μL H2SO4
Read Absorbance at 450 nm
A capture Antibody highly specific for IL-12p40 has been coated to the wells of the microtitre strip plate provided during manufacture. Binding of IL-12p40 samples and known standards to the capture antibodies and subsequent binding of the biotinylated anti-IL-12p40 secondary antibody to the analyte is completed during the same incubation period. Any excess unbound analyte and secondary antibody is removed. The HRP conjugate solution is then added to every well including the zero wells, following incubation excess conjugate is removed by careful washing. A chromogen substrate is added to the wells resulting in the progressive development of a blue coloured complex with the conjugate. The colour development is then stopped by the addition of acid turning the resultant final product yellow. The intensity of the produced coloured complex is directly proportional to the concentration of IL-12p40 present in the samples and standards. The absorbance of the colour complex is then measured and the generated OD values for each standard are plotted against expected concentration forming a standard curve. This standard curve can then be used to accurately determine the concentration of IL-12p40 in any sample tested. 950
Reagent Preparation

Bring all reagents to room temperature before use
8.1.Assay Design Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running zeros and standards. Each sample, standard, zero and control should be tested in duplicate. Remove sufficient Microwell Strips for testing from the aluminium pouch immediately prior to use. Return any wells not required for this assay with desiccant to the pouch. Seal tightly and return to 2-8 °C storage.
8.2.Preparation of Wash Buffer Dilute the (200x) wash buffer concentrate 200 fold with distilled water to give a 1x working solution. Pour entire contents (10 mL) of the Washing Buffer Concentrate into a clean 2,000 mLgraduated cylinder. Bring final volume to 2,000 mLwith glass-distilled or deionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2°-25 °C.
8.3.Preparation of Standard Diluent Buffer Add the contents of the vial (10x concentrate) to 225 mL of distilled water before use. This Solution can be stored at 2-8 °C for up to 1 week.
8.4.Preparation of Standard Depending on the type of samples you are assaying, the kit may include two standard diluents. Because biological fluids might contain proteases or cytokine-binding proteins that could modify the recognition of the cytokine you want to measure, you should reconstitute standard vials with the most appropriate Standard Diluent. For serum and plasma samples: use Standard Diluent - human serum. For cell culture supernatants: use Standard Diluent Buffer. Standard vials must be reconstituted with the volume of standard diluent shown on the vial immediately prior to use. This reconstitution gives a stock solution of 2000pg/mL of IL-12p40. Mix the reconstituted standard gently by aspirations/ejections. Serial dilutions of the standard are made directly in the assay plate to provide the concentration range from 2000 to 62.5pg/mL. A fresh standard curve should be produced for each new assay. Immediately after reconstitution add 200 μL of the reconstituted standard to well's A1 and A2, which provides the highest concentration standard at 2000pg/mL. Add 100 μL of appropriate standard diluent to the remaining standard wells B1 and B2 to F1 and F2. Transfer 100 μL from wells A1 and A2 to B1 and B2. Mix the well contents by repeated aspirations and ejections taking care not to scratch the inner surface of the wells. Continue this 1:1 dilution using 100 μL from wells B1 and B2 through to wells F1 and F2 providing a serial diluted standard curve ranging from 2000pg/mL to 62.5pg/mL. Discard 100 μL from the final wells of the standard curve (F1 and F2). Alternatively these dilutions can be performed in separate clean tubes and immediately transferred directly into the relevant wells.
8.5.Preparation of Controls Freeze-dried control vials should also be reconstituted with the most appropriate Standard Diluent to your samples. For serum and plasma samples: use Standard Diluent - human serum. For cells culture supernatants: use Standard Diluent Buffer. The supplied Controls must be reconstituted with the volume of Standard Diluent indicated on the vial. Reconstitution of the freeze-dried material with the indicated volume, will give a solution at the concentration stated on the vial. Do not store after use.
8.6.Preparation of Biotinylated anti-IL-12p40 It is recommended this reagent is prepared immediately before use. Dilute the biotinylated anti-IL-12p40 with the biotinylated antibody diluent in an appropriate clean glass vial using volumes appropriate to the number of required wells.
8.7.Preparation of Streptavidin-HRP It is recommended to centrifuge vial for a few seconds in a microcentrifuge to collect all the volume at the bottom. Dilute the 5 μL vial with 0.5 mL of HRP diluent immediately before use. Do not keep this diluted vial for future experiments. Further dilute the HRP solution to volumes appropriate for the number of required wells in a clean glass vial.

Sample Preparation

Cell culture supernatants, human serum, plasma or other biological samples will be suitable for use in the assay
Remove serum from the clot or red cells, respectively, as soon as possible after clotting and separation. Cell culture supernatants: Remove particulates and aggregates by spinning at approximately 1000 x g for 10 min
Serum: Use pyrogen/endotoxin free collecting tubes
Serum should be removed rapidly and carefully from the red cells after clotting. Following clotting, centrifuge at approximately 1000 x g for 10 min and remove serum. Plasma: EDTA, citrate and heparin plasma can be assayed
Spin samples at 1000 x g for 30 min to remove particulates. Harvest plasma. Storage: If not analyzed shortly after collection, samples should be aliquoted (250-500 μL) to avoid repeated freeze-thaw cycles and stored frozen at -70 °C
Avoid multiple freeze-thaw cycles of frozen specimens. Recommendation: Do not thaw by heating at 37 °C or 56 °C. Thaw at room temperature and make sure that sample is completely thawed and homogeneous before use. When possible avoid use of badly haemolysed or lipemic sera. If large amounts of particles are present these should be removed prior to use by centrifugation or filtration.

Assay Procedure

We strongly recommend that every vial is mixed without foaming prior to use. Prepare all reagents . Final preparation of Biotinylated Secondary Antibody and Streptavidin-HRP should occur immediately before use.
1.Addition Prepare Standard curve
2.Addition Add 100 μL of each, Sample, Standard, Control and zero (appropriate standard diluent) in duplicate to appropriate number of wells
3.Addition Add 50 μL of diluted biotinylated anti-IL-12p40 to all wells
4.Incubation Cover with a plastic plate cover and incubate at room temperature (18 to 25 °C) for 1 hour
5.Wash Remove the cover and wash the plate as follows: a) Aspirate the liquid from each well b) Dispense 0.3 mLof 1x washing solution into each well c) Aspirate the contents of each well d) Repeat step b and c another two times
6.Addition Add 100 μL of Streptavidin-HRP solution into all wells
7.Incubation Cover with a plastic plate cover and incubate at room temperature (18 to 25 °C) for 30 min
8.Wash Repeat wash step
5.9. Addition Add 100 μL of ready-to-use TMB Substrate Solution into all wells
10.Incubation Incubate in the dark for 12-15 minutes at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil.
11.Addition Add 100 μL of H2SO4:Stop Reagent into all wells Read the absorbance value of each well (immediately after step 11.) on a spectrophotometer using 450 nm as the primary wavelength and optionally 620 nm as the reference wave length (610 nm to 650 nm is acceptable).
Incubation time of the substrate solution is usually determined by the ELISA reader performance. Many ELISA readers only record absorbance up to 2.0 O.D. Therefore the colour development within individual microwells must be observed by the analyst, and the substrate reaction stopped before positive wells are no longer within recordable range.

Calculation of Results

Calculate the average absorbance values for each set of duplicate standards, controls and samples. Ideally duplicates should be within 20 % of the mean. Generate a linear standard curve by plotting the average absorbance of each standard on the vertical axis versus the corresponding IL-12p40 standard concentration on the horizontal axis. The amount of IL-12p40 in each sample is determined by extrapolating OD values against IL-12p40 standard concentrations using the standard curve.Every laboratory must produce a standard curve for each set of microwell strips assayed. Do not extrapolate the standard curve beyond the maximum standard curve point. The dose-response is non-linear in this region and good accuracy is difficult to obtain. Concentrated samples above the maximum standard concentration must be diluted with Standard diluent or with your own sample buffer to produce an OD value within the range of the standard curve. Following analysis of such samples always multiply results by the appropriate dilution factor to produce actual final concentration. The influence of various drugs on end results has not been investigated. Bacterial or fungal contamination and laboratory cross-contamination may also cause irregular results. Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells before dispensing fresh Washing Buffer, fill with Washing Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods. Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use. As with most biological assays conditions may vary from assay to assay therefore a fresh standard curve must be prepared and run for every assay.

Restrictions For Research Use only
Handling Advice Handling of reagents, serum or plasma specimens should be in accordance with local safety procedures, e.g.CDC/NIH Health manual: "Biosafety in Microbiological and Biomedical Laboratories" 1984.
The human serum included in this kit have been tested and found non reactive for HbsAg, anti HIV1 & 2 and anti VHC. Nevertheless, no known method can offer complete assurance that human blood derivatives will not transmit hepatitis, AIDS or other infections. Therefore handling of reagents, serum or plasma specimens should be in accordance with local safety procedures.
Laboratory gloves should be worn at all times.
Avoid any skin contact with H2SO4 and TMB. In case of contact, wash thoroughly with water.
Do not eat, drink, smoke or apply cosmetics where kit reagents are used.
Do not pipette by mouth.
When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles labels.
All reagents should be warmed to room temperature before use. Lyophilized standards should be discarded after use.
Once the desired number of strips has been removed, immediately reseal the bag to protect the remaining strips from deterioration.
Cover or cap all reagents when not in use.
Do not mix or interchange reagents between different lots.
Do not use reagents beyond the expiration date of the kit.
Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross contamination, for the dispensing of H2SO4 and substrate solution, avoid pipettes with metal parts.
Use a clean plastic container to prepare the washing solution.
Thoroughly mix the reagents and samples before use by agitation or swirling.
All residual washing liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells.
The TMB solution is light sensitive. Avoid prolonged exposure to light. Also, avoid contact of the TMB solution with metal to prevent colour development. Warning TMB is toxic avoid direct contact with hands. Dispose off properly.
If a dark blue colour develops within a few minutes after preparation, this indicates that the TMB solution has been contaminated and must be discarded. Read absorbance's within 1 hour after completion of the assay.
When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.
Follow incubation times described in the assay procedure.
Dispense the TMB solution within 15 min of the washing of the microtitre plate.
Storage 4 °C
Storage Comment Store kit reagents between 2 and 8 °C. Immediately after use remaining reagents should be returned to cold storage (2-8 °C). Expiry of the kit and reagents is stated on box front labels. The expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling.
Expiry Date 1 week
Product cited in: Vilisaar, Kawabe, Braitch, Aram, Furtun, Fahey, Chopra, Tanasescu, Tighe, Gran, Pothoulakis, Constantinescu: "Reciprocal Regulation of Substance P and IL-12/IL-23 and the Associated Cytokines, IFNγ/IL-17: A Perspective on the Relevance of This Interaction to Multiple Sclerosis." in: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, Vol. 10, Issue 3, pp. 457-67, 2015 (PubMed).

Sobiś, Rykaczewska-Czerwińska, Świętochowska, Gorczyca: "Therapeutic effect of aripiprazole in chronic schizophrenia is accompanied by anti-inflammatory activity." in: Pharmacological reports : PR, Vol. 67, Issue 2, pp. 353-9, 2015 (PubMed).

Lu, Nossent: "Thrombopoietin levels in systemic lupus erythematosus are linked to inflammatory cytokines, but unrelated to thrombocytopenia or thrombosis." in: Lupus, Vol. 24, Issue 1, pp. 18-24, 2014 (PubMed).

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