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- Mouse IL-12 p70 ELISA Kit
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Product Specification
| Usage | Need to bring your own test equipment 1. Microplate reader (can measure the absorption value of 450nm detection wavelength and 540nm or 570nm correction wavelength) 2. High precision liquid dispenser and disposable suction head 3. Distilled water or deionized water 4. Washing bottle (spray bottle), multi-channel plate washer or automatic plate washer 5. 500mL cylinder One, preparation before the experiment 1. Sample collection and storage ① Cell culture supernatant: particles should be removed by centrifugation; Test the sample immediately. If the sample is not tested in time after collection, it is recommended that the sample be divided according to the dosage and stored in the refrigerator at -20 ° C to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute. ② Serum: Samples were collected using a serum separation tube (SST) and samples were left at room temperature for 30 minutes. The samples were centrifuged at 1000g for 15 min. Serum was immediately removed and tested immediately. If the sample is not tested in time after collection, it is recommended to repack according to a single dosage and freeze in ≤ -20℃ refrigerator to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute. ③ Plasma: Plasma was collected using EDTA, heparin, or citric acid as an anticoagulant, centrifuged at 1000g for 15 min within 30 min of collection, and tested immediately. If the samples are not detected in time after collection, it is recommended to separate the samples according to the single dosage and freeze them in &le. -20℃ refrigerator to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute. 2 Reagent preparation (Place all reagents and samples at room temperature for 15 minutes before use. It is recommended that all experimental samples and standards do double hole detection ) 1× Preparation of washing solution: concentrated washing solution in the kit is 20× Mother liquor, diluted to 1× with distilled water before use; Working liquid. Example: Take 10mL concentrated washing solution +190mL distilled water to 200mL, the actual operation can be calculated first, then make up. ②1× Dilution with buffer preparation: concentrate dilution in the kit with buffer 10× Mother liquor, diluted to 1× with distilled water before use; Working solution. example: Take 3mL of concentrated dilution with buffer +27mL of distilled water to a constant volume of 30mL. In practical operation, the required dilution buffer can be calculated according to the dilution multiple of the sample, and then the preparation can be made. ③ Detection of antibody: the dry powder was centrifuged to the bottom of the tube, and 110uL dilution buffer (1×) was used. Dissolve and let stand at room temperature for 5 minutes to obtain 100× Mother liquor; Dilute to 1× before use; Working solution. Calculate the desired volume by using 100uL per well. example: 10 Wells were used, then take 10uL of 100 times the working concentration of the test antibody, using dilution buffer (1×) Constant volume to 1mL, get 1mL of 1× The working concentration of the detected antibody. ④SA-HRP: SA-HRP is 40× Mother liquor, use dilution buffer before use (1×) Dilute to make 1× Working solution, 100uL required per well. example: used 10 holes, then take 25uL of 40× Mother liquor +975uL dilution buffer (1×) Constant volume to 1mL to obtain 1× of 1mL; The working concentration of the detected antibody. ⑤ Chromogenic agent: according to 100uL per well, calculate the amount needed for this test, take out the corresponding volume of chromogenic agent, avoid light; The removed chromogenic agent is only used on the same day. ⑥ Standards: lyophilized standards with dilution buffer (1×) Redissolve, redissolve volume 1000uL, to obtain a concentration of 2500pg/mL standard mother liquor. Gently shake for at least 5 minutes, it is fully dissolved. Add 300uL dilution buffer (1×) to each dilution tube. . The standard mother liquor is diluted according to the picture below, and each tube must be fully mixed before pipetting to the next tube. The standard mother liquor without dilution can be used as the highest point of the standard curve (2500pg/mL), dilute with buffer (1×) Can be used as the zero point of the standard curve (0pg/mL).  2, operation steps 1. Prepare all required reagents and standards; 2 Remove the microplate from the sealed bag that has been balanced to room temperature, and put the unused slat back into the aluminum foil bag and re-seal it; 3. Add 300uL washing solution to the microplate, let it soak for 30 seconds, discard the washing solution and pat the microplate dry on absorbent paper, please use immediately do not let the microplate dry; 4. Add different concentrations of standard, experimental samples or quality control into the corresponding Wells, 100uL for each well. The Wells were sealed with plate adhesive and incubated at room temperature for 2 hours. 5 Suck the liquid out of the plate and wash the plate using a bottle washer, a multi-channel plate washer, or an automatic plate washer. Add 300uL of washing liquid to each well, and then suck the washing liquid out of the plate. Repeat 3 times. Every time you wash the plate, try to absorb the residual liquid to help you get a good test result. At the end of the last plate wash, please blot all the liquid in the plate or invert the plate and pat all the residual liquid in the absorbent paper; 6. Add 100uL detection antibody to each microwell. Seal the reaction Wells with sealer tape and incubate for 2 hours at room temperature; 7. Repeat the plate washing operation of step 5; 8. Add 100 ULSA-HRP to each microwell and incubate for 20 minutes at room temperature. Be careful to avoid light; 9. Repeat step 5 to wash the plate; 10. Add 100uL of color development solution to each microwell, incubate at room temperature for 5-30 minutes, pay attention to avoid light; 11. Add 50uL of termination solution to each microwell, and the color of the solution in the well will change from blue to yellow. If the color of the solution turns green or the color change is inconsistent, tap the microplate to mix the solution evenly; 12. Within 30 minutes after the termination solution is added, the absorbance value at 450nm is measured using a microplate reader and 540nm or 570nm is set as the correction wavelength. If the dual wavelength correction is not used, the accuracy of the results may be affected; 13 Calculation results: The corrected absorbance values (OD450-OD540/OD570), the compound reading were averaged for each standard and sample, and then the average zero standard OD value was subtracted. Standard curves were created by 4-parameter logic (4-PL) curve fitting using computer software. Alternatively, a curve can be generated by plotting the logarithm of the concentration of the standard against the logarithm of the corresponding OD value, and the best fit line can be determined by regression analysis. This process produces an adequate but less accurate fit to the data. If the sample is diluted, the concentration should be multiplied by the dilution.  3. Kit parameters 1. Recovery: Different levels of mouse IL-12 p70 were incorporated into the cell culture medium samples, and the recovery rate was determined. Recoveries ranged from 83 to 107%, with an average recovery of 93%. 2. Sensitivity: The minimum detectable dose (MDD) of murine IL-12 p70 was generally less than 2.24pg/mL. The lowest detectable value was calculated as the corresponding concentration based on the average of the zero absorbance values of 20 standard curves plus two standard deviations. 3. Correction: the ELISA kit by high purity recombinant e. coli expression correction by IL - 12 p70 protein in mice. 4. Linearity: Four different samples were mixed with high concentrations of mouse IL-12 p70, followed by dilution (1×). Linearity was determined by dilating the samples to within the detection range.
5. Specificity: the ELISA method to detect natural and restructuring IL - 12 p70 protein in mice. The following factors in diluent mixture 50 ng/mL (1 x) to detect the concentration of IL - 12 p70 cross reaction with mice. To the interference of 50 ng/mL factor added to the middle scope IL - 12 p70 reference substance in the restructuring of the mice, to detect the interference of IL - 12 p70 in mice. No significant cross-reactivity or interference was observed. The recombinant rat IL-12 p70 containing 1.25ng/mL had cross reactivity.
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| Theory | This kit with double antibody sandwich enzyme-linked immunosorbent detection technology. Specific anti mouse antibody of IL - 12 p70 pre package is on the high affinity enzyme label plate. After incubation, the IL-12 p70 in the sample was combined with the solid phase antibody and the detection antibody to form immune complexes. After washing to remove not combined with material, by adding horseradish peroxidase labeled chain mildew avidin (Streptavidin - HRP). After washing, adding chromogenic substrate, dark color. Join terminated liquid termination reaction, in the 450 - nm wavelength (reference calibration wavelength of 540 nm or 570 nm) determination of absorbance values. | |||||||||||||||||||||||||||
| Composition |
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| Background | Interleukin-12 (IL-12, also known as NKSF), a 70-75 kda heterodimer glycoprotein, belongs to the IL-12 heterodimer cytokine family. IL - 12 by 35 kda (p35) and 40 kda (p40) of two subunits, the two subunits by disulfide bond is linked together, no amino acid sequence homology between each other. The mature p35 subunit is 196 amino acids long, containing seven cysteines and a potential N-linked glycosylation site. Mature mouse p35 shares 63% and 86% amino acid identity with human and rat, respectively. Mature mice p40 subunits long 313 amino acids, including 13 cysteine and five potential glycosylation sites of N - connection. Some studies have reported that the p40 sequence is polymorphic, but this IL-12 p70 kit does not recognize these allelic products. mature mouse p40 has 72% and 93% amino acid identity with human and rat, respectively. Although p35 resembles a ligand for erythropoietin, p40 is more similar to the N-terminus of the erythropoietin receptor, with WSXWS domains, an immunoglobulin-like domain, and four conserved cysteines. This suggests that IL-12 may be a cytokine receptor mimetic, similar to the IL-6/ soluble IL-6R complex. It is worth noting that the p40 can exist in the form of monomer or homologous dimers, but never found p35 monomer form. p40 is the larger of the two subunits of interleukin 23 (IL-23). Although IL-12 has long been considered a secretory molecule, membrane-bound IL-12 has also been found in human and mouse cells. Il-12-producing cells include macrophages, dendritic cells, monocytes, Langerhans cells, neutrophils, keratinocytes, plasmacytoid dendritic cells, microglia, CD8+DC (mouse cells only), and non-germinal center (CD38-CD44+) B cells (human cells only). The high-affinity receptor for mouse IL-12 is composed of at least two type I transmembrane glycoproteins, similar to members of the cytokine receptor superfamily. Beta 1 first subunits (R) of 100 kda, with Kd = 1 nm and IL - 12. This receptor is the primary binding site for the p40 subunit; The second subunit (Rβ2) is 130kDa and has no amino acid homology with the Rβ1 subunit. The receptor is an important part of signal transduction, as a disulfide bond connected p30 - p40 dimers attachment points. As mentioned above, mouse p40 exists as a monomeric, dimeric form, and binds either at p35 to form IL-12 or at p19 to form IL-23. Both homodimer p40 and IL-23 can bind IL-12R and act as antagonists that do not transmit signals. Alternatively, p40 homodimer can also bind to Rβ1 and activate microglia and macrophages. Functionally, IL-12 has been shown to enhance cytotoxicity and induce interferon-γ production by NK cells, T cells, and dendritic epidermal T cells. It has been reported that IL-12 can induce the production of interferon-γ by macrophages. Together with its IL-12 family members such as IL-23 and IL-27, IL-12 can promote the progression of CD4+ Th1-type immune responses. In response to infection, IL-27 is secreted first to promote the excess of TH0 to THR0/1, followed by IL-12 secretion to generate Th1 effector cells. "Production together with IL-18 and IL-12 transforms effector cells into Th1 memory cells, which are then activated by IL-23." | |||||||||||||||||||||||||||
| General Notes | 1. Please use the kit within the validity period. 2. Components of different kits and different batch kits should not be mixed. 3. If the sample value is greater than the highest value of the standard curve, the sample should be diluted with (1×) The samples were diluted and retested. "If the cell culture supernatant sample needs to be diluted in a distributed manner, cell culture medium may be used for intermediate dilutions, except for the last step when diluent is used." 4. The difference of the test results can be caused by a variety of factors, including the operation of the experimenter, the use of the pipettor, the washing technique, the reaction time or temperature, the storage of the kit, etc. 5. The termination solution in the kit is acidic. Please protect your glasses, hands, face and clothes when using it. 6. For scientific research only, not for in vitro diagnosis. | |||||||||||||||||||||||||||
| Storage Temp. | Unopened kits were stored at 2-8 ° C. | |||||||||||||||||||||||||||
| Test Range | 39.06pg/mL-2500pg/mL |
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AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.
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