Mouse IL-2 ELISA Kit
Mouse IL-2 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 1, preparation before the experiment 1. Sample collection and storage ① Cell culture supernatant: particles should be removed by centrifugation; Test the samples 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 1000pg/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 (1000pg/mL), and the dilution 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 rate: Different levels of Mouse IL-2 were incorporated into the cell culture medium samples and the recovery rate was determined. Recoveries ranged from 84 to 107%, with an average recovery of 95%. 2. Sensitivity: Mouse IL - 2 the minimum detectable dose (MDD) is generally less than 3.5 pg/mL. Minimum measurable value is according to the 20 standard curve zero absorbance value plus two times the standard deviation of average calculated corresponding to the concentration. 3. Correction: the ELISA kit by e. coli expressed high purity recombinant Mouse IL - 2 protein correction. 4. Linearity: Four different samples were mixed with high concentrations of Mouse IL-2, followed by dilution (1×). Linearity was determined by dilutes the samples to the detection range.
5. Specificity: Both native and recombinant Mouse IL-2 protein could be detected by this ELISA. The following factors were diluted (1×) Configure to a concentration of 50ng/mL to detect cross-reactivity with Mouse IL-2. To detect interference with Mouse IL-2, 50ng/mL of interfering factor was incorporated into the intermediate range of recombinant Mouse IL-2 controls. No significant cross-reaction or interference was observed.
4, common problem resolution 1. The white board (no color), after the completion of color
2. Flower plate (blank, negative and positive controls were normal, but the OD value of sample Wells was significantly higher)
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| Theory | Double antibody sandwich enzyme-linked immunosorbent assay was used in this kit. Specific anti-mouse IL-2 antibody was precoated on a high-affinity microplate. The standard substance, test sample and biotinylated detection antibody were added into the microplate Wells. After incubation, the IL-2 present in the sample combined with the solid-phase antibody and detection antibody to form immune complexes. After washing to remove the unbound material, the horseradish peroxidase-labeled Streptavidin-HRP was added. After washing, the chromogenic substrate was added and the color was developed in the dark. The reaction was terminated by adding the termination solution, and the absorbance value was measured at 450nm wavelength (reference correction wavelength 540nm or 570nm). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Composition |
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| Background | Interleukin-2 (IL-2, also known as TCGF) is a 15-16 kda monomeric α-helical glycoprotein belonging to the type I cytokine family. Mouse IL-2 precursor is a 169-amino acid (AA) peptide containing a 20-amino acid signal sequence and a 149-amino acid mature chain. There is an additional potential site for O-linked glycosylation, as well as a polyglutamine region at residues 35 to 46. It is worth noting that mouse IL-2 has multiple alleles, resulting in differences in the number of glutamine at amino acids 21-46 and the number of TSSS repeats in the mouse IL-2 sequence (SwissProt#:P04351). The amino acid sequence of mature mouse IL-2 shared 56% and 73% identity with human and rat IL-2, respectively, with the major difference located in the polyglutamine region. There is some cross-reactivity between mouse and human IL-2. Functionally, IL-2 was originally recognized as a T-cell growth factor and, therefore, was used in the treatment of cancer and AIDS patients, as well as to enhance the immune response. However, these efforts have had limited effect. "Experimental studies have shown that mice lacking IL-2 or its receptor also develop lymphoproliferation and autoimmune diseases, suggesting that IL-2 is a growth-limiting rather than growth-inducing factor." Recent studies have shown that the real cause of lymphocyte proliferation is the inability to produce CD4+FoxP3+CD25/IL-2Rα+Treg cells. It is important to note that now IL - 2 is considered successful induction of CD8 + memory cells necessary for memory response. It is well known that mammalian cells expressing IL-2 include CD4+ and CD8+T cells, NK cells, NKT cells, and dendritic cells. The IL-2 receptor is complex and consists of three different subunits. The two subunits can be combined with ligands, called IL - 2 r alpha and beta IL - 2 r. IL-2Rα is 55kDa and binds IL-2 with low affinity. IL - 2 beta for 75 kda, is part of the IL - 15 receptors, moderate affinity with IL - 2. Signal transduction is mediated by IL-2Rβ and a 64kDa common γ chain (γc). Gamma c and IL - 4, 7, 9, 15-21 and Shared receptors. Signal transducerγc does not bind to IL-2 but heterodimerizes with IL-2Rβ to form a functional IL-2 receptor. Heterologous trimer alpha beta gamma receptor may be combined with IL - 2 c, forming R alpha beta complex R. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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, except for the last step, diluent (1×) is used. "In addition to dilution, cell culture medium may be used for other intermediate dilutions." 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. |
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| Storage Temp. | Unopened kit, 2-8° C Storage | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Test Range | 15.6pg/mL-1000pg/mL |
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Immunohistochemistry
