Mouse interleukin-1 β

Mouse interleukin-1 β ELISA kit

Background Introduction：

IL-1 is divided into two types: IL-1a and IL-1 β. The human and mouse IL-1 genes are located on chromosome 2 and both contain seven exons. ProIL-1 precursor (ProIL-1) is 31 kDa and is cleaved by proteolytic enzymes to form mature IL-1 molecules. IL-1 has high homology among different species. At the amino acid level, the homology of IL-1 α and IL-1 β in different species is 60% to 70% and 75% to 78%, respectively; IL-1 α and IL-1 β have a molecular weight of approximately 17.5kDa, with isoelectric points of 5 and 7, respectively, and a homology of 28%.Mature mouse IL-1 β with a molecular weight of 17kDa shares 90% amino acid sequence identity with IL-1 β from cotton mice and rats, and 65% -78% identity with IL-1 β from dogs, horses, cats, humans, pigs, and monkeys.

IL-1 β is mainly produced by monocytes and macrophages in the blood, as well as astrocytes, oligodendrocytes, adrenal cortex cells, NK cells, endothelial cells, keratinocytes, megakaryocytes, platelets, neurons, neutrophils, osteoblasts, Schwann cells, trophoblast cells, T cells, and fibroblasts. IL-1 has a wide range of immunomodulatory effects and has thermogenic and inflammatory mediating effects.

Detection Principle：

SolarbioSolarby ®)The ELISA kit adopts enzyme-linked immunosorbent assay detection technology based on double antibody sandwich method.resistancemouseIL-1BMonoclonal antibodies are coated on enzyme-linked immunosorbent assay (ELISA) plates; Add gradient diluted standard and pre diluted sample separately, and add them to the standard and samplemouseIL-1BWill be coated with antibodies on the enzyme-linked immunosorbent assay (ELISA) platefullycombination;After washing the boardAdd biotinylated antibodiesmouseIL-1BantibodyThe antibody will interact with the mouse IL-1 in the standard sample and sample captured by the antibody on the boardBSpecific binding occurs; After washing the boardAdding horseradish peroxidase (HRP) labeled streptavidin will cause a reaction between biotin and streptavidinHigh strength non covalentcombination;After washing the boardAdd chromogenic substrate TMB，If there are samples of different concentrations in the reaction wellmouseIL-1BHRP will cause colorless TMB to turn into blue substances of different shades (positively correlated), and the reaction well will turn yellow after adding the termination solution; Afterwards, at λ max=450 nm（OD=450 nm）Measure the absorbance of the reaction well sample（OD）, in the samplemouseIL-1BConcentration andODDirectly proportional, it can be calculated by drawing a standard curve and using four parameter fitting softwareoutIn the samplemouseIL-1BThe concentration.

schematic diagram:

Notes:※※※

• The reagent kit should be used within its validity period, please do not use expired reagents.
• The reagent kit should be stored in 2-8 when not in use℃ refrigerator, already dissolvedbutPlease discard any unused standard products.
• Before using the reagent kitPleaseRestore at room temperature for 30 minutes min，And fully mix the various components in the reagent kit andpreparedSample.
• In the experiment, it is recommended to perform double well testing on the standard and sample, and the order of adding reagents should be kept *.
• To avoid cross contamination, please use a disposable test tube and gun tip in the experiment，Sealing film（※）And clean plastic containers..
• The volume of concentrated biotinylated antibody and concentrated enzyme complexes is relatively small, and trace amounts of liquid may stick to the tube wall and bottle during transportation

Cover and centrifuge before use (5-10 seconds is sufficient), so that the liquid on the tube wall is concentrated at the bottom of the tube. When taking it out, please carefully blow it several times with a pipette.

• Except for the concentrated washing solution in the reagent kitandTermination solution can be used universally, please do not use reagent kits from other sourcesContains reagentsReplace a single component in this kit.
• To ensure accurate results, every test is conductedneedCreate a standard curve.

Safety Tip:The termination solution in the reagent kit is an acidic solution,The operator isWhen usingPleaseWear gloves and pay attention to protection;During the operation process, it is also necessary toAvoid contact between the reagent and the skin and eyes. If accidentally touched, rinse thoroughly with plenty of water; When testing blood samples and other bodily fluid samples, please follow the relevant management regulations of national biological laboratory safety protection.

Composition and storage of reagent kit：

Self provided experimental equipment (not provided, can be purchased on behalf of others)

• ELISA reader (main wavelength 450nm, reference wavelength 630nm)
• High precision pipette and disposable tip: 0.5-10, 2-20, 20-200, 200-1000 μ l
• Washing machine or bottle washing machine
• 37℃hatchcultivatebox
• Double distilled water, deionized water, measuring cylinder, etc
• Dilute polypropylene test tube

Sample collection and storage:

• Cell culture supernatant:

Transfer the cell culture medium into a sterile centrifuge tube and incubate at 4 ℃ for 1000 hours×Centrifuge for 10 minutes, then equally divide the supernatant into small EP tubes and transfer them-2Store at 0 ℃(Can be stored at 2-8 ℃ for testing within 24 hours)Avoid repeated freezing and thawing.

• Serum sample:

After 20 minutes of natural coagulation of blood at room temperature, 1000 at 4 ℃×Centrifuge for 10 minutes, then equally divide the supernatant into small EP tubes and transfer them-2Store at 0 ℃ (can be stored at 2-8 ℃ for testing within 24 hours). If there is precipitation during storage, please centrifuge again to avoid repeated freezing and thawing.

• Plasma sample:

Collect whole blood into a tube containing anticoagulant, select EDTA, sodium citrate, or heparin as anticoagulant according to the actual requirements of the specimen, mix for 20 minutes, and perform 1000 tests at 4 ℃×Centrifuge for 10 minutes, then equally divide the supernatant into small EP tubes and transfer them-2Store at 0 ℃ (tested within 24 hours and stored at 2-8 ℃) to avoid repeated freeze-thaw cycles.

※Note:Avoid using hemolysis and hyperlipidemia samples in serum and plasma samples to avoid affecting the test results；If in the sampleTarget substanceIf the detection concentration is higher than the high value of the standard, please dilute the sample by an appropriate multiple before testing. It is recommended to conduct a pre experiment before the formal experiment to determine the dilution factor.

Reagent preparation：

• Reagent reheating: First, 30 minutes before the experimenttest kitto be testedsamplePlaced inroom temperaturedownIf crystallization occurs in the concentrated washing solution, please place it in a 37 ℃ bath until all crystals are dissolved.
• prepare, compound, formulateWashing liquid:Pre calculate the volume of diluted detergent to be used, and thenUse double distilled water or deionized water to20 timesDilute concentrated washing solution into1 timesApply the concentrated washing solution that has not been used up and put it in4℃Store in refrigerator.

3. reference standardgradient dilution：Add standard products/sampleDilution solution（SR1）1mlIn the freeze-dried standard, let it stand for 15 minutes until it * dissolves, then gently mix well (concentration:2000pg/ml)， thenaccording toThe following concentrations:2000Dilute at 1000, 500, 250, 125, 62.5, 31.25, and 0 pg/ml. Dissolved standard stock solution（2000pg/ml）Unused items should be discarded or packaged according to a single use as needed, and stored in a -20 to -80 ℃ freezer,as followspicture.

4. Biotinylated antibody working solution:Pre calculatedThe required amount for the experiment is diluted with testing solution（SR2）Concentrate 100 fold antibody solutiondiluteDouble the application of working fluid（Mix thoroughly before dilution），PleaseWithin 30 minutesAdd it to the reaction well.

Specific biotinylated antibody working solutiondilution methodAs follows:

5. Enzyme conjugate working solution: prepared according to the required amount for each experiment, diluted with enzyme conjugate solution（SR3) Dilute 100 fold concentrated enzyme conjugate to1x applicationworking fluid（Before dilutionCentrifugal)Please use within 30 minutes.

Enzyme conjugate working solution specificdilution methodAs follows:

6. Washing method:

• Automatic plate washing: Shake off the liquid in the wells of the enzyme-linked immunosorbent assay (ELISA) plate, pat dry on a thick layer of absorbent paper, and inject the washing solution to 300ul/hole, with a 30 second interval between injection and suction, wash the plate 5 times.
• Manual board washing: Shake off the liquid in the enzyme labeled plate holes, pat dry on thick absorbent paper, and add washing solution 3 to the washing bottle00ul/well, let it stand for 30 seconds, then shake off the liquid in the wells of the enzyme-linked immunosorbent assay plate, pat dry on thick absorbent paper, and wash the plate 5 times.

Testing steps:

result judgment：

1. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at a wavelength of 450 nm. Choose dual wavelength detection with a reference wavelength of 630 nm. If dual wavelength detection is not possible, please use a 450 nm detectorODmeasured valuesubtract630 nmODMeasurement value.

2. Calculate the average OD value of standards and samples: The OD value of each standard and sample should be subtracted from the OD value of the zero well

3. Using standard concentration as the horizontal axis and absorbance OD value as the vertical axis, use software to draw a standard curve for the samplemiddleIL-1BThe content can be determined throughcorrespond toThe OD value is converted from the standard curve to the corresponding concentration.

If the OD value of the specimen is higher than the upper limit of the standard curve, it should bedoAfter appropriate dilutionRetestWhen calculating the concentration, multiply it by the dilution factor.

parameter characterization：

1. Data and standard curve

This figure is for reference only and should be calculated based on the standard curve drawn from the current test standard samplemouseIL-1BSample size

2. Sensitivity:

Low detectabilitymouseIL-1Bconcentration reaches15pg/ml，

Calculate the corresponding detectable concentration by adding two standard deviations to the average OD of 20 zero standard concentrations.

3. Specificity:

Not withmouseIL-2、34. 67, 10、IL-1a、IL-1RaWaiting for a response

4. Repeatability:

Within the board, the coefficient of variation between boards is less than 10%

5. rate of recovery:

In the selectedhealthmouseplasmaCell culture supernatantAdd three different concentration levels ofmouseIL-1BCalculate the recovery rate.

6. Linear dilution：

Selected separately4 healthy portionsIn mouse plasma and cell culture supernatantAdd high concentrationmouseIL-1BDilute within the standard curve dynamics range and evaluate linearity.

References：

1. Dinarello, C.A. (1994) Eur. Cytokine Netw.5:517.

2. March, C.J. et al. (1985) Nature315:641.

3Sims, J.E. et al. (1989) Proc. Night. Acad. Ski. United States86:8946.

4Sims, J.E. et al. (1988) Science241:585.

5. Huang, J. et al. (1997) Proc. Natl. Acad. Sci. USA94:12829.

6Wesche, H. et al. (1997) J. Biol. Chem.272:7727.

7Sims, J.E. et al. (1993) Proc. Night. Acad. Ski. United States90:6155.

8. Giri, J.G. et al. (1994) J. Immunol.153:5802.

9. Symons, J.A. et al. (1995) Proc. Natl. Acad. Sci. USA92:1714.

10. Wewers, M.D. et al. (1997) J. Immunol.159:5964.

11. Gonzales-Hernandez, J.A. et al. (1995) Clin. Exp. Immunol.99:137.

Common problems and solutions：

Mouse interleukin-1 β ELISA kitOrdering instructions:

1. The vast majority of products are in stock and can generally be ordered and shipped on the same day.
2. Some non essential products require a 1-2 day advance booking, while overseas futures require a 3-6 week advance booking.
3. Some product prices may change due to factors such as delivery time and batch. If there are any changes, the new price on the day of ordering shall prevail.
4. The daily order deadline is 16:00 sharp, and in some cities it can be until 17:00 sharp. If the deadline is exceeded and the shipment cannot be made on the same day, it will be arranged for the next day.
5. After processing the payment, please notify us by fax, text message, etc. of the receipt and the recipient's address, name, etc.

Transportation instructions:

Ultra low temperature products: Dry ice is added during the transportation of extremely low temperature products. Wrap the product with dry ice, seal it with a foam box, glue the foam box layer by layer with adhesive tape, put it into the box of SOLEBAR, and then deliver it to the cooperative express delivery, which will deliver it safely, quickly, efficiently, and confidently to the customers, ensuring the stability of the product's nature and escorting your experiment.
Low temperature products: During the transportation of low-temperature products, add Solaibao ice packs for transportation. Wrap the product with ice bags in advance, then seal it with foam boxes, seal the foam boxes with adhesive tape, and put it into the boxes of SOLEBAR (the heat preservation effect can last for a week if it is less), and then deliver it to the cooperative express, which can deliver it safely, quickly, efficiently and confidently to the customers, ensuring the stability of the product properties and escorting your experiment.
Normal temperature products: Normal temperature products do not require ice or special packaging during transportation. The products are quickly distributed by the company's warehouse personnel, and accurate, fast, and efficient express delivery ensures that the products are delivered to you quickly.