Mouse interleukin-1aELISA kit

Mouse interleukin-1aELISA kit

Background introduction:

IL-1 has two different molecular forms, one is IL-1 α, which is composed of 159 amino acids; Another type is called IL-1 β, which contains 153 amino acids; Both are encoded by different genes. The IL-1 gene in both humans and mice is located on chromosome 2 and contains 7 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; However, within the same genus, the homology between IL-1 α and IL-1 β is only 25%. IL-1 α and IL-1 β have a molecular weight of approximately 17.5kDa, isoelectric points of 5 and 7, respectively, and a homology of 28%. IL-1 α and IL-1 β bind with the same affinity to the same moleculecell surfaceReceptors play the same biological role.

Detection Principle：

SolarbioSolarby ®)The ELISA kit adopts enzyme-linked immunosorbent assay (ELISA) detection technology based on double antibody sandwich method. Coat the anti mouse IL-1a monoclonal antibody onto an enzyme-linked immunosorbent assay (ELISA) plate; Add gradient diluted standards and pre diluted samples separately, and the mouse IL-1a in the standards and samples will fully bind to the coated antibodies on the enzyme-linked immunosorbent assay (ELISA) plate; After washing the plate, biotinylated anti mouse IL-1a antibodies are added, which will specifically bind to the mouse IL-1a in the standard and sample samples captured by the antibodies on the plate; After washing the plate, horseradish peroxidase (HRP) labeled streptavidin is added, and biotin and streptavidin undergo high-intensity non covalent binding; After washing the plate, add the chromogenic substrate TMB. If there are different concentrations of mouse IL-1a in the sample in the reaction well, HRP will cause colorless TMB to turn into blue substances of different shades (positively correlated). After adding the termination solution, the reaction well will turn yellow; Afterwards, at λ max=450 Measure the absorbance (OD) of the reaction well sample at nm (OD=450 nm), and the concentration of mouse IL-1a in the sample is proportional to OD. By drawing a standard curve and using four parameter fitting software, the concentration of mouse IL-1a in the sample can be calculated.

schematic diagram:

Notes:※※※

• The reagent kit should be used within its validity period, please do not use expired reagents.
• When the reagent kit is not in use, it should be stored in a refrigerator at 2-8 ℃. Standard samples that have been dissolved but not used up should be discarded.
• Please restore the reagent kit to room temperature for 30 minutes before use min， And thoroughly mix the various components and prepared samples in the reagent kit.
• 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, gun tip, and sealing film in the experiment（※）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.

• Please do not use reagents from other sources other than the concentrated washing solution and termination solution in the reagent kit, which can be used interchangeably

Replace a single component in this kit with a reagent.

• To ensure accurate results, a standard curve is required for each test.

Safety Tip:The termination solution in the reagent kit is an acidic solution. Operators should wear gloves and pay attention to protection when using it; During the operation, it is also necessary to avoid contact between the reagent and the skin and eyes. If accidentally touched, please rinse 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℃Incubation box
• Double distilled water, deionized water, measuring cylinder, etc
• Dilute polypropylene test tube

Sample collection and storage:

• Cell culture supernatant:

Transfer the cell culture medium to a sterile centrifuge tube and centrifuge at 1000 × g for 10 minutes at 4 ℃. Then, equally divide the supernatant into small EP tubes and store at -20 ℃ (2-8 ℃ can be used for testing within 24 hours) to avoid repeated freezing and thawing.

• Serum sample:

After natural coagulation of blood at room temperature for 20 minutes, centrifuge at 1000 × g for 10 minutes at 4 ℃. Then, equally divide the supernatant into small EP tubes and store at -20 ℃ (2-8 ℃ can be used 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 tubes containing anticoagulants, select EDTA, sodium citrate, or heparin as anticoagulants according to the actual requirements of the specimen, mix for 20 minutes, centrifuge at 1000 × g at 4 ℃ for 10 minutes, and then equally divide the supernatant into small EP tubes and store at -20 ℃ (stored at 2-8 ℃ within 24 hours for testing) to avoid repeated freezing and thawing.

※Note:Avoid using hemolysis and hyperlipidemia samples in serum and plasma samples to avoid affecting the test results; If the detection concentration of the target substance in the sample is higher than the high value of the standard, please dilute the sample by an appropriate factor 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 experiment, place the reagent kit and the sample to be tested at room temperature. If crystallization occurs in the concentrated washing solution, please place it in a 37 ℃ bath until all crystals dissolve.
• Preparation of washing solution: Calculate the volume of diluted washing solution in advance, then dilute 20 times the concentrated washing solution with double distilled water or deionized water to 1 times the application solution. Store the unused concentrated washing solution in a refrigerator at 4 ℃.

3. Gradient dilution of standard: Add 1ml of standard/sample diluent (SR1) to the freeze-dried standard, let it stand for 15 minutes until it * dissolves, then gently mix (concentration of 2000pg/ml), and dilute according to the following concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 0 pg/ml. The standard solution (2000pg/ml) that has not been used up after reconstitution should be discarded or divided into batches according to the required dosage, and stored in a -20 to -80 ℃ refrigerator, as shown in the following figure.

4. Biotinylated antibody working solution: Calculate the required amount for the experiment in advance, dilute the 100 fold antibody concentrate with the detection diluent (SR2) to 1 fold of the application working solution (mix thoroughly before dilution), and add it to the reaction well within 30 minutes.

Specific biotinylated antibody working solutiondilution methodAs follows:

5. Enzyme conjugate working solution: Prepare according to the required amount for each experiment, dilute 100 fold concentrated enzyme conjugate with enzyme conjugate diluent (SR3) to 1 fold application working solution (centrifuge before dilution), please use within 30 minutes.

Enzyme conjugate working solution specificdilution methodAs follows:

6. Washing method:

• Automatic plate washing: Shake off the liquid in the enzyme labeled plate well, pat dry on thick absorbent paper, inject washing solution at 300ul/well, with a 30 second interval between injection and suction, and wash the plate 5 times.
• Manual 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, add 300ul of washing solution per well using a washing bottle, let it stand for 30 seconds, shake off the liquid in the wells of the ELISA plate, pat dry on the thick layer of 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, subtract the OD measurement at 630 nm from the OD measurement at 450 nm.

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, draw a standard curve using software. The IL-1a content in the sample can be converted to the corresponding concentration from the standard curve based on the corresponding OD value.

If the OD value of the specimen is higher than the upper limit of the standard curve, it should be diluted appropriately and retested. When calculating the concentration, it should be multiplied by the dilution factor.

parameter characterization：

1. Data and standard curve

This figure is for reference only, and the sample content of mouse IL-1A should be calculated based on the standard curve drawn from the current experimental standard

2. Sensitivity:

The low detectable concentration of IL-1a in mice reached 15pg/ml,

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

3. Specificity:

Not reacting with mouse IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 12, 13, rat IL-1BWaiting for a response

4. Repeatability:

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

5. rate of recovery:

Add three different concentrations of mouse IL-1a to the selected healthy mouse plasma and cell culture supernatant, and calculate the recovery rate.

6. Linear dilution：

Add high concentrations of mouse IL-1a to four selected healthy mouse plasma and cell culture supernatants, dilute within the standard curve kinetic range, and evaluate linearity.

References：

1. Greenfeder, S.A. et al. (1995) J. Biol. Chem.270:13575.

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

3. Sims, J.E. et al. (1994) Clin. Immune. Immunopathology.72:9.

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

5. Colotta, F. and A. Mantovani (1994) Trends Pharmacol. Sci.14:138.

6. Svenson, M. et al. (1993) Cytokine5:427.

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

Common problems and solutions：

Mouse interleukin-1aELISA 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.