Mouse interleukin-1 β (IL-1 β) ELISA kit

1.Sample type and collection:

serum：Place the whole blood sample collected in the serum separation tube at room temperature0.5-2Hour or4℃Overnight, then1000×gcentrifugation20 Minutes, take the supernatant, or place the supernatant in-20℃or-80℃Save, but avoid repeated freezing and thawing.
plasma：useEDTAOr heparin can be used as an anticoagulant to collect specimens, and the specimens should be collected after30Within minutes2-8℃ 1000×gcentrifugation15Minutes, take the supernatant for detection, or place the supernatant in-20℃or-80℃Save, but avoid repeated freezing and thawing.

Tissue homogenate:Using pre cooledPBS (0.01M, pH=7.4)Rinse the tissue, remove residual blood (lysed red blood cells in the homogenate can affect the measurement results), weigh the tissue and cut it into pieces according to the corresponding volumePBS(Generally according to1:9The weight to volume ratio, for example1gCorresponding organizational samples9mLofPBSThe specific volume can be adjusted appropriately according to experimental needs and recorded. Recommended atPBSAdd protease inhibitor to the glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or subjected to repeated freeze-thaw cycles. Finally, mix the homogenate with5000×gcentrifugation5~10minute,The supernatant can be taken for detection,Or place the supernatant in-20℃ or-80Store at ℃, but avoid repeated freeze-thaw cycles.

cell lysate: Discard the culture medium and useWash the cells once with PBS (0.01 M, pH 7.4). Scrape off the cells and add 2-5 mL of PBS (0.01 M, pH 7.4). Suspended cells can be omitted. Collect cell suspension, centrifuge at 1000 × g at 4 ℃ for 10 minutes, discard the culture medium, and rinse three times with pre cooled PBS. Add an appropriate amount of pre cooled PBS or non denaturing cell lysis buffer (protease inhibitor added before use) to resuspend the cells. Typically, 150-250 μ L of PBS is required for each well of a 6-well plate. Place the sample at -20 ℃ or -80 ℃, freeze it, and then thaw it at room temperature. Repeat this process three times to fully lyse the cells. Alternatively, the sample can be sonicated to achieve lysis. Centrifuge at 10000 × g at 4 ℃ for 10 minutes, remove cell debris, and take the supernatant for detection,Or place the supernatant in-20℃ or-80Store at ℃, but avoid repeated freeze-thaw cycles.

Cell culture supernatant or other biological specimens:Please1000×gcentrifugation20Minutes, take the supernatant for detection, or place the supernatant in-20℃ or-80Store at ℃, but avoid repeated freezing and thawing.

2.Sample preservation and stability

The sample is2-8℃Under certain conditions, it can be stored72h， in- 20℃or-80℃can be stored6 months andaboveAfter sample collection, it is not tested in one go. Please pack and freeze the samples according to a single dose to avoid repeated freeze-thaw cycles.

Steps:

1. Number: Number the corresponding micropores of the sample in sequence, and each plate should have a negative control2 wells, positive control 2 wells, blank control 1 well (no sample or enzyme-linked immunosorbent assay added to the blank control well, all other steps are the same)）.

2. Sample addition: Add negative control and positive control to the negative and positive control wells respectively50μl。 Then add 40 μ l of sample diluent to the test sample well, followed by 10 μ l of the test sample. Add the sample to the bottom of the enzyme-linked immunosorbent assay plate well, avoiding touching the well wall as much as possible, and gently shake and mix well.

3. Incubation: Seal the plate with a sealing film and place it on the back37Incubate at 3 ℃0minute.

4. Liquid preparation: willDilute 30 times the concentrated washing solution with distilled water and set aside for later use.

5. Washing: Carefully remove the sealing film and discard the liquid,spin-dryFill each hole with detergent and let it standDiscard after 30 seconds, repeat this 5 times, and pat dry.

6. Enzyme addition: Add enzyme labeled reagent to each well50Ml， Excluding blank holes.

7. Incubation: Seal the plate with a sealing film and place it on the back37Incubate at 3 ℃0minute.

8. Washing: Carefully remove the sealing film and discard the liquid,spin-dryFill each hole with detergent and let it standDiscard after 30 seconds, repeat this 5 times, and pat dry.

9. Color development: Add color developer to each hole firstA50MlAdd color developer againB50MlGently shake and mix well,37Color development in the dark at 10 ℃-20minute

10.Termination: Add termination for each holeliquid50μlTerminate the reaction(At this moment, blue turns to yellow).

11.Measurement: Zero with blank hole,450nmMeasure the wavelengths of each hole in sequenceabsorb lightDegree（ODValue)The measurement should be conducted after adding the termination solution15Conducted within minutes.

Note:

1. Reagent preparation: The reagent kit should be taken out of the refrigerated environment and equilibrated at room temperatureIt can only be used after 15-30 minutes. Prepare the enzyme immunoassay strips required for one experiment, and the others can be removed from the microplate, sealed, and stored according to the instructions for future use.

2. Sample addition: Please use disposable sterilization tips during experimental operations to avoid contamination. When adding the sample, be careful not to generate bubbles. Add the sample to the bottom of the enzyme-linked immunosorbent assay plate, try not to touch the well wall, and gently shake and mix well. As with the addition of reaction reagents, the time interval between the first and last wells during the sample addition process should be kept as small as possible (generally controlled withinWithin 10 minutes, if it is too large, it will result in different "pre incubation" times, which will significantly affect the accuracy and repeatability of the measurement values. For the accuracy of measurement, it is recommended to set up multiple holes for experimentation.

3. Incubation: To prevent sample evaporation, please place the enzyme-linked immunosorbent assay (ELISA) plate with a lid or film in a wet box during the experiment to avoid liquid evaporation. After washing the plate, the next step should be carried out as soon as possible. At all times, the ELISA plate should be kept dry, and the given incubation time and temperature should be strictly followed.

4. wash:Concentrated detergent solutionpossiblethere will becrystallizationPrecipitation can be dissolved by heating in a water bath during dilutionWashing does not affect the results. Thoroughly washing is very important, and during each washing process, the detergent should be shaken dry. The residual washing solution in the reaction well during the washing process should be dried on absorbent paper. Do not directly put the absorbent paper into the reaction well to absorb water. At the same time, gently wipe off the residual liquid and fingerprints at the bottom of the plate to avoid affecting the final reading of the enzyme-linked immunosorbent assay. If using an automatic washing machine, please use it proficiently before using it in the formal experimental process.

5. Control of reaction time: After adding the substrate, please observe the color change of the reaction well regularly. If a darker color is observed, please add a stop solution in advance to terminate the reaction and avoid the reaction being too strong, which may affect the optical density reading of the enzyme-linked immunosorbent assay.

6. Substrate: Please store the substrate in the dark and avoid direct exposure to strong light during storage and incubation.