Porcine IL-4 ELISA Kit

1. Sample processing and requirements:
1、The detection range of the kit is not equivalent to the concentration range of the test substance in the sampleIt is recommended to estimate the concentration of the test substance in the sample through relevant literature before the experiment and determine the actual concentration of the sample through pre-experiment. If the concentration of the test substance in the sample is too high or too low, please dilute or concentrate the sample appropriately.
2. If the sample tested is not among the sample types listed in the instructions, it is recommended to conduct pre-experiments to verify its detection effectiveness.
3、Serum:Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or 2-8 ℃ overnight, then centrifuge at 1000 × g for 20 minutes, take the supernatant, or store the supernatant at-20 ℃ or-80 ℃, but avoid repeated freezing and thawing.
4、Plasma:Collect samples with EDTA or heparin as anticoagulants, and centrifuge the samples at 2-8 ℃ 1000 × g for 15 minutes within 30 minutes after collection. Take the supernatant for detection, or place the supernatant at-20 ℃ or-80 ℃ for storage, but avoid repeated freezing and thawing.
5、Tissue homogenate:The tissue was washed with pre-cooled PBS (0.01 M, pH = 7.4) to remove residual blood (lysed red blood cells in the homogenate would affect the test results), and the tissue was weighed and cut into pieces. Combine the chopped tissue with the corresponding volume of PBS (generally according to the weight-to-volume ratio of 1: 9, for example, 1g of tissue sample corresponds to 9mL of PBS. The specific volume can be appropriately adjusted according to the experimental needs and recorded. It is recommended to add protease inhibitor to PBS) add to a glass homogenizer and grind thoroughly on ice or grind in a homogenizer. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was centrifuged at 5000 × g for 5-10 minutes, and the supernatant was taken for detection.
6、Cell culture supernatant:Please centrifuge at 1000 × g for 20 minutes, take the supernatant for detection, or store the supernatant at-20 ℃ or-80 ℃, but avoid repeated freezing and thawing.
7、Cell lysate: Adherent cells were gently washed with pre-cooled PBS, then digested with trypsin, centrifuged at 1000 × g for 5 minutes, and the cells were collected; The suspended cells can be collected directly by centrifugation. The collected cells were washed 3 times with pre-cooled PBS, and 150-200uL PBS was added to every 1 × 10 ^ 6 cells for resuspension (it is recommended to add protease inhibitors to PBS; if the content is very low, the PBS volume can be appropriately reduced) and the cells were disrupted by repeated freezing and thawing or ultrasound. The extract was centrifuged at 2-8 ℃, 1500 × g for 10 minutes, and the supernatant was taken for detection.
8、Other biological samples:Centrifuge at 1000 × g for 20 minutes, and take the supernatant for detection.
9、Sample Appearance:The sample should be clear and transparent, and the suspension should be removed by centrifugation.
10、Sample Preservation:If the sample is tested within 1 week after collection, it can be stored at 4 ℃. If it cannot be tested in time, please pack it according to the amount used once and store it frozen at-20 ℃ (test within 1 month) or-80 ℃ (test within 6 months) to avoid repeated freezing and thawing. Hemolysis of the sample will affect the final test results, so hemolyzed samples are not suitable for this test.

Sample dilution protocol:
Please estimate the concentration range of the sample in advance. If your test sample needs to be diluted, refer to the dilution plan as follows:
100-fold dilution:One step dilution. Take 5uL sample into 495uL universal diluent and dilute it 100 times;
1000-fold dilution:Two-step dilution. Take 5uL sample into 95uL universal diluent and do a 20-fold dilution, then take 5uL 20-fold dilution sample into 245uL universal diluent and do a 50-fold dilution, diluting a total of 1000 times;
Dilution 100000 times:Three-step dilution. Take 5uL sample into 195uL universal diluent and do 40-fold dilution, then take 5uL 40-fold dilution sample into 245uL universal diluent and do 50-fold dilution, and finally take 5uL 2000-fold dilution sample into 245uL universal diluent and do 50-fold dilution, diluting 100,000 times in total;
During each dilution step, the amount of liquid taken shall not be less than 3uL, and the dilution factor shall not exceed 100 times. Each step of dilution should be mixed evenly to avoid foaming.
2. Self-prepared test equipment required for the experiment:
1. Microplate reader (450nm)
2. High-precision pipette and tip: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL
3. 37 ℃ incubator
4. Distilled water or deionized water
3. Preparation work before testing:
1. Please take out the kit from the refrigerator 10 minutes in advance and balance it to room temperature.
2. Preparation of standard gradient working solution: Add 1mL of universal diluent to the freeze-dried standard, let it stand for 15 minutes until it is completely dissolved, and then gently mix (the concentration is 100pg/mL), and then follow the following concentrations: 100pg/mL, 50pg/mL, 25pg/mL, 12.5 pg/mL, 6.25 pg/mL, 3.12 pg/mL, 1.56 pg/mL, 0pg/mL for dilution.
Double dilution method: Take 7 EP tubes, add 500μL of universal diluent to each tube, draw 500μL of 100pg/mL standard working solution into the first EP tube and mix well to prepare 50pg/mL standard working solution, according to this step, draw and mix well in sequence. The last tube is directly used as a blank hole, so there is no need to suck liquid from the penultimate tube, as shown in the figure below.

3、Preparation of biotinylated antibody working solution:The concentrated biotinylated antibody was centrifuged at 1000 × g for 1 minute 15 minutes before use, and 100 × concentrated biotinylated antibody was diluted to 1 × working concentration (example: 10 uL concentrate + 990 uL universal diluent) with a universal diluent, which was prepared for ready use.
4、Preparation of enzyme conjugate working solution:Fifteen minutes before use, 100 × concentrated enzyme conjugate was centrifuged at 1000 × g for 1 minute, and 100 × concentrated HRP enzyme conjugate was diluted to 1 × working concentration in a universal diluent (example: 10 uL concentrate + 990 uL universal diluent), which was prepared for ready use.
5、1×Wash liquid preparation: Take 10mL of 20 × washing liquid into 190mL distilled water (the concentrated washing liquid taken out of the refrigerator may have crystals, which is a normal phenomenon. It can be left at room temperature and prepared after the crystals are completely dissolved).
4. Operation steps:
1. Take out the required slats from the aluminum foil bag after equilibration at room temperature for 10 minutes, and seal the remaining slats with a ziplock bag and put them back to 4 °C.
2. Sample addition: Add the sample or standard of different concentration to the corresponding well according to 100μL per well, and add 100μL of universal diluent to the blank well. Incubate at 37 °C for 60 minutes after covering the plate sealing film. (Recommendation: Dilute the sample to be tested with a universal diluent at least 1 times and then add it to the enzyme labeled plate for testing. This reduces the influence of matrix effect on the test results. Finally, the sample concentration needs to be multiplied by the corresponding dilution factor when calculating. It is recommended to set up double wells for all samples and standards to be tested during testing).
3. Add biotinylated antibody: take out the enzyme labeled plate, discard the liquid, and do not wash it. 100 uL of biotinylated antibody working solution was directly added to each well, and the plate sealing membrane was covered and incubated at 37 °C for 60 minutes.
4. Plate washing: Discard the liquid, add 300uL 1x washing liquid to each hole, let it stand for 1 minute, throw away the washing liquid, pat dry on absorbent paper, and repeat washing the plate 3 times (you can also use a plate washing machine to wash the plate).
5. Add enzyme conjugate working solution: Add 100uL of enzyme conjugate working solution to each well, cover the sealing membrane and incubate at 37 °C for 30 minutes.
6. Wash the plate: Discard the liquid and wash the plate 5 times according to the washing method in step 3.
7. Add substrate: Add 90uL of substrate (TMB) to each well, cover with a plate sealing film, and incubate at 37 °C in the dark for 15 minutes.
8. Add stop solution: Take out the enzyme plate, directly add 50uL of stop solution to each well, and immediately measure the OD value of each well at a wavelength of 450nm.
5. Calculation of experimental results:
Result judgment：
1. Calculate the average OD value of the standard and sample duplicate well and subtract the OD value of the blank well as the correction value. Taking concentration as abscissa and OD value as ordinate, the standard curve of four-parameter logic function is drawn on double logarithmic coordinate paper.
2. If the OD value of the sample is higher than the upper limit of the standard curve, it should be properly diluted and retested and multiplied by the corresponding dilution factor when calculating the sample concentration.
Typical data and reference curves:
The following data and curves are for reference only, and experimenters need to establish standard curves according to their own experiments.

attention : This picture is for reference only , the specimen content should be calculated from the standard curve drawn by the same test standard.

6. Kit performance:
1. Repeatability: The intra-plate coefficient of variation is less than 10%, and the inter-plate coefficient of variation is less than 10%.
2. Recovery rate: Three different concentrations of porcine IL-4 were added to the serum, plasma and cell culture supernatant of selected healthy pigs, and the recovery rate was calculated.