Porcine Interleukin----4 (IL-4) Enzyme Linked Immunoassay (ELISA)

Porcine interleukin-4 (IL-4) enzyme-linked immunoassay (ELISA)
Kit Instructions for Use This reagent is for research purposes only: This kit is used to determine the content of interleukin-4 (IL-4) in pig serum, plasma and related liquid samples.

Experimental principle:
This kit uses the double antibody sandwich method to determine the level of porcine interleukin-4 (IL-4) in the specimen. Microporous plates were coated with purified porcine interleukin-4 (IL-4) antibody to make solid-phase antibodies. Interleukin-4 (IL-4) was added to the microwells coated with mAb in turn, followed by The HRP-labeled goat anti-pig antibody binds to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with interleukin-4 (IL-4) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the content of porcine interleukin-4 (IL-4) in the sample was calculated by a standard curve.

Kit composition:
Kit composition 48-well configuration 96-well configuration Preservation manual 1 part 1 part sealing film 2 pieces (48) 2 pieces (96)
Sealed bag 1 x 1 enzyme-coated plate 1 × 48 1 × 96 2-8 ℃ Store standard: 90 ng / L 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ℃ Store standard dilution 1.5ml × 1 vial 1.5ml × 1 vial at 2-8 ° C for 3 ml × 1 vial × 1 vial at 6-8 × 1 vial at 2-8 ° C for sample dilution 3 ml × 1 vial for 6 ml × 1 vial at 2-8 ° C Toner A solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃ Store developer B solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ℃ Storage stop solution 3ml × 1 bottle 6ml × 1 bottle 2 Store the concentrated washing solution at -8 ℃ (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle at 2-8 ℃

Sample processing and requirements:
1. Serum: The blood will naturally coagulate at room temperature for 10-20 minutes and centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage.
2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation.
4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the concentration of cell 2 will reach about 1 million / ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.
5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ℃. Add a certain amount of PBS (PH7.4) and homogenize the specimen by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use.
6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.
7. The samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).

Steps:
1. Dilution and loading of standard products: set 10 standard wells on the enzyme-coated plate, add 100 μl of standard products in the first and second wells, and then add in the first and second wells Standard diluent 50 μl, mix well; then take 100 μl from the first and second wells and add them to the third and fourth wells respectively, then add standard diluent to the third and fourth wells 50 μl, mix well; then take 50 μl each in the third and fourth wells and discard, then add 50 μl each to the fifth and sixth wells, and then in the fifth and sixth wells Add 50ul of standard diluent separately and mix well; after mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively, then add standards to the seventh and eighth wells respectively 50 μl of the product dilution, mix 50 μl from the seventh and eighth wells to the ninth and tenth wells, and add 50 μl of the standard dilutions to the ninth and tenth wells, After mixing, take 50 μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50 μl, and the concentration is 60 ng / L, 40 ng / L, 20 ng / L, 10 ng / L, 5 ng / L respectively).
2. Add sample: set up blank wells separately (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same), and the sample wells to be tested. Add 40 μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10 μl of the test sample (the final dilution of the sample is 5 times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, gently shake to mix.
3. Incubation: seal the plate with the sealing film and incubate at 37 ℃ for 30 minutes.
4. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing solution with distilled water 30 times (20 times of 48T) and then reserve.
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let stand for 30 seconds and then discard, repeat 5 times, pat dry.
6. Add enzyme: add 50 μl of enzyme label reagent to each well, except for blank wells.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add color developer A50 μl to each well, then add color developer B50μl, gently shake to mix, and develop color at 37 ℃ in the dark
15 minutes.
10. Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color turns to yellow at this time).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner zero and 4 50 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.

Precautions:
1 . The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes before use. If the enzyme-coated plate is unsealed after opening, the strip should be stored in a sealed bag.
2 . Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
3. The sample adder should be used in each step of sample addition, and its accuracy should be regularly checked to avoid experimental errors. It is best to control the time of one sample addition within 5 minutes.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute it with a certain multiple (n times) of the sample diluent and then determine it. Multiple (× n × 5).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined by the microplate reader.
8 . All samples, washing liquids and various wastes should be treated as infectious agents.
9 . The components of different batches of this reagent shall not be mixed.
10. If there is any difference with the English manual, the English manual shall prevail.

Calculation:
Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the graph paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; multiply by the dilution factor; or use the concentration of the standard Calculate the linear regression equation of the standard curve with the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor to obtain the actual concentration of the sample.
(This picture is for reference only)

Kit performance:
1. The correlation coefficient R between the linear regression of the sample and the expected concentration is above 0.95.
2. In-batch and approval should be less than 9% and 11% respectively
examination range:
1n g / L-80 ng / L
Storage conditions and validity period:
1. Store the kit:; 2-8 ℃.
2 . Validity period: 6 months 4

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