Manual of Porcine Interleukin-8 (IL-8) ELISA Kit
This kit is for research use only
Intended application
ELISA method was used to quantitatively determine the content of IL-8 in pig serum, plasma, cell culture supernatant, lung alveolar lotion and other related liquids.
Overview
Interleukin (IL-8) is a basic heparin binding protein that was first isolated and purified from the supernatant of porcine mononuclear cells stimulated by lipopolysaccharide in 1987. It has endogenous leukocyte chemotaxis and activation. It was named as monocyte-drvived neutrophils chemotactic factor (MDNCF), and it was officially named as neutrophil-activating peptide / IL-8 (NAP / IL-8) in 1998. IL-8 belongs to the CXC type subfamily, and it is called ELR + -CXC type chemokine because of the glutamic acid-leucine-arginine before the first cysteine ​​of its amino acid. IL-8 is a multifunctional chemokine that can be derived from multiple cells. Its biological function is not species-specific. In addition to its specificity, neutrophils leave the bloodstream to migrate to inflammatory tissues, degranulate, and produce Oxygen anions, in addition to causing respiratory bursts, also show pro-angiogenic activity and can participate in the metastasis and angiogenesis of various malignant tumors. In addition, IL-8 also has chemotactic and activation effects on lymphocytes and other immune cells.
Experimental principle
This kit uses double antibody sandwich enzyme-labeled immunoassay to determine the level of IL-8 in the specimen. The microtiter plate was coated with purified antibody to make a solid-phase antibody. IL-8 antigen, biotinylated anti-porcine IL-8 antibody, and HRP-labeled avidin were added to the monoclonal antibody-coated microwells in sequence. After thorough washing, color was developed with substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with IL-8 in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.
Kit composition
1. ELISA plate: one piece (96 wells)
2. Standard product (lyophilized product): 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and its concentration is 1000pg / mL after full mixing. After serial dilutions, each is diluted to 500 pg / mL , 250 pg / mL, 125 pg / mL, 62.5pg / mL, 31.2pg / mL, 15.6 pg / mL, the sample dilution is directly used as the standard concentration of 0 pg / mL, prepared within 15 minutes before use.
3. Sample diluent: 1 × 20ml / bottle.
4. Test the diluent A: 1 × 10ml / bottle.
5. Test diluent B: 1 × 10ml / bottle.
6. Test solution A: 1 × 120ul / bottle (1: 100) is diluted with test diluent A1: 100 before use.
7. Detection solution B: 1 × 120ul / bottle (1: 100) is diluted with detection diluent B1: 100 before use.
8. Substrate solution: 1 × 10ml / bottle.
9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop solution: 1 × 10ml / bottle (2N H2SO4).
Collection and preservation of specimens
1. Supernatant of cell culture: collect the supernatant after centrifugation, and store the specimen at -20 ℃, and avoid repeated freezing and thawing.
2. Serum: Please leave the specimen at room temperature for 2 hours and centrifuge at 1000 xg for 30 minutes. Take the supernatant for testing, or store the specimen at -20 ℃, but avoid repeated freezing and thawing.
3. Plasma: Specimens can use heparin, EDTA or citrate as anticoagulants. Centrifuge at 1000 xg for 15 minutes within 30 minutes after sample collection. Take the supernatant for testing, or store the sample at -20 ℃, but avoid repeated freezing and thawing.
Steps
Each reagent is equilibrated to room temperature before use.
1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. In addition to the blank wells, add 100ul of the standard solution or the sample to be tested to the remaining wells and mix gently, at 37 ℃ for 120 minutes.
2. Discard the liquid and spin dry without washing.
3. Add 100ul of detection solution A working solution to each well at 37 ℃ for 60 minutes. Wash the plate 3 times, 350ul / per well, spin dry.
4. Add 100ul of detection solution B working solution to each well at 37 ° C for 60 minutes, wash the plate 5 times and spin dry.
5. Add 90ul of substrate solution to each well in sequence, and develop color at 37 ° C in the dark for 30 minutes.
6. Add 50ul of stop solution to each well in sequence to stop the reaction. The optical density (OD value) of each well was measured sequentially with an enzyme-linked instrument at a wavelength of 450 nm.
Note:
1. One hole is left for each experiment as a blank zero-adjusting hole. No reagents are added to this hole, only the substrate solution and 2NH2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.
2. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test.
Plate washing method Manual plate washing method: aspirate (do not touch the wall) or shake off the liquid in the enzyme plate; place a few layers on the experimental table
Absorb the paper with the microtiter plate down and force it several times; inject at least 0.4ml of the recommended washing buffer into the well and soak for 1-2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Specificity
The kit can simultaneously detect recombinant or natural porcine IL-8.
Calculation
Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate 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 standard concentration and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor to obtain the actual concentration of the sample.
Sensitivity
This kit determines the minimum detectable detection concentration (MDD) of pig IL-8, and its value is generally about 0.28 pg / mL
Precautions
1. The washing process is very important, inadequate washing is easy to cause false positives.
2. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
3. Please make a standard curve at the same time of each measurement, it is best to make a double hole.
4. If the content of IL-8 in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.
5. When preparing the standard and the working solution of the test solution, please prepare it with the corresponding diluent, not to be confused.
examination range:
15.6 pg / mL – 1000 pg / mL
Explanation
1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).
2. Validity: 6 months
3. The concentrated washing liquid will have salt precipitation, and it can be heated and dissolved in the water bath when diluted.
4. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
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