Shanghai Hengyuan Company is one of the leading R&D agents and agents of China's leading ELISA kits. At the same time, it provides better familiarity and mastery of ELISA technology for the majority of laboratories. Shanghai Hengyuan has specially analyzed and compiled the six major ELISA kits for pigs. Stage, for teachers and classmates to observe!
â— Stage 1: Capture antibody coated plates;
1. Centrifuge the capture antibody slightly to concentrate the antibody on the bottom of the tube;
2. Resuspend the capture antibody to the concentration required in the instructions with sterile water and let stand for 10 minutes to completely dissolve the antibody;
3. Resuspend the antibody at the highest speed for 3 minutes;
4. Dilute the capture antibody in 1x PBS to the concentration required on the instructions;
5. Gently invert or shake to mix, do not create bubbles;
6. Immediately add 100 uL of capture antibody to each ELISA plate well;
7. Firmly cover the sealing film on the ELISA plate, taking care not to splash the antibody on the sealing film;
Incubate overnight at 8.25 Â° C, or incubate for 2-4 hours at 37 Â° C;
9. When washing the plate, the liquid should be drained and patted on a clean absorbent paper;
10. Add 300 ul of wash solution to each well and then remove the liquid;
11. This step is repeated 3 times for a total of 4 washes;
12. After the last wash, invert the plate to remove the liquid and pat dry on a clean absorbent paper;
13. There are several other washing methods for ELISA plates;
14. Regardless of which method is used, please be consistent throughout the ELISA experiment.
â— Phase II: Closing non-specific binding
1. Bovine serum albumin acts as a blocking reagent to block the site of any unbound protein in the pores of the plastic;
2. Add 300 ul of blocking solution to each well;
3. Seal the plate and incubate at 25 Â°C for at least 1 hour.
â— Stage 3: Specific binding of experimental antigens in pig ELISA kits
1. Before the end of the previous incubation process;
2. Centrifuge the standard product slightly;
3. Resuspend in sterile water to the required concentration;
4. Allow to stand for at least 10 minutes to allow the standard to dissolve completely;
5. After resuspension, remove the plate and wash 4 times;
6. Resuspend the standard at the highest speed for 3 minutes;
7. Dilute to the desired concentration, gently invert or shake to mix;
8. The area of â€‹â€‹the standard curve on the ELISA plate is dedicated to the detection of protein standards of known concentration;
9. If using a multi-channel pipette, add 100 ul of diluent to all standard curve wells except the first column;
10. Then, add 200 ul of standard solution to the first column of the standard curve well;
11. Repeated dilution, the standard has at least 6 concentrations;
12. The final volume of the liquid in each standard curve well should be 100ul;
13. The standard has at least 6 concentrations, each concentration is 3 duplicate wells, and then 6 blank wells, which contain only the diluent;
14. Blank wells are required and they will be used to evaluate the efficacy of the kit for detecting samples;
15. Then, the sample to be tested is added to the remaining holes, and each sample is provided with 3 duplicate holes;
16. If the concentration of the antigen to be tested is too high in the sample, the sample may need to be diluted to obtain the best results.
17. Seal the plate and incubate at 25 Â° C for 2 hours.
â€¢ Stage 4: Add biotin-labeled detection antibody to form a sandwich
1. Before the end of the incubation, the antibody is detected by centrifugation and resuspended in sterile water for 10 minutes;
2. After the incubation is completed, the plate is taken out and washed 4 times;
3. Centrifuge at maximum speed for 3 minutes;
4. Dilute the test antibody to the desired concentration with a diluent;
5. Mix gently and immediately add 100 ul of detection antibody solution to each well;
6. Seal the plate and incubate for 2 hours at 25 Â°C.
â— Stage 5: Adding enzyme-linked avidin to the sandwich
1. When the plate is incubated, remove a tube of Avindin-HRP (6 ul) from the refrigerator to melt it;
2. After the plate is incubated, wash 4 times;
3. Dilute Avidin-HRP with 1:2000 dilution;
4. Mix gently and immediately add 100ul to each well;
5. Seal the plate and incubate at 25 Â° C for 30 minutes;
6. To reduce background interference, make sure the incubation time is exactly 30 minutes.
â— Stage 6: Substrate color development
1. Wash the plate immediately after the incubation. Add 100 ul of ABTS substrate solution per well;
2. Avidin-HRP can only be used with ABTS;
3. If used in combination with TMB or other substrate solutions, it will significantly improve the background color;
4. Incubate the color at room temperature;
5. Read the plate once every 5 minutes, and monitor for up to 60 minutes;
6. Set the appropriate correction wavelength on the microplate reader;
7. The difference in EDK and the different reading times required in the manual may result in differences in OD values.
Shanghai Hengyuan related pig ELISA kit experimental equipment supply! Welcome everyone to inquire about the quotation.
1. Microplate reader: Finland (Labsystems Multiskan MS)
Instrument model: 352 type
2. Washing machine: Finland (Thermo Labsystems)
Instrument model: AC8
3. Centrifuge: micro high speed centrifuge (domestic)
Instrument model: TG16W
4. Incubator: Water-proof constant temperature incubator (domestic)
Instrument model: GNP-9080
5. Electron microscopy instrument: Phenom, desktop scanning electron microscope, FEI, USA
Instrument model: TY-HRC
Ningbo Vinny International Trading Co., Ltd , https://www.vinnystationery.com