At present, there are more and more personnel establishing ELISA methods in the laboratory. In order to reduce experimental errors caused by personnel operation, avoid interference between instruments and reagents due to shared use, and facilitate the analysis and evaluation of experimental results, we now share with you the norms that need to be followed in Elisa experimental operations.
1. Package:
Prepare a clean tank, take 10 Nml of 1 * coating solution into the tank according to the number of enzyme-linked immunosorbent assay plates to be coated (N), place the protein to be coated on an ice box, and aspirate the required volume of protein (using a low adsorption tip with a filter cartridge). Pay special attention to not inserting the tip too deeply into the liquid surface, only about 1 millimeter into the liquid surface, to ensure that the protein is aspirated. If it is found that the tip is dipped in too much protein during this step, aspirate again. Before adding the protein to the coating solution, the Tip tip is slightly docked on the inner wall of the protein storage tube to allow a small amount of protein dipped on the tip tip of the Tip tip to adsorb onto the inner wall of the protein storage tube.
After adding the protein to the coating solution, it should be repeatedly blown and mixed several times. Then, a large volume pipette should be used to blow and mix (with a filter cartridge on the gun or tip). Finally, a pipette should be used to blow and mix a few more times before adding it to the enzyme-linked immunosorbent assay (ELISA) plate. Each well should be filled with 100 microliters. When pouring out the liquid, the tip should be placed as close to the bottom of the ELISA plate as possible and added vertically. At this point, the protein begins to coat on the alcohol labeled plate. It should be immediately sealed with a high-quality clean sealed bag and placed in the designated location of the refrigerator. After the coating process is completed, remember to put the coating solution back into the refrigerator in a timely manner.
2. Closure:
After removing the enzyme-linked immunosorbent assay (ELISA) plate that has been coated, shake off the liquid in the well and try to pat the plate dry. Then wash the plate twice with detergent, with 110 microliters per well and soaking for about 1 minute. For the first time, place the plate upside down and add detergent from left to right, and for the second time, invert it upside down and add detergent from left to right. After two washes, try to pat the board dry as much as possible. Add 110 microliters of sealing solution SB to each hole (the pipette technique should be consistent each time the SB is suctioned and blown. SB has a certain degree of adsorption and is easy to hang on the nozzle wall. After the first blow, it can be blown again after a short period of time, or the blowing speed can be reduced appropriately to allow it to flow downwards under the action of gravity). Seal it with a sealed bag and place it flat in a 37 ℃ temperature box.
After sealing for 1 hour, use a multi-channel pipette to suck out the sealing solution SB and transfer it to a clean tank. Use moderate force to pat the plate several times to remove the SB dipped in the hole wall (SB has a certain degree of adsorption, but there will still be some at the bottom of the hole), and then invert it and dry it in a 37 ℃ oven for 2 hours. After the time is up, take out the sealed bag and add a packet of desiccant, seal and store at 4 ℃.
The extracted SB is recycled into a dedicated bottle and labeled as having been used once. SB can be reused, but only to block the same protein, and the number of blocks should not be too many, preferably no more than 4 times. SBs that have been reused different times are stored in different containers and labeled with the processed protein and the number of times they have been reused.
3. Detection operation:
Serum diluent, standard samples, and other detection reagents such as enzyme-linked immunosorbent assay (ELISA) plates should be taken out and warmed in advance, and gently shaken before use.
A. Sample dilution: Regardless of the number of samples, they should be diluted on a serum dilution plate first (in the ratio of adding 1 microliter of serum to 100 microliters of serum dilution), and the serum diluent should be taken according to the required amount. The amount added to the dilution well should be at least 20 microliters more than the actual required amount. If it is a deep well plate, at least 50 microliters more should be added. Before adding it to the detection plate, be sure to blow and mix the diluted serum sample evenly. Remember to use different tips for different samples to prevent cross contamination. When transferring the sample to the detection well, the use of the pipette should be consistent (including the rhythm of suction and blowing, as well as the number of blows), ensuring that the amount added to each well is as equal as possible. After sealing the detection plate with a high-quality sealed bag, incubate it in a 37 ℃ incubator. After using the serum dilution plate, rinse it with tap water in a timely manner, then rinse it with distilled water three times, shake it dry, cover it with a lid, and store it in a sealed bag (no need to seal it).
B. Washing and plating: After incubation with serum, shake off the liquid in the well and wash it four times with washing solution. The washing volume should be between 250-300 microliters, and the tip should be suspended to prevent the washing solution added to the well from coming into contact with the tip. The soaking time for each washing is about 1 minute. To ensure that the soaking time for each hole is as consistent as possible, the washing method is reversed in order. Don't pat dry every time you shake off the liquid, just try to pat dry as much as possible for the last time. For other intermediate steps, just shake a few more times, and the book used for the plate should have a smooth surface on copperplate paper.
C. Selection and addition of enzyme-linked immunosorbent assay (ELISA): There are various types of ELISA secondary antibodies, and different detection items require different concentrations. There are also pure dilutions that do not contain ELISA secondary antibodies. Before use, be sure to pay attention to the markings on the bottle, and choose according to your desired concentration, or prepare directly with the diluent. If you find that the tip of each concentration is suspended to prevent cross contamination. The incubation process of enzyme-linked immunosorbent assay is the same as serum incubation, and the washing and plate making methods after incubation are also the same.
D. Substrate addition and color development: The substrate must be removed before addition (i.e. after the plate has been dried), and the tank must be clean. The amount taken should be about 500 microliters more than the actual amount needed, so that the gun can smoothly extract the last column. The amount added to each well is 50 microliters. The substrate also has a certain adsorption capacity and is not easy to blow clean. When adding, it can be briefly stopped or rotated around the upper edge of the well. The rhythm and interval of substrate addition in each column should be consistent, ensuring that the amount added and the duration of action are as consistent as possible. After adding the substrate, there is no need to seal it in a sealed bag. Simply place it in a temperature chamber to avoid light and color development. At this point, turn on the enzyme-linked immunosorbent assay (ELISA) reader to preheat. The substrate color development usually takes 10 minutes, which can be adjusted according to one's own experimental results. Immediately clean the tank that has been filled with substrate, otherwise the substrate will remain on it, causing the tank to change color and be difficult to remove.
E. Termination and reading: After color development, remember not to shake off the liquid in the hole, but directly add termination solution to terminate the color reaction. Before the color development is completed, a termination solution should be prepared so that it can be terminated immediately after the color development is finished. The amount of termination solution is the same as the substrate, and the rhythm and interval are kept consistent when adding each column. After termination, readings should be taken immediately. If the interval is long, the values of some samples with high 0D values will significantly decrease. If there are large bubbles in the hole before reading, use a needle to puncture them.
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