Analysis and solutions of common problems affecting the effect of ELISA test

Analysis and solutions of common problems affecting the effect of ELISA test
The ELISA test has been widely used in clinic with the characteristics of high sensitivity and good specificity, but each link in the operation has a great influence on the detection effect of the test. And other results. I summarize the causes and solutions of the problems that often occur in various links of the operation in order to bring some inspiration to the peers and improve the quality of the experiment.
Steps
possible reason
Solution
Choose reagents
Choose good quality detection reagents, strictly follow the reagent instructions, and equilibrate the reagents at room temperature for 30-60 minutes before operation.
Loading
1. Sampling of serum or plasma samples is not good;
2. In manual operation, too many sample plates cause too long waiting time after adding samples to the incubator (especially when the indoor temperature is high);
3. When the sample is added and the enzyme reagent is added, the enzyme splashes out of the hole.
1. The specimen is serum: it is best to store the blood naturally for 1-2 hours, and then centrifuge it at 3000 rpm for 15 minutes; the specimen is plasma: the blood specimen collection tube containing anticoagulant must be used, and the blood collection tube must be reversed immediately after blood collection 5-10 times, after a period of time, centrifuge at 3000 rpm for 15 minutes; if it is tested within a few days, it can be placed in a 2-8 ℃ refrigerator, if it is to be stored, it is placed in a -20 ℃ low temperature refrigerator.
2. After adding the sample, put it in the incubator in time.
3. After adding the enzyme reagent, gently blot the surface of the enzyme plate with absorbent paper to dry.
4. If you use AT or other fully automatic sample addition, it is best to choose FAME or other post-processing equipment plus enzyme reagents.
5. When there are many specimens, please operate in batches.
Incubation
1. Without incubation or cover during incubation, the specimen or diluent will evaporate and adsorb to the wall of the well, making it difficult to clean thoroughly;
2. The incubation time is artificially extended, resulting in non-specific binding tightly around the reaction well, which is difficult to clean thoroughly.
1. Paste the seal or cover;
2. Follow the instructions to strictly control the operating time.
Wash board
1. Manually wash the plate, and the liquid between the holes crosses.
2. When using a semi-automatic plate washer to wash the plates, the amount of washing liquid is insufficient, resulting in incomplete plate washing; the plate washing needle is blocked and the suction is incomplete; the plate washing is not smooth, resulting in poor plate washing effect.
3. Too many reaction plates cause long waiting time for plate washing.
1. Make sure that the washing liquid is filled into the holes and the plate washing needle is unobstructed. After washing the plate, it is best to pat dry on absorbent paper (choose clean, no or less dust absorbent material);
2. Reasonable arrangement, or use more washing machines.
Color rendering
1. The developer has been left for a long time after preparation, or used expired developer;
2. Spilled out of the hole when adding the developer, causing liquid backflow.
1. The developer is prepared as soon as possible before use, insisting that no expired developer is used, and the light blue TMB developer is visible to the naked eye;
2. Keep the developer from flowing out when adding samples;
3. Liquid A and B should avoid contact with metal instruments.
termination
More bubbles are generated when the stop solution is added, resulting in an increase in false positives.
Avoid adding air bubbles when adding stop solution.
Reading
The bottom of the board is not clean during reading.
The microplate should be clean.
The whole process
1. Ensuring that the microplate is not in contact with hypochlorous acid during the entire operation
2. Realize the automation of ELISA testing standards, effectively improve the testing quality.
In actual operation, in addition to selecting good reagents, you must strictly follow the operation steps, at the same time make indoor quality control, inter-room quality assessment, and strict work style to test each specimen to ensure the quality of the test. At present, a considerable number of domestic units have automatic microplate readers, which plays an important role in realizing standardized ELISA testing and improving the quality of testing.

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