Elisa analyzes the reasons affecting the results in the experiment
1. Select reagents Select the detection reagents of good quality, strictly follow the reagent instructions, and equilibrate the reagents at room temperature for 30-60 minutes before operation.
2 Possible reasons for sample addition: â‘ Serum or plasma specimens are separated if the sample is not well separated; â‘¡ In manual operation, too many sample plates cause too long waiting time after the sample is placed in the incubator (especially when the indoor temperature is high) ; â‘¢ When the sample is added and enzyme reagent is added, the enzyme splashes out of the hole.
Solution: ①The specimen is serum: it is best to store the blood naturally for 1-2 hours, and then centrifuge at 3000rmp for 15 minutes; the specimen is plasma: you must use a blood specimen collection tube containing anticoagulant, and the blood must be collected upside down Mix blood vessels 5-10 times, centrifuge at 3000 rpm for 15 minutes after standing for a period of time; if it is tested within a few days, it can be placed in a refrigerator at 2-8 ° C, and if it is to be stored, it should be placed in a low temperature refrigerator at -20 ° C. ② Put the sample into the incubator in time. ③After 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. ⑤When there are many specimens, please operate in batches.
3. Possible reasons for incubation: â‘ No seal or cover was added during the incubation, which caused the specimen or dilution to evaporate and adsorb to the wall of the well, which was difficult to clean thoroughly; It is difficult to clean thoroughly.
Solution: â‘ Stick the sealing sheet or cover; â‘¡ strictly control the operation time according to the instructions. 4. Possible reasons for washing the plate: â‘ Manually wash the plate, and the liquid between the holes crosses. â‘¡ When using a semi-automatic plate washing machine to wash the plates, the amount of washing liquid is insufficient, which results 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. â‘¢ Excessive reaction plates cause long waiting time for washing plates.
Solution: â‘ Ensure that the washing liquid is filled in each hole, 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); â‘¡ Reasonably arrange, or use more Several washing machines.
5. Possible causes of color development: â‘ The developer is left for too long after preparation or the expired developer is used; â‘¡ When the developer is added, it splashes out of the hole and causes the liquid to return.
Solution: â‘ The developer should be prepared as soon as possible before use, and insist on not using the expired developer. The light blue TMB developer should not be used by the naked eye; â‘¡ Keep the developer not outflow when adding samples; â‘¢ A and B should be Avoid contact with metal instruments.
6. The possible reasons for termination are that more bubbles are generated when the termination solution is added, leading to an increase in false positives. Therefore, avoid adding bubbles when adding the stop solution.
7. Reading the board, such as the bottom of the board is not clean when reading. The microplate should be clean.
Therefore, ensure that the enzyme-labeled plate is not exposed to hypochlorous acid during the entire operation process; as far as possible, the ELISA detection standard should be automated to effectively improve the detection 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 rigorous 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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