Common problems and solutions in all aspects of ELISA

The elisa test has been widely used in clinical practice because of its high sensitivity and good specificity. However, all the links in the operation have a great influence on the test results. If you do not pay attention, it may lead to incomplete coloration and flower plate. Wait for the result. Now I will summarize the reasons and solutions for the problems that often occur in each part of the operation, in order to bring some inspiration to the peers and improve the quality of the test.

The following is an analysis of the reasons that may affect the results in the Elisa test operation, and the corresponding solutions are given. 141Q150G-0.gif

First, select the reagent

Select a good quality test reagent, strictly follow the reagent instructions, and equilibrate the reagent at room temperature for 30-60 minutes before the operation.

Second, the sample loading

possible reason:

1) If the serum or plasma specimens are not well separated, the sample is loaded; 2) In the manual operation, if the sample plate is too much, the waiting time is too long before the sample is put into the incubator (especially when the indoor temperature is high); When the specimen is added and the enzyme reagent is added, the enzyme spills out of the pore.


1) The specimen is serum: it is best to store the blood naturally for 1-2 hours, then centrifuge at 3000rmp for 15 minutes; the specimen is plasma: blood specimen collection tubes containing anticoagulant must be used, and the blood collection must be reversed immediately after blood collection. 5-10 times, after standing for a while, centrifuge at 3000 rpm for 15 minutes; if it is tested within a few days, it can be placed in a refrigerator at 2-8 °C. If it is to be stored, it should be placed in a low temperature refrigerator at -20 °C. 2) Put it into the incubator in time after loading. 3) After adding the enzyme reagent, use a blotting paper to gently blot and dry on the surface of the microplate. 4) If using AT or other fully automatic loading, it is best to choose FAME or other post-processing instruments plus enzyme reagents. 5) When there are many specimens, please operate in batches.

Third, incubate

possible reason:

1) When the incubation is not attached or capped, the specimen or diluent is evaporated and adsorbed on the pore wall, which is difficult to clean thoroughly; 2) The incubation time is artificially extended, resulting in non-specific binding surrounding the reaction well, which is difficult to clean thoroughly.


1) Attach the cover or cover; 2) Strictly control the operation time according to the instructions.

Fourth, wash the board

possible reason:

1) Wash the plate by hand and cross the liquid between the hole and the hole. 2) When washing the plate with a semi-automatic washer, the amount of washing liquid is insufficient, resulting in incomplete washing; the washing plate is clogged, the suction is not complete; the washing plate is not smooth, resulting in poor washing effect. 3) Excessive reaction plates cause long waiting time for washing.


1) Ensure that the washing liquid fills the holes, and the washing plate needle is unblocked. After washing the plate, it is best to pat dry on the absorbent paper (choose a clean, no or little dust-absorbing material); 2) arrange it reasonably, or use a few more Washing machine.

Five, color development

possible reason:

1) The developer is left for too long after the preparation of the developer or the expired developer is used; 2) When the developer is added, the liquid is recirculated outside the hole.

Solution: 1) The coloring agent should be prepared as far as possible before use. It should not be used without expiring the color developer. The light blue TMB developer is not visible to the naked eye. 2) Keep the color developer out of the flow when loading; 3) A. Liquid B should avoid contact with metal equipment.

Sixth, termination

Possible causes are that more bubbles are generated when the stop solution is added, resulting in an increase in false positives. Therefore, bubbles should be avoided when adding the stop solution.

Seven, read the board

If the board is not clean when reading the board, etc. The microplate should be cleaned. Therefore, the enzyme labeling plate is not exposed to hypochlorous acid during the whole operation process; the ELISA detection standard is automated as much as possible, and the detection quality is effectively improved.

In the actual operation, in addition to selecting excellent reagents, it is necessary to strictly follow the operation steps, and at the same time, make indoor quality control and inter-room quality assessment, and test each specimen with strict working style to ensure the quality of inspection. At present, a considerable number of units in China have automatic microplate readers, which plays an important role in achieving standardized detection of ELISA and improving the quality of detection.

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