Analysis of the causes of common problems affecting the ELISA test results and solutions

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. 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.

Steps

possible reason

Solution

Selection 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.

Loading

1. If the serum or plasma specimens are not well separated, the sample is loaded;

2. In manual operation, too much sample plate will cause too long waiting time before loading into the incubator (especially when the indoor temperature is high);

3. When the specimen is added and the enzyme reagent is added, the enzyme splashes 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. Add the sample to the incubator in time.

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.

Incubate

1. When the incubation is not attached or capped, the specimen or diluent is evaporated and adsorbed on the wall of the hole, which is difficult to clean thoroughly;

2. The incubation time is artificially prolonged, resulting in non-specific binding surrounding the reaction well, which is difficult to clean thoroughly.

1. affixing or capping;

2. Follow the instructions to strictly control the operating time.

Washing board

1. Wash the board by hand, and the liquid crosses between the hole and the hole.

2. When the plate is washed by the semi-automatic washing machine, 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 (select clean, no or less dusty absorbent material);

2. Reasonable arrangements, or use more than one washing machine.

Color

1. The developer is left for a long time after preparation or use an expired color developer;

2. When the developer is added, the liquid is recirculated outside the hole.

1. The coloring agent should be prepared as far as possible before use. It is not necessary to use the expired color developing agent, and the light blue TMB color developing agent is not visible to the naked eye;

2. Keep the developer from flowing out while loading;

3. A, B liquid should avoid contact with metal equipment.

End

More bubbles are generated when the stop solution is added, resulting in an increase in false positives.

Avoid air bubbles when adding stop solution.

Reading board

The bottom of the board is not clean when reading the board.

The microplate should be cleaned.

The whole process

1. Ensure that the microplate is not exposed to hypochlorous acid during the whole operation;

2. Realize the automation of ELISA test standards and effectively improve the quality of testing.

    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.