Analysis of the causes of common problems affecting the ELISA test results and solutions From Transhold Disposable Plastic Aprons,Plastic Aprons,Disposable Aprons,Disposable Polythene Aprons Surgimed Medical Supplies Co.,Ltd , https://www.surgimedcn.com
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The ELISA test has been widely used in scientific research because of its high sensitivity and good specificity. However, all the links in the operation have a great influence on the detection effect of the test. If it is not noticed, it may lead to incomplete coloration and flower board. Wait for the result.
The following are the reasons and solutions for the problems that often occur in each part of the operation:
Steps:
table of Contents
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1 Select reagents:
2 Specimen collection and preservation:
3 Preparation and precautions in the use of reagents:
4 Addition:
5 Temperature Education:
6 Washing board:
7 color:
8 End:
9 reading board:
10 The whole process:
11 ELISA experiment results judgment and data analysis
Select reagents:
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.
Refer to the correct procedure for preparing an ELISA experiment.
Specimen collection and preservation:
Avoid using macroscopic hemolyzed specimens, high-fat specimens, and turbid specimens;
Specimens can be placed for 24-48 hours at 2-8 °C and placed at -20 °C for long-term storage.
Please avoid repeated freezing and thawing.
Please refer to:
Collection and processing of ELISA test specimens:
Preservation of ELISA specimens:
Preparation and precautions in the use of reagents:
There are hints in the kit, generally need to be prepared:
Washing liquid; prepared before use; some kits will be marked, can be placed for one month under 4 degrees; if not, try to use fresh, do not prepare more.
Color developing solution (with liquid A and liquid B), prepared in the first 15 minutes;
Standards: Some kits have been prepared and stored at 4 degrees; some are ready to be prepared; follow the instructions;
Concentrated biotin-conjugated secondary antibody and HRP: If it needs to be prepared, the kit does not indicate how long it can be stored, and it should be used as soon as possible (prepared 15 minutes before use);
The principle is:
If the storage method of the preparation is not indicated on the kit, it should be used now; do not be bothered;
Also, the tube of the vial should be centrifuged before opening the lid to prevent the total amount from being insufficient after the lid is adhered.
Loading:
Reagents incubated at room temperature, especially those with relatively short incubation times, have a greater impact on the data. Pay special attention to the problem of loading.
Bad operation reasons:
The sampler will not be used correctly;
If the serum or plasma specimens are not well separated, the sample is loaded;
In manual operation, too many sample plates cause too long waiting time before loading into the incubator (especially when the indoor temperature is high);
When the specimen is added and the enzyme reagent is added, the enzyme splashes out of the pore;
Solution:
Familiar with the use of the sampler, and use the water to practice the fast and accurate loading before the experiment;
Before the test, the specimen should be slowly warmed to room temperature. If the specimen is serum, the frozen serum will be partially concentrated and unevenly distributed. It should be thoroughly mixed and avoid bubbles. The turbid or precipitated serum samples should be centrifuged or filtered before clarification;
Add the sample to the bottom of the ELISA plate hole when loading, avoid adding it to the upper part of the hole wall, and be careful not to spill;
Put it into the incubator in time after loading;
After adding the enzyme reagent, use a blotting paper to gently blot and dry on the surface of the microplate;
If using AT or other fully automatic loading, it is best to choose FAME or other post-processing instruments plus enzyme reagents;
When there are many specimens, please operate in batches. It is best to complete the sample in two to three minutes. It is best to add three rows to one when adding the standard. Timing each time the sample is added;
Reference ELISA# loading
Warming:
Bad operation reasons:
When the incubation is not sealed or capped, the specimen or the diluent is evaporated and adsorbed on the wall of the hole, which is difficult to clean thoroughly;
The incubation time is artificially prolonged, resulting in non-specific binding surrounding the reaction well, which is difficult to clean thoroughly.
Solution:
Patching or capping: In order to avoid evaporation, the plate should be covered. Plastic sealing paper or plastic wrap can be used to cover the hole. The reaction plates should not be stacked to ensure that the temperature of each plate can be quickly balanced.
Follow the instructions to strictly control the operating time.
It is recommended to use an air bath for incubation.
Reference incubation in ELISA experimental procedures (incubation)
Washing board:
Possible reasons for poor results
The plate is washed by hand, and the liquid crosses between the hole and the hole.
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 and the suction is not complete; the washing plate is not smooth, resulting in poor washing effect.
Excessive reaction plates cause long waiting time for washing.
In the indirect method, the background is higher.
Washing is the most important key technique in ELISA operations, especially when operating:
Make sure that the washing liquid fills the holes, and the washing needle is unblocked. After washing the board, it is best to pat dry on absorbent paper or towel (choose clean, no or less dusty water-absorbing material) (if using easy-to-slag paper, Paper scraps will remain in the hole of the plate, resulting in high values ​​due to the presence of oxidants in the paper dust.);
Note that the lotion of the various kits should not be mixed. If the lotion needs to be diluted, it should be diluted as required. The conductivity of the water used should be below 1.5us/cm. If the crystallization is to be crystallized, it should be prepared after melting.
Ensure that the washing time of the washing plate is about 40 seconds, the cleaner the liquid in the hole is absorbed by the washing machine, and the washing effect is better. The hand washing plate prevents the washing liquid from forming bubbles in the hole.
Make reasonable arrangements or use a few more washing machines.
In the indirect method, if the background is higher, the number of washings or the soaking time can be increased.
Reference ELISA# washing
Color:
Possible reasons for poor results
The developer is left for a long time after preparation or uses an expired color developer;
When the developer is added, the outside of the hole is splashed to cause liquid backflow.
Solution:
The coloring agent should be prepared as far as possible before use, and the coloring agent is not used, and the light blue TMB coloring agent is not visible to the naked eye;
Keep the developer out of the flow when loading, and the amount of sample should be accurate, the order can not be reversed.
A, B liquid should avoid contact with metal equipment.
Can refer to ELISA# color and colorimetric
End:
Possible reasons for poor results:
More bubbles are generated when the stop solution is added, resulting in an increase in false positives.
Solution:
Avoid adding bubbles when adding stop solution and stop color development in time.
Reading board:
Possible reasons for poor results:
The bottom of the plate is opaque, with water droplets, scratches, and irregular surfaces when the plate is read.
The microplate should be cleaned.
In addition, the microplate reader should not be placed under sunlight or strong light. The room temperature should be 15~30°C during operation. Preheat the instrument for 15-30 minutes before use. The test results are more stable.
The whole process:
Ensure that the microplate is not exposed to hypochlorous acid during the whole operation;
Realize the automation of ELISA test standards and effectively improve the quality of testing.
Rigorous design, quality reagents, proper operation and good instrumentation are essential for ELISA. In the actual operation, it is necessary to strictly follow the prescribed operation, and each specimen can be tested with strict style to ensure the test effect.
Results judgment and data analysis of ELISA experiment
Please refer to the instruction manual of the kit.
Example:
A quantitative kit: