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Common problems in ELISA experiment operation

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ELISA, or enzyme-linked immunosorbent test, is an experimental method commonly used in hospital laboratory and scientific research laboratories. It has high sensitivity and specificity and is widely used in clinical practice. Mainly used for qualitative or quantitative determination of antibody or antigen concentration. The experimental operation includes several main steps such as sample loading, plate washing, enzyme addition, plate washing, color development, termination, and plate reading. The various links and steps in the experimental operation process have a greater impact on the final experimental results. If you are not careful, it may lead to incomplete color rendering (false negative), flower board (false positive) and other results.

In this article, we summarize and analyze the factors that may affect the experimental results in each step of Elisa’s operation based on actual work experience, and share the points that need to be paid attention to during the experimental process. (Take horseradish peroxidase (HRP) as the marker and hydrogen peroxide (H2O2) and tetramethylbenzidine (TMB) as the chromogenic substrate)

Preparation of specimens

The most commonly used specimen in ELISA testing is serum. What we need to pay attention to is:

(1) Avoid producing filaments. (After the blood has spontaneously coagulated and then centrifuged as required, false positives are likely to occur if there are fibers);

(2) Avoid severe hemolysis. (Because hemoglobin has peroxidase-like activity, if there is severe hemolysis, the free hemoglobin during incubation is easy to adsorb to the solid carrier, and finally the color substrate reacts, prone to false positives) In addition, high blood lipids and high bilirubin Hyperemia also has a certain impact on the results;

(3) Avoid bacterial contamination. (The enzymes produced by the bacteria will cause non-specific interference to the test results);

(4) Specimens can be stored at 2-8 degrees Celsius for short-term storage. For long-term storage, they need to be stored at -15 to -20 degrees Celsius or below. Before use, equilibrate the samples at room temperature for more than 30 minutes. Frozen samples need to be mixed. use;

(5) Do not use sodium azide for preservation of specimens (sodium azide will inhibit enzyme activity and result in false negative results);

(6) Avoid repeated freezing and thawing. (After repeated freezing and thawing, the molecular structure of the protein will be destroyed, and false negatives are prone to appear).

Preparation of reagents

(1) Before the ELISA test, the reagent should be taken out of the refrigerator in advance, and it should be equilibrated at room temperature for at least 30 minutes before use. (If the reagent is used directly after being taken out of the refrigerator, the temperature of the reagents and reaction wells of the kit is low, which is prone to false negative results);

(2) Shake the lotion well before disposing (if crystals are fully dissolved before disposing the lotion);

(3) It is recommended to label the 96-well plates before the test to avoid mixing up the sequence after the plate is washed off.

Add sample, add enzyme

(1) While adding the sample, observe again whether there are filaments in the specimen;

(2) When using the sample gun, the sample addition speed should not be too fast. (the sample addition accuracy and uniformity decrease);

(3) Avoid the generation of bubbles (bubbles are likely to make the interface of the reaction liquid different);

(4) Avoid adding to the upper part of the hole wall (the upper part of the hole wall is a non-coated area, which may cause false positives if it is non-specifically adsorbed, or if the positive specimen is not in contact with the coated area, it may cause false negatives);

(5) Avoid splashing. (Easily contaminate other specimens)


(1) Put the reaction plate in the incubator, the most commonly used incubation temperature is 37℃, and strictly control the incubation time according to the instructions (too long time is easy to form non-specific adsorption, too short time to react incompletely );

(2) Seal the plate with sealing film for incubation to prevent the liquid in the hole from being adsorbed on the hole wall after evaporation.

Wash the board

(1) When washing the plates by hand, pay attention to the water flow when the washing liquid is injected slowly, not too quickly. (Too rapid water flow may cause the antibody or antigen bound to the solid phase carrier to fall off and cause false negatives);

(2) Make sure that the washing needles are unblocked when washing the plates of the automatic plate washer;

(3) Make sure that the lotion fills the holes and soak for 30-60s at the same time and then wash again (If the lotion is not enough or the washing time is too short, it will easily lead to insufficient washing, Make the result false positive);

(4) Wash 5 times (if the number of times of washing liquid is small, it is easy to cause insufficient washing, which may cause “drawing board” and make the result false positive);

(5) After washing the plate, it is best to gently pat dry on absorbent paper or a clean towel. (If there is liquid residue, the result will be false positive).

Color rendering

(1) Chromogenic reagents should be prepared before application. Do not use expired or unsealed (configured) color reagents (such as reagent H2O2 that is too long to decompose and will cause false negative results. If TMB is too long, it is easy to show Light blue leads to false positive results or invalid results of the whole plate experiment);

(2) After adding the color reagent, mix it on the shaker in time. (If it is not mixed, the result will be false negative);

(3) Strictly control the color development time (too long time is prone to false positives).


(1) Avoid bubbles when adding stop solution;

(2) After adding the stop solution, mix it on the shaker in time;

(3) Go on the machine in time to make the measurement.

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