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Details of success or failure in ELISA experiment operation

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Enzyme-linked immunosorbent assay (ELISA) is widely used in the determination of various antigens and antibodies in clinical testing because of its high sensitivity and strong specificity. Although the ELISA operation is simple, there are also big articles in the small experiment. Each link of the ELISA operation has a great influence on the detection effect of the experiment, and a little carelessness will produce false positive or false negative results. Now Xiaoyu will tell you some common sense that 818 should know when applying ELISA.

ELISA classification and specific process

Generally, ELISA can be divided into the following four categories: direct ELISA, indirect ELISA, sandwich ELISA, and competitive inhibition ELISA. Other ELISAs belong to these four types of ELISA or are derived from a combination of these four types of ELISA.
The following analyzes the reasons that may affect the results in the ELISA experiment operation, and gives the corresponding solutions.


Sample collection and preservation

The most commonly used clinical specimen for ELISA determination is serum (plasma). Care should be taken to avoid severe hemolysis, because hemoglobin contains a heme group with similar peroxide activity, which is easily adsorbed on the solid phase during incubation and reacts with HRP substrate to produce false positives.

During sample collection and serum separation, care should be taken to avoid bacterial contamination. On the one hand, enzymes secreted by bacteria may decompose proteins such as antigens and antibodies. On the other hand, bacterial endogenous enzymes such as E. coli β-galactosidase can affect The corresponding enzyme-labeled assay method produces non-specific interference.

The sample should avoid repeated freezing and thawing. The mechanical shearing force produced by repeated freezing and thawing has a damaging effect on the protein and other molecules in the specimen, causing false negative results. In addition, when the sample is mixed, do not shake it vigorously, just reverse and mix it repeatedly.

Generally speaking, if the test is performed on the day the sample is collected, the sample can be stored at 4°C for future use; and the sample to be tested on the next day should be aliquoted and frozen at -20°C for use. If the sample is to be stored for a long time, It is best to freeze it at -70°C.

Specific steps of the experiment

Reagent preparation

Before the start of the experiment, take the kit out of the refrigerator and place it at room temperature for 20 minutes, and then perform the measurement, so that the kit is balanced with room temperature before use, and the temperature in the reaction well during incubation can reach the required quickly The height to meet the determination requirements.

Secondly, the washing liquid in the current commercial ELISA kits needs to be diluted and prepared during the experiment. Therefore, the quality of the distilled or deionized water used in the dilution should be guaranteed. In addition, the substrate reaction solution should be prepared and used before the reaction and color development. At the same time, in order to ensure the uniformity of the experiment, all reagents in the experiment must be shaken before adding samples.


Due to the high sensitivity of ELISA, the serum should be diluted to an appropriate multiple as required to reduce non-specific reactions and fully reflect specific antigen-antibody reactions.

When adding samples, the single-hole dosage requirement: ≥20ul/index, if 2 multiple holes are needed, the blood volume ≥60ul/index. If the amount is sufficient, it is best to provide 50ul/hole/index. The specific dosage also needs to be determined according to the requirements of the kit.

When aspirating samples, the pipette tip should not adhere to excess liquid; do not add liquid to the hole at 90 degrees during sample addition, otherwise the liquid will remain on the tip and the sample will be inaccurate. The correct method of adding the sample should be 45 degrees, and the tip should be attached to the junction of the hole wall and the liquid surface.

The sample loading speed should not be too fast, otherwise the accuracy and uniformity of the micro sample addition cannot be guaranteed; it is necessary to avoid non-specific adsorption by adding the sample to the upper part of the well wall. Do not spill the sample to avoid contamination of adjacent holes.


Generally, the incubation time is inversely proportional to the temperature, that is, the higher the temperature, the shorter the time required. The most commonly used incubation temperature is 37°C for 1-2h.

Mounting or capping should be applied during incubation to prevent the sample or diluent from evaporating and adsorbing on the wall of the well, making it difficult to clean. During incubation, the reaction plates should not be stacked to ensure that the temperature of each plate can be quickly balanced. At the same time, the incubation time should be strictly controlled to avoid non-specific binding close to the reaction well due to too long time.

When incubating, try to eliminate the “edge effect”, that is, the color of the peripheral holes of the 96-well plate is deeper than the center hole. The reason is that the surface or thermodynamic characteristics of the peripheral hole and the center hole of the 96-well plate are different. Therefore, you can use a water bath or heat the plate and the solution to the incubation temperature (37°C) when the reaction solution is added to the wells of the plate.

Wash plate

In order to ensure the specificity of the ELISA assay, washing the plate can remove non-specific binding substances, reduce the background signal, and increase the signal-to-noise ratio of the analysis.

When washing the plate by hand, the clapboard should be vertical to avoid cross-contamination, and the force should not be too strong to prevent the antigen-antibody complex from detaching. When using semi-automatic plate washing, you should always check whether the flushing head is unobstructed. If it is blocked by debris, you can use a syringe needle to pick it out. In order to achieve a better washing effect, the laboratory can use a combination of machine and manual washing, that is, manual washing 1-2 times after machine washing.

The plate washing solution used in the ELISA kit with HRP as the labeled enzyme is generally neutral PBS containing 0.05% Tween20. Among them, the concentration of Tween 20 can be between 0.05% and 0.2%. When it is higher than 0.2%, it can be Desorb the antigen or antibody coated on the solid phase and reduce the sensitivity of the experiment.

Color rendering

In the current commercial ELISA kits that use HRP as the labeled enzyme, if TMB is used as the substrate, the substrates provided are two bottles of application solutions A and B; if OPD is used as the substrate, the kit provides OPD tablets or powders , Prepare before use. The developer should be discarded for too long time after preparation (when the light blue TMB is visible to the naked eye).

Before adding the substrate to start the color reaction, it is best to check the effectiveness of the substrate solution. You can add a drop of each of the two solutions A and B to a clean empty plate hole or eppendorf tube to observe whether there is any evidence. If the color appears, it indicates that the substrate has deteriorated.

The color reaction conditions are generally 37°C or room temperature for 15-30 minutes. When adding stop solution, avoid false positives caused by bubbles.

Plate reading

Make sure that the microplate reader is clean when reading the plate, and also pay attention to whether the wavelength of the microplate reader has been adjusted to the right or whether the filter used is correct.

Generally, the measured value of single-wavelength plate reading generally refers to the colorimetric measurement at the wavelength with the maximum absorption for color development, such as 450nm or 492nm; while the measured value of dual-wavelength plate reading is generally the absorbance value of sensitive wavelengths such as 450nm and insensitive wavelengths. The difference in absorbance at wavelengths such as 630nm can eliminate the influence of internal organs.

At the same time, since there is a certain degree of uncertainty in the non-specific absorption of a single blank hole in the ELISA measurement, that is to say, the difference in the position of the blank hole in each measurement or the same measurement may result in different absorbance measurement values. Therefore, in the ELISA measurement For color comparison, it is best to use dual wavelength color comparison.

Data analysis

ELISA measurement results can be divided into two types: qualitative measurement and quantitative measurement. The former only needs to be determined as negative or positive; the latter requires specific values. In quantitative determination, the absorbance value obtained by the standard product is often drawn on a standard curve.

ELISA intractable diseases

1) Light color rendering, low sensitivity

2) There are many false positives, high background and even slate

3) Poor repeatability

4) Whiteboard

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Indirect ELISA procedure

Elisa experimental service (enzyme-linked immunosorbent test)

ELISA standard operation and common problem analysis

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