ELISA, also known as enzyme-linked immunosorbent assay, is a commonly used immunoenzymatic technique. The main method is to adsorb known antigens or antibodies onto the surface of a solid carrier, then incubate them with enzyme labeled (conjugated) antibodies or antigens, add color reagents for color development, measure the difference in color between the test substance and the standard substance using an enzyme-linked immunosorbent assay (ELISA) reader, and draw an enzyme activity curve to determine the concentration of the test substance.

ELISA can be used to detect antigens as well as antibodies.

There are three necessary reagents in ELISA experiments:

① Solid phase antigen or antibody (used to bind to a solid phase carrier)

② Marked antigen or antibody (marker)

③ Substrate of action (chromogenic agent, used for color development)

Four common ELISA methods

1. Direct ELISA

Fix the antigen onto an ELISA plate and then directly detect the antigen using enzyme-linked immunosorbent assay (ELISA) antibodies.

Compared to other types of ELISA experiments, direct ELISA experiments have fewer steps, faster detection speed, do not require secondary antibodies, avoid cross reactions, and are less prone to errors in measurement results. However, due to the non-specific fixation of antigens in direct ELISA, target proteins and other impurity proteins in the sample will bind to the ELISA plate, resulting in a higher experimental background. Moreover, direct ELISA requires the preparation of primary antibodies that can specifically bind to each target protein, making the experiment less flexible. In addition, due to the absence of secondary antibodies, the signal was not amplified, which reduced the sensitivity of the measurement.

2. Indirect ELISA

First, bind the antigen to the ELISA plate, and then perform the detection in two steps: first, add the detection antibody to specifically bind to the antigen, and then add the enzyme-linked secondary antibody for detection and use the substrate for color development.

Compared with direct ELISA, indirect ELISA uses enzyme-linked secondary antibodies, which have higher sensitivity, require fewer labeled antibodies, and are more economical. Indirect ELISA also provides greater flexibility as different primary antibodies can be used together with a single labeled secondary antibody. The disadvantage of indirect ELISA experiments is the possibility of cross reactivity (direct binding of enzyme-linked secondary antibodies to antigens), which may increase the background. At the same time, compared with direct ELISA, indirect ELISA experiments have an additional step of secondary antibody incubation and a longer experimental period.

3. Sandwich ELISA

First, fix the captured antibody in the ELISA plate well, then add the sample, and finally add the detection antibody. If the detected antibody is an enzyme-linked immunosorbent assay (ELISA), it can be referred to as a direct sandwich ELISA; If the detection antibody does not have a label, it is necessary to use an enzyme-linked immunosorbent assay (ELISA) to bind with the detection antibody, which is called indirect sandwich ELISA.

The sensitivity of sandwich ELISA is high, and it is 2-5 times more sensitive than direct or indirect ELISA; At the same time, sandwich ELISA uses two specific antibodies to bind to antigens and has high specificity. In addition, sandwich ELISA can detect directly or indirectly, with high flexibility. The disadvantage of sandwich ELISA is that it requires high levels of paired antibodies. If there are no standardized test kits or paired antibodies that have already been tested, it is necessary to customize and optimize the paired antibodies, as reducing cross reactions between capture antibodies and detection antibodies is crucial.

4. Competitive ELISA method

Pre coat the antigen onto a solid-phase carrier and add enzyme labeled specific antibodies. During the experiment, the test antigen (or antibody) is added. If the test substance is an antigen, the test antigen competes with the antigen pre coated on the solid-phase carrier to bind to the enzyme-linked antibody; If the test substance is an antibody, the test antibody competes with the existing enzyme-linked antibody in the system to bind to the antigen encapsulated on the solid-phase carrier. Wash off the enzyme-linked antibodies that have been competitively bound by washing, and finally add substrate for color development. It should be noted that the color result is inversely proportional to the amount of antigen (or antibody) to be tested.

Competitive ELISA is relatively more complex compared to the three methods introduced above, but all three types of ELISA are applicable to the form of competitive ELISA. Its main advantages are the ability to detect impure samples and high data reproducibility, but there are issues with overall sensitivity and specificity.

Common Problems and Solutions of ELISA

1. Positive results appear in the negative control group

① Samples and reagents are contaminated, or improper handling during sample addition can cause cross contamination due to solution splashing between adjacent wells - replace reagents with caution.

② Incomplete washing of enzyme-linked immunosorbent assay (ELISA) plates - Before washing the plates, pour the antibody solution clean first, and then fill the plate holes with the cleaning solution to ensure thorough washing.

③ Excessive antibody dosage leads to non-specific binding - use the recommended amount of antibody according to the instructions and dilute the antibody to the appropriate concentration.

2. The overall background of the enzyme-linked immunosorbent assay (ELISA) plate is high

① Non specific binding of antibodies - it should be ensured that the plate wells are closed and appropriate blocking solution is used to prevent non-specific binding.

② Excessive substrate binding concentration - Dilute the substrate appropriately.

③ Reaction time too long - When the enzyme-linked immunosorbent assay (ELISA) plate has enough color development for absorbance reading, immediately use a stop buffer to terminate the reaction and shorten the color development time appropriately.

④ Substrate solution contamination - Normal substrate solution should be clear and transparent. If yellow or other colors appear, it indicates contamination and should be replaced with a new substrate solution.

⑤ The substrate incubation process is not light shielded - substrate incubation should be carried out under light shielded conditions.

3. Poor repeatability between multiple pores

① The sample quantity is not neat, and the sample addition time varies - try to keep the sample addition time as close as possible to the first time when increasing the pore size.

② Inconsistent sample volume - The sample should be thoroughly mixed before dilution and the same pipette should be used.

③ Inconsistent washing conditions and operators - When retesting samples, the operating conditions and personnel should be as consistent as possible with the last time.

4. High or low absorbance value

① Too low a content of the antigen to be tested in the sample can lead to low measurement results - you can try increasing the sample usage.

② Adding an inappropriate amount of antibodies can result in lower or higher results - use the recommended amount of antibodies or adjust the most suitable amount of antibodies as much as possible to improve the results.

③ The incubation time is too short, resulting in low detection results - appropriately extending the incubation time of antibodies or antigens to ensure that the test sample can fully bind to the detection antibody.

④ The incubation temperature is not suitable - the antibody should be incubated under the most suitable conditions (usually at 37 ℃ for 1 hour).