All you need to know about the ELISA experiment is here. ELISA, the full name of enzyme-linked immunosorbent assay, is a commonly used immunoassay method;
The known antigen or antibody is adsorbed on the surface of the solid phase carrier, the antigen or antibody is labeled with enzyme, and the antigen or antibody is incubated on the solid phase surface, and the unknown antibody or antigen in the liquid is detected by adding substrate luminescence and color.
Highly specific: maintain the specificity of the antigen-antibody reaction
High sensitivity: high efficiency of enzyme-catalyzed substrate color development, pg level trace detection
Qualitative and quantitative detection: the color depth or optical density value of the reaction solution
ELISA types include direct method, indirect method, double antibody sandwich method and competition method;
The so-called direct and indirect refer to whether the enzyme-labeled antibody is introduced “directly” or “indirectly”. Direct ELISA means that the antigen is coated on a solid carrier, the enzyme-labeled antibody is added after blocking, the unbound enzyme-labeled antibody is washed away, and the substrate is added to develop color. The color depth is proportional to the amount of coated antigen and the amount of enzyme-labeled antibody. The so-called “direct” refers to adding the enzyme-labeled antibody to the solid-phase carrier, directly adding the substrate to develop the color, and directly refers to whether the process of introducing the enzyme is “direct”. This is the simplest ELISA, often used for the detection of antigen activity or the titer and quality of labeled antibodies.
The difference between indirect ELISA and direct ELISA is that after the antigen is coated on the solid carrier, the enzyme-labeled antibody is not directly added, but the antibody is added first, and then the enzyme-labeled antibody (enzyme-labeled secondary antibody) is added after washing, and then the substrate is added to the substrate after washing. color. After the introduction of the enzyme-labeled secondary antibody, the detection signal will be amplified dozens of times.
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Lindström and Wager published the ELISA indirect method for the first time in 1978. Compared with the ELISA direct method, it uses an enzyme-labeled secondary antibody for detection (Figure 2), because the primary antibody has multiple epitopes that can bind to it. Improved the detection sensitivity and accuracy, just like in a dark room, the ELISA direct method has only one 50W bulb, while the ELISA indirect method can connect two 50W bulbs in parallel, so the brightness of the room is different, the ELISA indirect method The difference between the operation and the direct method of ELISA is the addition of secondary antibody incubation.
The first step is also to passively adsorb the antigen to the surface of the multiwell plate, wash and seal the excess area, add the analysis sample containing the antibody to be tested, incubate until the specific binding is complete, then wash and incubate the secondary antibody, and finally add the substrate Perform color reaction detection (Figure 4), so this method is mostly used for quantitative analysis of antibodies. It is very obvious that the specificity of the reaction is improved compared to the direct method, and it is also more economical, because it is not necessary to design an enzyme-labeled antibody for each specific antigen. Of course, the introduction of a second antibody will also bring about cross-activity. This may cause some background noise.
Sandwich ELISA is to put the object to be tested in the middle, and the double antibody sandwich method is the most widely used. The solid-phase carrier is coated with an antibody (Capture Antibody), and washed after blocking, adding antigen to react, and then adding another antibody (Detection Antibody) after washing. The detection antibody needs to be labeled with enzyme. The antigen in the double antibody sandwich method should contain at least 2 or more epitopes.
The antibody against the small molecule antigen is coated on the ELISA plate, and the sample to be tested is added during detection, so that the antigen to be tested in the sample is combined with the antibody on the ELISA plate. Then add the HRP enzyme-labeled antigen, this antigen can also be combined with the antibody on the ELISA plate, because the number of antibodies fixed on the ELISA plate is limited, so when the amount of antigen in the sample is more, the HRP enzyme-labeled antigen can bind The less the coating antibody is, the two antigens compete to bind the coating antibody, so it is called the competition method.
As we all know, the direct method of ELISA only requires one detection antibody, while the detection process of the indirect ELISA method is divided into two steps, incubating twice, and two antibodies are required to complete the specific binding. Now let’s take a look at the overall method. The difference helps us better understand and use.
Table 1 The difference between the direct method and the indirect method of ELISA:
| ELISA direct method | ELISA indirect method | |
| Ø Comparison of method sensitivity | Low sensitivity | Higher sensitivity |
| Ø Time consumption | Less steps and less time consumption | There are many steps and need to incubate the secondary antibody for a long time |
| Ø Antibody usage | Only one type of antibody is required | Requires primary and secondary antibodies |
| Ø Enzyme-labeled antibody | Enzyme linked to primary antibody | Enzyme linked to secondary antibody |
| Ø Cross activity | Only one antibody, no cross activity | There is cross-activity, which will increase background noise |
| Ø Signal | Relatively weak signal | The detection signal is amplified and the signal is high |
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