AxisPharm Services

Enzyme-linked immunosorbent assay (ELISA) classification methods and key points of operation

Enzyme-linked immunosorbent assay (ELISA or ELASA) refers to a qualitative and quantitative detection method that binds soluble antigens or antibodies to solid-phase carriers such as polystyrene, and uses antigen-antibody specific binding to carry out immunoreaction. Enzyme-linked immunosorbent assay (ELISA) is a classic experiment in immunology.

ELISA Principle

In 1971, Engvall and Perlmann published an article on the quantitative determination of IgG using enzyme-linked immunosorbent assay (ELISA), which made the enzyme-labeled antibody technology for antigen localization developed in 1966 to be used for the detection of trace substances in liquid samples. test methods.
The rationale for this approach is:
① Bind the antigen or antibody to the surface of a certain solid phase carrier and maintain its immunological activity.
② The antigen or antibody is linked with a certain enzyme to form an enzyme-labeled antigen or antibody, which retains both its immune activity and the activity of the enzyme.
During the measurement, the test sample (the antibody or antigen in it) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the surface of the solid phase carrier according to different steps. The antigen-antibody complex formed on the solid-phase carrier is separated from other substances by washing, and the amount of enzyme bound to the solid-phase carrier is proportional to the amount of the tested substance in the specimen. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme into a colored product, and the amount of the product is directly related to the amount of the tested substance in the sample, so it can be qualitatively or quantitatively analyzed according to the depth of the color reaction. Due to the high catalytic efficiency of the enzyme, the reaction effect can be greatly amplified, so that the assay method can achieve high sensitivity.

ELISA Classification

Commonly used ELISA can be divided into the following five categories: ①Direct ELISA; ②Indirect ELISA; ③Sandwich ELISA; ④Competitive ELISA; ⑤Competitive inhibition ELISA. Other ELISAs belong to or are derived from combinations of these five ELISAs.

1. direct ELISA

The method is to dilute the antigen according to a certain ratio and coat it on the solid phase carrier, then add the diluted specific enzyme-labeled antibody, and after incubation, add the substrate to develop the color and interpret the result. The operation of direct ELISA is very simple and the steps are relatively concise, but its application range is still very limited. An important reason is that this ELISA only undergoes one step of signal amplification (enzyme amplification), so its sensitivity is not very high; in addition, The object of its determination is also very limited, and it can only measure the molecules labeled with enzymes.

2. indirect ELISA

The difference between indirect ELISA and direct ELISA is that the non-enzyme-labeled antibody binds to the coated antigen in indirect ELISA, and a second antibody (ie, secondary antibody) is introduced. The secondary antibody is enzyme-labeled, which can specifically bind to the first antibody, and finally the substrate is added to develop the color and interpret the result. Since the secondary antibody is generally a polyclonal antibody, a primary antibody molecule can bind multiple secondary antibody molecules, and at the same time, a secondary antibody molecule can be labeled with multiple enzyme molecules, so when the antibody to be tested is a polyclonal antibody, the signal passes through The two-step amplification ultimately increases the sensitivity of the detection. In addition, since the preparation of the secondary antibody is relatively easy and commercialization has been started very early, the operator does not need to carry out the enzyme labeling of the primary antibody, which greatly reduces the workload. In the process of detecting antibody titer, serum titer and screening of monoclonal antibodies, indirect ELISA is a very important experimental process. In clinical diagnosis, indirect ELISA is also an important means of detecting marker antibodies.

3. Sandwich ELISA

Sandwich ELISA can be generally divided into two types, direct sandwich ELISA and indirect sandwich ELISA.
Direct sandwich ELISA is divided into double antibody sandwich ELISA and double antigen sandwich ELISA.
The method of double-antibody sandwich ELISA is to coat the first antibody (capture antibody) on a solid phase carrier, add the antigen to be detected after blocking, and add the second antibody (detection antibody) after incubation. The capture antibody and detection antibody can be It is two kinds of monoclonal antibodies against different epitopes, or one kind of monoclonal antibody and one kind of polyclonal antibody against the same antigen, but the detection antibody needs to be labeled with enzyme.
The principle and operation of double-antigen sandwich ELISA is basically the same as that of double-antibody sandwich ELISA.
For double-antibody sandwich ELISA, the object to be tested must include two or more epitopes, otherwise the detection antibody cannot bind to the antigen to be detected. For example, haptens and small molecule antigens cannot be detected by double-antibody sandwich ELISA. For double-antigen sandwich ELISA, the operation is basically the same as that of indirect ELISA, but the specific antigen is used instead of the enzyme-labeled secondary antibody, so the specificity is better than the indirect method. In addition, since the secondary antibody used in indirect ELISA generally only recognizes IgG, and any similar immunoglobulin can be detected in double antigen sandwich ELISA, double antigen sandwich ELISA is also more sensitive than indirect ELISA.
Indirect sandwich ELISA is a sandwich ELISA based on antibodies from two different species. After washing, add another species-specific antibody (non-enzyme-labeled, as a detection antibody), and finally add an enzyme-labeled secondary antibody (specifically recognize the detection antibody), and then add the substrate for color development. Compared with the direct double-antibody sandwich ELISA, the indirect sandwich ELISA introduces an enzyme-labeled secondary antibody that specifically recognizes the detection antibody, which is equivalent to a one-step amplification system for the signal of the entire system, so the final result is more sensitive than the direct double-antibody sandwich ELISA. . At the same time, because the enzyme-labeled secondary antibody in the indirect sandwich ELISA can only recognize the detection antibody, but not the capture antibody, the specificity of the system is also guaranteed.

4. Competitive ELISA

In this method, the specific antibody is first adsorbed on the surface of the solid phase carrier, and after washing, it is divided into two groups: one group adds the mixture of enzyme-labeled antigen and the tested antigen; the other group only adds the enzyme-labeled antigen, and then after incubation and washing Add substrate to develop color, and the difference between the two groups of substrate degradation amounts is the amount of unknown antigen to be determined. The antigen determined by this method only needs to have one binding site. Therefore, this method is commonly used for the determination of small molecule antigens such as hormones and drugs.

5. Competitive inhibition ELISA

The antigen is pre-coated on the solid phase carrier, and the diluted antigen (or antibody) to be detected is added during the experiment, and then the specific antibody and the enzyme-labeled secondary antibody corresponding to the antibody are added in turn. The antigen (or antibody) in the sample to be tested competes with the antigen (or antibody) bound on the solid phase carrier in the pre-preparation system for binding to the specific antibody (or the antigen bound on the solid phase carrier). The competing specific antibody is washed away, and finally the substrate is added to develop the color. The final color result is inversely proportional to the amount of antigen (or antibody) to be detected.

Experiment design ideas and basic steps

No matter what kind of ELISA, its operation is composed of the following basic operations: ①Adsorb antigen or antibody to the solid phase carrier; ②Add the sample to be tested and subsequent reagents; ③Incubate; ④Wash and remove Free unbound reactants; ⑤ Add enzymes to detect substrates; ⑥ Interpretation of results.

Sandwich method

The sandwich method is often used to detect macromolecular antigens. The general operation steps are:

The specific antibody is fixed (coating) on ​​the plastic well plate, and the excess antibody is washed off after completion;
Add the sample to be tested. If the sample contains the antigen to be tested, it will specifically bond with the antibody on the plastic well plate;
Wash off the excess sample to be tested, add another primary antibody specific for the antigen, and bond with the antigen to be tested;
Wash off excess unbonded primary antibody, add secondary antibody with enzyme, and bond with primary antibody;
Wash off excess unbound secondary antibody, add enzyme substrate to make the enzyme color, read the color result with naked eyes or instrument.

Indirect method

The indirect method is often used to detect antibodies. The general operation steps are:

Fix the known antigen on the plastic well plate, and wash off the excess antigen after completion;
Add the sample to be tested. If the sample contains the primary antibody to be tested, it will specifically bond with the antigen on the plastic well plate;
Wash off the excess sample to be tested, add secondary antibody with enzyme, and bond with the primary antibody to be tested;
Wash off the excess unbonded secondary antibody, add enzyme substrate to make the enzyme color, measure the absorbance value (OD value) in the plastic plate with an instrument (ELISA reader) to evaluate the content of the colored final product to measure the antibody to be tested content.

Competition law

The competition method is a less commonly used ELISA detection mechanism, which is generally used to detect small molecule antigens. The operation steps are as follows:

Fix the specific antibody on the plastic well plate, and wash off the excess antibody after completion;
Add the sample to be tested, so that the antigen to be tested in the sample is specifically bound to the antibody on the plastic well plate;
Add the antigen with enzyme, and this antigen can also be specifically bound to the antibody on the plastic well plate. Since the number of antibodies fixed on the plastic well plate is limited, when the amount of antigen in the specimen is more, the more The antigen with the enzyme has fewer fixed antibodies that can be bound, that is, both antigens compete to bind with the antibodies on the plastic well plate, which is the origin of the so-called competition method;
Wash away the sample and the antigen with the enzyme, and add the enzyme substrate to make the enzyme color. When the amount of antigen in the sample is more, it means that the less antigen with the enzyme is left in the plastic well plate, and the color will be stronger. shallow.

When it is necessary to detect antigens for which more than two unique antibodies cannot be obtained, or it is difficult to obtain enough purified antibodies to immobilize on the well plate, the use of competition ELISA is generally considered.


Avidin is a glycoprotein, and one molecule of avidin can have a specific affinity with four biotin small molecules, similar to the affinity of antigen and antibody. The force between them is the strongest known non-covalent interaction. After the 1980s, with the discovery of the biotin-avidin system (BAS), this affinity was applied to ELISA technology, which greatly improved the sensitivity and specificity of the latter reaction. Biotin is easily covalently bound to proteins (such as antibodies, etc.). In this way, the avidin molecule combined with the enzyme reacts with the biotin molecule combined with the specific antibody, which not only plays a multi-stage amplification effect, but also changes color due to the catalytic effect of the enzyme when it encounters the corresponding substrate, achieving detection. The purpose of the unknown antigen (or antibody) molecule.

Result Judgment

Qualitative determination

The result of qualitative determination is to give a simple answer of “yes” or “no” to whether the tested sample contains the antigen or antibody to be tested, which is expressed as “positive” and “negative” respectively. “Positive” indicates that the specimen responded in the assay system. “Negative” means no response. Semi-quantitative results can also be obtained by the qualitative judgment method, that is, the titer is used to express the intensity of the reaction, and its essence is still a qualitative test. In this semi-quantitative assay, the sample is tested after a series of dilutions, and the highest dilution that gives a positive reaction is the titer. According to the titer, the reactivity of the sample can be judged, which is more quantitatively meaningful than judging the strong positive and weak positive by observing the color depth of the undiluted sample.
In indirect and sandwich ELISA, positive wells were darker than negative wells. In a competitive ELISA, on the contrary, negative wells are darker than positive wells.

Quantitative determination

The ELISA operation steps are complex, and there are many factors that affect the reaction. In particular, the coating of the solid-phase carrier is difficult to achieve consistency between individuals. Therefore, in the quantitative determination, a series of reference standards with different concentrations must be used for each batch of tests. A standard curve was made under the conditions. Sandwich ELISA for the determination of large molecular weight substances, the range of the standard curve is generally wide, and the absorbance at the highest point of the curve can be close to 2.0. The semi-log value is often used for drawing, taking the concentration of the test substance as the abscissa and the absorbance as the ordinate. The concentration values ​​are connected point by point, and the obtained curve is generally S-shaped, the head and tail curves tend to be flat, and the central part that is more straight is the most ideal detection area.
Determination of small molecular weight substances commonly used competition method, the absorbance in the standard curve is negatively correlated with the concentration of the tested substance. The shape of the standard curve varies slightly depending on the mode used in the kit. Note that the abscissa in the standard curve of the ELISA assay is a logarithmic relationship, which is more conducive to the expression of the assay system.


1. Very light or no color

(1) Missing addition of substrate components, substrate failure, and incorrect calculation of substrate preparation concentration;
(2) There is an antigen or antibody mismatch during the entire ELISA process;
(3) Excessive dilution of the sample during the entire ELISA process;
(4) The dilution system is wrong, such as using a protein-containing reagent for coating.

2. Full plate positive

(1) The concentration of enzyme-labeled antigen or antibody is too high, try to reduce the concentration;
(2) The blocking reagent used is incorrect or not blocked;
(3) The enzyme-labeled antigen or antibody is bound to other reagents in the system;
(4) The substrate is contaminated with enzyme-labeled antigen or antibody.

3. uneven color development

(1) If there is a quality problem of the ELISA plate, re-check the homogeneity of the ELISA plate;
(2) In the process of coating or sample addition, some holes are leaked or added less, which may be because the operator is not careful, or the pipette leaks;
(3) Too many bubbles are injected into the pipette when adding samples;
(4) The purchased ELISA plate is incorrect, and the plate for other purposes may be selected by mistake;
(5) The plates are stacked too thick during the incubation;
(6) The reagents are not fully mixed during the process of sample addition or dilution, or the dilution gradient is calculated incorrectly;
(7) The plate was washed incorrectly, some wells were not filled with washing liquid or the washing liquid was not mixed well after adding detergent, or air bubbles were brought into the washing process.

4. Color is too fast

(1) The concentration of enzyme-labeled antigen or antibody is too high;
(2) The concentration of a certain reagent is too high.

5. Color is too slow

(1) The concentration of enzyme-labeled antigen or antibody is too low, or the labeling efficiency is low, or the immune activity is affected after labeling;
(2) The reagent is contaminated, such as HRP enzyme-labeled antigen or antibody is contaminated with sodium azide;
(3) The reaction temperature is too low;
(4) The pH of the substrate buffer is incorrect.

6. dark background

(1) The concentration of enzyme-labeled antigen or antibody is too high;
(2) Non-specific binding of antibodies;
(3) Impure antigen or antibody;
(4) Species cross-recognition of secondary antibodies.

In Vitro Diagnostic (IVD) Research and Development