What Is an Enzyme-Linked Immunosorbent Assay?

Enzyme-linked immunosorbent assay refers to the use of the characteristics of antibody molecules that can specifically bind to antigen molecules, combining free heteroproteins with target proteins bound to a solid-phase carrier, and qualitatively or quantitatively analyzing them with special labels. A detection method. The principle is: the antigen or antibody can be physically adsorbed on the solid phase surface and maintain its immune activity; the antigen or antibody can form an enzyme conjugate with the enzyme through a covalent bond, while maintaining the respective immune activity or enzyme activity; enzyme binding After binding a substance to the corresponding antigen or antibody, the color reaction of the substrate can be used to determine the occurrence of an immune response, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen. ^[1]

Enzyme-linked immunosorbent assay

Make the antigen or antibody bind to the surface of a certain solid carrier and keep it

ELISA can be used to determine antigens and antibodies. This method is suitable for the determination of samples in cell culture supernatant, serum, plasma and interstitial fluid, and the level of cytokine (or receptor) in nanograms per milliliter can be measured with small interference. There are three necessary reagents in this assay: solid phase antigen or antibody, enzyme-labeled antigen or antibody, and substrate for enzyme action. Depending on the source of the reagents and the characteristics of the specimen and the conditions for the detection, various types of detection methods can be designed.

Double antibody sandwich

The double antibody sandwich method is the most commonly used method for detecting antigens, and the steps are as follows:

(1) Connect the specific antibody to the solid phase carrier to form a solid phase antibody: wash to remove unbound antibody and impurities.

(2) Block with skimmed milk powder or BSA. Wash to remove excess skim milk or BSA.

(3) Add the test specimen: contact the solid-phase antibody for a period of time to allow the antigen in the specimen to bind to the antibody on the solid-phase carrier to form a solid-phase antigen complex. Wash to remove other unbound material.

(4) Add enzyme-labeled antibody: bind the antigen on the solid-phase immune complex with the enzyme-labeled antibody. Wash unbound enzyme-labeled antibodies thoroughly. At this time, the amount of the enzyme carried on the solid-phase carrier is positively related to the amount of the test substance in the specimen.

(5) Substrate: The enzyme in the sandwich complex catalyzes the substrate to become a colored product. Depending on the degree of the color response, the antigen is qualitatively or quantitatively quantified.

According to the same principle, the solid antigen and the enzyme-labeled antigen conjugate are prepared separately from the macromolecular antigen, and the antibody in the specimen can be determined by the dual antigen sandwich method.

Two-site one-step method

In the double-antibody sandwich method for determining antigens, if monoclonal antibodies against two different epitopes on the antigen molecule are used as solid-phase antibodies and enzyme-labeled antibodies, respectively, the addition of specimens and enzyme-labeled antibodies can be performed during the assay. Two steps and one step (Figure 15-5). This double-site not only simplifies the operation and shortens the reaction time in one step. For example, the use of high-affinity monoclonal antibodies can significantly improve the sensitivity and specificity of the assay. The use of monoclonal antibodies has raised the level of ELISA for antigen detection to a new level.

In the one-step measurement, attention should be paid to the hook effect, which is similar to the phenomenon of excess banding of antigen in the precipitation reaction. When the concentration of the test antigen in the specimen is quite high, the excess antigen is bound to the solid-phase antibody and the enzyme-labeled antibody, respectively, without forming a sandwich complex, and the result obtained will be lower than the actual content. False negative results can even occur when the hook effect is severe.

Indirect detection of antibodies

The indirect method is the most commonly used method for detecting antibodies. Its principle is to use an enzyme-labeled anti-antibody to detect the test antibody that has bound to the solid phase, so it is called the indirect method. The steps are as follows:

(1) Connect the specific antigen to the solid phase carrier to form a solid phase antigen: wash to remove unbound antigen and impurities.

(2) Diluted test sera: the specific antibodies are bound to the antigen to form a solid phase antigen-antibody complex. After washing, only specific antibodies remain on the solid support. Impurities in other immunoglobulins and serum are washed away during washing because they cannot bind to the solid phase antigen.

(3) Enzyme-labeled anti-antibody: binds to the antibody in the solid phase complex, so that the antibody is indirectly labeled with the enzyme. After washing, the amount of enzyme on the solid support represents the amount of specific antibodies. For example, to test human antibodies to a certain disease, an enzyme-labeled goat anti-human IgG antibody can be used.

(4) Substrate-based color development: The color depth represents the amount of test antibody in the specimen.

As long as this method replaces different solid-phase antigens, an enzyme-labeled anti-antibody can be used to detect various antibodies corresponding to the antigens.

Competition law

Competitive methods can be used to determine antigens as well as antibodies. Taking the determination of the antigen as an example, the test antigen and the enzyme-labeled antigen compete with the solid-phase antibody for binding, so the amount of the enzyme-labeled antigen bound to the solid phase is inversely proportional to the amount of the test antigen. The steps are as follows:

(1) Connect the specific antibody to the solid phase carrier to form a solid phase antibody. washing.

(2) Add a mixed solution of the test specimen and a certain amount of enzyme-labeled antigen to the test tube to make it react with the solid-phase antibody. If there is no antigen in the test specimen, the enzyme-labeled antigen can smoothly bind to the solid-phase antibody. If the test specimen contains an antigen, it will bind to the solid-phase antibody at the same opportunity as the enzyme-labeled antigen, competitively taking up the opportunity of the enzyme-labeled antigen to bind to the solid-phase carrier, and binding the enzyme-labeled antigen to the solid-phase carrier The amount is reduced. Only the enzyme-labeled antigen is added to the reference tube. After incubation, the binding of the enzyme-labeled antigen to the solid-phase antibody can reach the most sufficient amount. washing. (3) Substrate coloration: The reference tube is the darkest because it has the most enzyme-labeled antigen bound. The difference between the color depth of the reference tube and the color depth of the tube to be tested represents the amount of antigen in the test specimen. The lighter the color of the tube to be tested, the more antigen content in the specimen.

Capture IgM antibody

Specific IgM and certain IgGs for certain antigens are often present in the serum, the latter will interfere with the determination of IgM antibodies. Therefore, to determine the IgM anti-multiple capture method, all serum IgM (including heterosexual IgM and non-specific IgM) were first fixed on the solid phase, and the specific IgM was determined after removing IgG. The steps are as follows:

(1) The anti-human IgM antibody is connected to a solid-phase carrier to form a solid-phase anti-human IgM. washing.

(2) Diluted serum samples were added: IgM antibodies in serum were captured by solid-phase antibodies after incubation. Wash to remove other immunoglobulins and serum impurities.

(3) Add specific antigen reagent: it only binds to specific IgM on the solid phase. washing.

(4) Add a specific enzyme-labeled antibody: make it react with the antigen bound to the solid phase to bind. washing.

(5) Substrate coloration: If there is a color, it means that the specific IgM antibody in the serum sample is present, which is a positive reaction.

ELISA with avidin and biotin

Avidin is a glycoprotein that can be extracted from egg whites. With a molecular weight of 60kD, each molecule consists of 4 subunits and can be intimately combined with 4 biotin molecules. Streptavidin extracted from Streptomyces is used more now. Biotin, also known as vitamin H, has a molecular weight of 244.31 and is found in egg yolk. Derivatives made by chemical methods, biotin-hydroxysuccinimide (BNHS) can form biotinylated products with many types of molecules, including proteins, sugars and enzymes. Although the binding of avidin and biotin is not an immune response, it has strong specificity and high affinity. Once the two are combined, they are extremely stable. Since one avidin molecule has four binding sites for biotin molecules, more biotinylated molecules can be connected to form a lattice-like complex. Therefore, coupling avidin and biotin with ELISA can greatly improve the sensitivity of ELISA.

The avidin-biotin system is used in ELISA in various forms, which can be used for indirect coating or for final reaction amplification. Avidin can be pre-coated on the solid phase. The antibody or antigen that was originally coated on the solid phase by adsorption method is combined with biotin, and the biotinylated antibody or anti-phase is made through the avidin-biotin reaction. . This coating method can not only increase the amount of antibody or antigen adsorbed, but also fully expose its binding site. In addition, the enzyme-labeled antibody in a conventional ELISA can also be replaced with a biotinylated antibody, and then the avidin-enzyme conjugate is connected to amplify the reaction signal.

ELISA is commonly used as a non-radioactive isotope bonding assay. In this method, standard ligands are usually immobilized, and they are bonded by adding a solution-phase receptor or protein. By adding antibodies that specifically react with the receptor To quantify the binding receptors, and the amount of the initial antibody is measured by adding a second antibody that can develop color. The second antibody can recognize the end of the antibody, the alkaline phosphate or peroxidase at the end and The enzyme reacts, causing the solution to develop a color.