Brief Introduction of ELISA Principles
ELISA( Enzyme linked immunosorbent assay) is a test in which a certain concentration of antibody or antigen is coated on the microwells surface of a polystyrene microtiter plate through physical absorption method. Then unknown samples are added to indicate the presence of substance or the amount of the substance according to the color change in the substrate. As one type of the immunolabelling techniques, the main features of ELISA are its rapidity(sensitivity), qualitative analysis, rapid quantitative analysis or even telltale mark. Now it has found its wide application in research and clinic experiment.
The most commonly used methods of ELISA are double antibody sandwich method, competitive method, indirect method, double antigen sandwich method, capturing method. The following texts will explain these methods in detail.
1. Double antibody sandwich method
The basic principle of the method is that an amount of coated antibody is immobilized on the microwells of a polystyrene microtiter plate through physical absorption method. After add ing non-targeted protein to block unbinded sites.， add unknown samples , detecting antibody and enzyme-linked secondary antibody in sequence, the TMB substrate exhibits color. After the reaction is terminated, the stronger color it exhibits, the higher OD value will be. Thus, it signals the higher concentration of the sample.
This method applies to macromolecular substances with multiple antigen epitopes. Since this method needs two kinds of antibody, it involves the match of antibodies. Generally, there are three common ways of match: 1. The coated antibody is monoclonal antibody, while the detection antibody is polyclonal antibody. 2. The coated antibody is monoclonal antibody, while the detection antibody is also monoclonal antibody. However, these two monoclonal antibodies target different antigen epitopes. 3. The coated antibody is polyclonal antibody, while the detection antibody is also polyclonal antibody. In general, these two antibodies are from different species
when the method is applied in the enzyme labeled secondary antibody, we should pay attention to one principle.:the secondary antibody must only target detection antibody, but not coated antibody. considering the limitation of the enzyme labeled secondary antibody, most manufacturers introduce Biotin-Avidin—System. The system not only contributes to the enhancement of reaction sensitivity but also brings convenience.
Instead of marking each of heterogeneous secondary antibody through enzyme labeling, we
just apply biotin labeling to detecting antibody and HRP labeling to avidin. Therefore, the whole process can be simplified.
2. Competitive method
Competitive method can be divided into direct competitive method and indirect competitive method. Here we explain the procedure of direct competitive method. 1. Immobilize a certain amount of coated antibody on the microwells of a polystyrene microtiter plate. 2. Add irrelative protein carrier to block unbinded sites. 3. Add unknown samples and allow them to compete for binding with biotin-labeled antigen. 4.Wash the plate after it is given time to incubate at appropriate temperature.5. Add HRP labeled streptavidin for reaction and the TMB substrate will show chromogenic signal. The concentration of the samples is then determined by comparing the O.D. of the samples to the standard curve
The method is often used to detect micro-molecule substance with fewer epitopes. It can also be used to detect macromoleculed antigen or even antibody.when we detect the macromoleculed antigen, the stereo-hindrance effect will result in a lower sensitivity compared with the double antibody sandwich method. Due to the heterogeneous competitive antibodies, it may give rise to lower homoplasy and less reliable testing results.
3. Indirect method
Indirect method is generally used to detect antibody. The procedure is explained as follows: 1. Coat a certain amount of antigen on the microwells of a polystyrene microtiter plate. 2.Add irrelative protein carrier to block unbinded sites. 3.Add unknown samples and enzyme-labeled secondary antibody. 4.After incubating and washing, add TMB substrate to show chromogenic signal. Generally, due to the limitation of the enzyme-labeled secondary antibody, the detected antibody is the total IgG.
4. Double antigen sandwich method
Double antigen sandwich method is similar to the double antibody sandwich method. The procedure is explained as follows: 1.Coat a certain concentration of antigen on the microwells of a polystyrene microtiter plate. 2.Add irrelative protein carrier to block the unbinded sites. 3.Add biotin-labeled antigen and HRP-labeled streptavidin. 4.After adding the substrate solution and stop solution, the sample concentration can be calculated according to the data from microplate reader. Double antigen sandwich method and indirect method both can detect antibody. But the former targets to all variations of antibody, including IgG, IgM, IgA，IgD, IgE. This can’t be achieved by indirect method.
5. Capturing method
Capturing method is often used to detect IgM antibodies. The procedure is explained as follows:
1.Coat the anti- IgM antibody on solid phase of a polystyrene microtiter plate.
2.Add irrelative protein carrier to block the unbinding sites.
3.Add unknown samples and antigen.
4.Add specific and biotinylated antibody which targets to the antigen, and then add HRP-labeled streptavidin
5. At last add the substrate solution and stop solution, the sample concentration can be measured ,Since high concentration of IgG antibody coexists with IgM in the serum, IgG and IgM will compete to bind with antigen on the solid phase As IgG largely outnumbers the IgM, the igM that can be conjugated successfully is very few. Therefore, it will result in a false-negative.
The most commonly used methods of ELISA are double antibody sandwich method, competitive method, indirect method, double antigen sandwich method, capturing method. The following texts will explain these methods in detail.
1. Double antibody sandwich method
The basic principle of the method is that an amount of coated antibody is immobilized on the microwells of a polystyrene microtiter plate through physical absorption method. After add ing non-targeted protein to block unbinded sites.， add unknown samples , detecting antibody and enzyme-linked secondary antibody in sequence, the TMB substrate exhibits color. After the reaction is terminated, the stronger color it exhibits, the higher OD value will be. Thus, it signals the higher concentration of the sample.
This method applies to macromolecular substances with multiple antigen epitopes. Since this method needs two kinds of antibody, it involves the match of antibodies. Generally, there are three common ways of match: 1. The coated antibody is monoclonal antibody, while the detection antibody is polyclonal antibody. 2. The coated antibody is monoclonal antibody, while the detection antibody is also monoclonal antibody. However, these two monoclonal antibodies target different antigen epitopes. 3. The coated antibody is polyclonal antibody, while the detection antibody is also polyclonal antibody. In general, these two antibodies are from different species
when the method is applied in the enzyme labeled secondary antibody, we should pay attention to one principle.:the secondary antibody must only target detection antibody, but not coated antibody. considering the limitation of the enzyme labeled secondary antibody, most manufacturers introduce Biotin-Avidin—System. The system not only contributes to the enhancement of reaction sensitivity but also brings convenience.
Instead of marking each of heterogeneous secondary antibody through enzyme labeling, we
just apply biotin labeling to detecting antibody and HRP labeling to avidin. Therefore, the whole process can be simplified.
2. Competitive method
Competitive method can be divided into direct competitive method and indirect competitive method. Here we explain the procedure of direct competitive method. 1. Immobilize a certain amount of coated antibody on the microwells of a polystyrene microtiter plate. 2. Add irrelative protein carrier to block unbinded sites. 3. Add unknown samples and allow them to compete for binding with biotin-labeled antigen. 4.Wash the plate after it is given time to incubate at appropriate temperature.5. Add HRP labeled streptavidin for reaction and the TMB substrate will show chromogenic signal. The concentration of the samples is then determined by comparing the O.D. of the samples to the standard curve
The method is often used to detect micro-molecule substance with fewer epitopes. It can also be used to detect macromoleculed antigen or even antibody.when we detect the macromoleculed antigen, the stereo-hindrance effect will result in a lower sensitivity compared with the double antibody sandwich method. Due to the heterogeneous competitive antibodies, it may give rise to lower homoplasy and less reliable testing results.
3. Indirect method
Indirect method is generally used to detect antibody. The procedure is explained as follows: 1. Coat a certain amount of antigen on the microwells of a polystyrene microtiter plate. 2.Add irrelative protein carrier to block unbinded sites. 3.Add unknown samples and enzyme-labeled secondary antibody. 4.After incubating and washing, add TMB substrate to show chromogenic signal. Generally, due to the limitation of the enzyme-labeled secondary antibody, the detected antibody is the total IgG.
4. Double antigen sandwich method
Double antigen sandwich method is similar to the double antibody sandwich method. The procedure is explained as follows: 1.Coat a certain concentration of antigen on the microwells of a polystyrene microtiter plate. 2.Add irrelative protein carrier to block the unbinded sites. 3.Add biotin-labeled antigen and HRP-labeled streptavidin. 4.After adding the substrate solution and stop solution, the sample concentration can be calculated according to the data from microplate reader. Double antigen sandwich method and indirect method both can detect antibody. But the former targets to all variations of antibody, including IgG, IgM, IgA，IgD, IgE. This can’t be achieved by indirect method.
5. Capturing method
Capturing method is often used to detect IgM antibodies. The procedure is explained as follows:
1.Coat the anti- IgM antibody on solid phase of a polystyrene microtiter plate.
2.Add irrelative protein carrier to block the unbinding sites.
3.Add unknown samples and antigen.
4.Add specific and biotinylated antibody which targets to the antigen, and then add HRP-labeled streptavidin
5. At last add the substrate solution and stop solution, the sample concentration can be measured ,Since high concentration of IgG antibody coexists with IgM in the serum, IgG and IgM will compete to bind with antigen on the solid phase As IgG largely outnumbers the IgM, the igM that can be conjugated successfully is very few. Therefore, it will result in a false-negative.
