Enzyme-linked immunosorbent assay, also known as ELISA, is the most widely used technique in enzyme immunoassay technology. In 1971, Engvall et al. published an article on the quantitative measurement of IgG using ELISA, which developed the enzyme-labeled antibody technique used for antigen localization in 1966 into a method for quantifying trace substances in liquid samples.
The principle of ELISA
The principle of ELISA involves immobilizing an antigen or antibody on a solid-phase carrier, utilizing the specific binding between antigen and antibody. During the detection process, unbound substances are typically removed by washing the plate, and the color of the substrate after enzymatic reaction is used for qualitative and quantitative analysis of proteins in the sample.
Common main reagents used in ELISA
1. Antibodies: Monoclonal antibodies have strong specificity, while polyclonal antibodies exhibit high binding affinity. High-end assay kits require efficient pairing.
2. Antigens: Mostly recombinant proteins, cytokines, and test samples. Efficient standard materials require calibration by NIBSC/WHO.
3. Chromogenic systems: Initially, horseradish peroxidase (HRP) forms a chromogenic system with common substrates like TMB and OPD. Additionally, alkaline phosphatase (AP) utilizes p-nitrophenyl phosphate as a substrate to produce a yellow color. Glucose oxidase (GOD) generates a specific color through the glucose oxidase method. Finally, β-D-galactosidase (β-D-Gal) utilizes 4-methylumbelliferyl-β-D-galactopyranoside (4MUG) as a substrate to generate high-intensity fluorescence.
Figure 1: Schematic Diagram of Common Reagents Used in ELISA
Types of ELISA
According to the source of reagents, the nature of the samples, and the specific conditions of the assay, various types of detection methods can be designed. Among the most commonly used methods in the market, based on principles and operations, are:
1. Indirect ELISA
This method is most commonly used for antibody detection. The principle involves using an enzyme-labeled secondary antibody to detect the primary antibody bound to the solid phase, followed by colorimetric detection. The advantage is that the primary antibody being tested does not need to be labeled, making the process rapid and convenient. Direct ELISA is similar to indirect ELISA, but with the primary antibody labeled with an enzyme. The detection incubation also primarily involves the antigen. However, direct ELISA has limited sensitivity compared to indirect ELISA and requires careful consideration of its use.
Figure 2: Schematic Diagram of Indirect ELISA
2. Sandwich ELISA
It is further divided into Double Antigen Sandwich ELISA and Double Antibody Sandwich ELISA, with similar principles used to detect antibody or antigen levels. Taking Double Antibody Sandwich ELISA as an example, the captured antibody is coated on a solid-phase carrier, and the antigen or test sample is added to bind. Subsequently, the detection antibody, which binds to the antigen, is added to form a sandwich structure. Double Antibody Sandwich ELISA is the most common and convenient method used in the market for analysis.
Figure3：Double Antibody Sandwich ELISA Diagram
3. Competitive ELISA
Competitive ELISA is commonly used for the detection of small molecule antigens and haptens. The more antigen present in the sample, the less enzyme-labeled antigen is bound, leading to a competitive negative correlation system. As a result, there will be fewer colorimetric substances. The antigen content in the sample can be fitted based on the ratio of colorimetric substances.
Figure 4：Competitive ELISA Schematic
4.Other methods
a. Immunoinhibition ELISA: The degree of inhibition of color development of the substrate by the test sample in ELISA is directly proportional to the amount of antigen contained in the sample. The difference between the two is the amount of antigen to be tested, similar to competitive ELISA.
b. Blocking ELISA: Also known as competitive ELISA, this method is used to detect specific antibodies. The principle involves antigen coating on a solid phase, followed by competition incubation between the test sample and enzyme-labeled primary antibody. The more antibodies present in the test sample, the lighter the color development, and vice versa. This method is involved in the African Swine Fever Virus antibody detection kit.
c. Additionally, there are other ELISA variations including antibody capture ELISA for detecting IgM antibodies, Dot-ELISA (using nitrocellulose membrane) and C-ELISA (using polyester fabric) which involve modifications in the solid phase carrier, as well as technical classifications such as TRFIA and multiplex detection methods.
Assessment of ELISA
The quality of an ELISA kit is determined by multiple indicators. Excellent kits require evaluation of the following six major criteria, as analyzed below:
1. Accuracy
a. Recovery Rate: Calibration of the kit's data accuracy. Abnormally high or low rates may lead to inaccuracies in sample analysis.
b. Linearity: Accuracy of the kit's dilution solution and sample microenvironment. Deviations from the expected values can indicate inaccuracies.
2. Precision
a. Intra-Assay Precision: Evaluation of differences between replicate wells of the same known concentration in a single experiment.
b. Inter-Assay Precision: Evaluation of differences between multiple experiments of the same sample.
3. Sensitivity
Lower Limit of Detection: The minimum signal value that significantly distinguishes the blank signal, indicating the kit's sensitivity. Lower values indicate higher sensitivity.
4. Specificity
a. Cross-Reactivity: Assessment of specificity by measuring the binding ability of different molecules to the kit's antibodies. Higher cross-reactivity indicates poorer specificity.
b. Anti-Interference Ability: Evaluation of interference from homologous substances and endogenous substances on the detection system.
5. Natural Sample Detection
Analysis of detection of natural samples and appropriate sample types. Determination of sample types to avoid false positive or false negative results.
6. Stability
The shelf life and stability of the kit are crucial for ensuring accurate detection. Higher stability allows for longer storage time of the kit.
Figure 5: Classification of ELISA assay kit products
The application of ELISA
The ELISA technique is the gold standard for quantifying specific target proteins, providing rapid, stable, and analytically accessible results. It can be applied to the quantitative analysis of biomolecules/small molecules, evaluation or screening of affinity, and comparative analysis of the activity of recombinant proteins or cytokines. It is commonly used in the detection of cytokines, chemokines, growth factors, and other targets, and is also applied to targets in research areas such as cancer, stem cells, immunology, neurology, and signal transduction.
Figure 6: Technical applications of ELISA
In biomedical research, bacterial diseases, and clinical diagnostics, ELISA serves as a mature immunological detection method for quantitatively measuring the desired substances such as cytokines and antigens. Its application is very extensive. Understanding ELISA and selecting the appropriate assay kit can greatly streamline research efforts and yield significant results.
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