Enzyme immunoassay (EIA) is an immunoassay technique that combines the specificity of antigen-antibody reaction with the specificity of enzyme-catalyzed reaction. It is a combination of enzyme and antigen or antibody synthetase to label the antigen or antibody, and after the specific reaction between the enzyme labeled antigen or antibody and the corresponding antibody or antigen is completed, the corresponding substrate of the enzyme is added, and the antigen or antibody is located, qualitative and quantitative determination and analysis through the chromogenic reaction between the enzyme and the substrate. The commonly used enzymes are horseradish peroxidase (HRP), alkaline phosphatase (ALP) and β-galactosidase (β-Gal).
As a classic immunolabeling technique, enzyme immunoassay has the advantages of high sensitivity, strong specificity, good accuracy, long validity period of enzyme markers, low reagent price and simple operation. In recent years, with the development of monoclonal antibody technology, chemiluminescence technology, biotin-avidin technology and other related technologies, enzyme immunoassay technology has been continuously improved and updated, and its sensitivity, specificity and automation have been further improved, and its application in the field of medicine and biology has been increasingly extensive.
According to whether bound enzyme markers and free enzyme markers need to be separated during the reaction process, there are two types of homogeneous enzyme immunoassays and heterogeneous enzyme immunoassays. The homogeneous enzyme immunoassay mainly includes enzyme amplification immunoassay and clonal enzyme donor immunoassay.
According to whether solid material is used to adsorb antigens or antibodies, heterogeneous enzyme immunoassay can be divided into liquid phase enzyme immunoassay and solid phase enzyme immunoassay. The representative of solid phase enzyme immunoassay is enzyme-linked immunosorbent assay (ELISA).
Bioconjugation refers to the process of chemically linking biomolecules, such as proteins, antibodies, or enzymes, to enhance their functionality and specificity in various applications, including EIA. The effectiveness of these assays relies heavily on the successful bioconjugation of enzymes to antibodies or other biomolecules, which is crucial for signal generation and amplification.
Antibodies: In EIAs, antibodies serve as the primary recognition element, binding specifically to the target analyte. The bioconjugation of enzymes to antibodies enables the conversion of a non-visual binding event into a measurable signal.
Enzymes: Commonly used enzymes include horseradish peroxidase (HRP) and alkaline phosphatase (AP), known for their high sensitivity and rapid kinetics. The requirements of the enzyme used for labeling: High purity, strong activity, and high catalytic reaction efficiency. It is easy to bind antigen and antibody, and the enzyme activity is maintained after labeling, which does not affect the reactivity of antigen and antibody. Specificity, the enzyme activity is not affected by the composition of the sample, and there is no endogenous enzyme or corresponding inhibitor of the same as the labeled enzyme in the tested tissue or body fluid. Homogeneous immunoassay requires that the enzyme activity is changed after the antibody binds to the enzyme labeled antigen. The product after the enzyme catalyzed substrate is easy to judge or measure, and the method is simple, sensitive and repeatable. Enzymes, cofactors and their substrates are harmless to the human body and the environment, enzyme substrates are easy to configure and preserve, and enzymes and substrates are cheap and easy to obtain.
Linkers: The choice of linker is critical in bioconjugation, as it can affect the stability, activity, and orientation of the enzyme-antibody complex. Linkers can be homobifunctional or heterobifunctional, enabling the selective attachment of enzymes to specific sites on antibodies. Common linkers include NHS-ester, maleimide, and amino-reactive groups that can facilitate covalent bonding.
Bioconjugation techniques: Several strategies can be employed for effective bioconjugation. Common methods include:
Direct conjugation techniques
This methods involve the straightforward covalent attachment of enzymes to biomolecules without intermediates. One prominent technique is amine coupling, where amine groups on enzymes or antibodies react with activated carboxylic acids, such as N-hydroxysuccinimide (NHS) esters. This reaction yields stable conjugates that retain enzymatic activity, making amine coupling a widely employed method in various immunoassays.
Another effective direct method is thiol-maleimide conjugation, which leverages the specificity of thiol groups on cysteine residues in proteins. In this process, maleimide-activated enzymes selectively bind to thiol-containing antibodies, producing stable and functional enzyme-antibody conjugates. This technique is advantageous for maintaining the biological activity of the conjugates while ensuring a robust attachment.
Indirect conjugation techniques
Indirect conjugation techniques often utilize intermediates or scaffolds to enhance specificity and stability. One of the most versatile methods is click chemistry, particularly azide-alkyne cycloaddition. This reaction is highly selective and occurs under mild conditions, making it ideal for conjugating sensitive biomolecules. For instance, researchers can modify antibodies with azide groups and then couple them to alkyne-activated enzymes, yielding stable and effective conjugates suitable for various immunoassay applications.
The biotin-streptavidin system exemplifies another indirect approach that capitalizes on the high affinity between biotin and streptavidin. In this technique, antibodies are biotinylated and subsequently linked to streptavidin-enzyme conjugates. This system not only enhances assay sensitivity through the strong binding interaction but also simplifies the detection process, as multiple biotin-streptavidin interactions can amplify the signal in immunoassays.
Antibody-enzyme conjugate
Antibozyme conjugate is the most basic form of EIA, usually using horseradish peroxidase (HRP) or alkaline phosphatase (AP) bound to specific antibodies. The conjugated compound generates a measurable signal through the specific binding of the antibody to the target molecule and the enzyme-catalyzed reaction. This structure has high sensitivity and selectivity, and is suitable for the analysis of a variety of biological samples.
Biotinized antibody and streptavidin-enzyme
Biotinylated antibody is the introduction of biotin molecules into the structure of the antibody. After forming biotinylated antibody, it can bind to streptavidin. Streptavidin is a protein with high affinity that can bind to a variety of biotinylated molecules. When biotinylated antibody is combined with streptavidin-enzyme, this combination not only improves the sensitivity of detection, but also enhances the stability of signal.
Biotinylated antibody, biotinylase and streptavidin
In some applications, it is possible to bind a biotinylated antibody to a biotinylase, which is subsequently linked using streptavidin. This method utilizes the high affinity between biotin and streptavidin to achieve efficient signal amplification. This design is particularly suitable for increasing the detection sensitivity of target molecules in complex samples.
Antibody-particle or antibody-surface conjugate
Antibody-particle couplings are often used to immobilize antibodies on the surface of microspheres or particles. In this way, the antibody can efficiently capture the target molecule and then conduct an enzyme reaction to generate a signal. This structure not only improves the reaction efficiency, but also facilitates the subsequent separation and purification. In addition, antibody-surface couplings can also be used as solid phase supports to enhance the throughput of the assay.
Antibody-polymer-enzyme
The antibody-polymer-enzyme conjugate forms a polypolymer structure by connecting the antibody to the polymer chain. This coupling can increase the number of binding sites, thereby increasing the strength and sensitivity of the signal. The antibody-polymer-enzyme combination is particularly suitable for low concentration analysis and can effectively increase the detection sensitivity.
Streptavidin-polymer-enzyme and biotinylated antibody
In some highly sensitive assays, streptavidin can bind to polymers and enzymes, and then form complexes by biotinizing antibodies. This structure not only enables multiple binding, but also significantly enhances signal strength and is suitable for applications requiring high sensitivity, such as the detection of early tumor markers.
Immobilized antigen or affinity ligand
In EIA, immobilized antigens or affinity ligands can bind to antibodies to form a stable detection platform. This method ensures the effective capture of target molecules and enhances the signal strength through enzyme-catalyzed reaction, which is suitable for quantitative analysis. The use of immobilized antigens can also reduce background interference and improve the specificity of detection.
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In the practical application of EIA, bioconjugators play a crucial role. These conjugates are typically made of antibodies bound to enzymes that can specifically recognize the target molecule and provide a detectable signal through enzyme-catalyzed reactions.
In drug monitoring, enzyme immunoassay is widely used to detect drug concentration. Phenobarbital is often measured by enzymes that bind to anti-phenobarbital antibodies, such as HRP. The method is highly sensitive and can detect extremely low concentrations in blood samples, helping doctors adjust drug dosages for optimal treatment.
In the diagnosis of infectious diseases, enzyme immunoassay is often used to detect specific antigens or antibodies. Enzyme-immunoassay for human immunodeficiency virus (HIV) can effectively identify antibody levels in serum samples using HRP coupled with HIV antibodies. This test is not only sensitive, but can also identify infections at an early stage and help intervene early.
In food safety testing, EIA is used to identify potentially harmful substances such as allergens and pesticide residues. The combination of anti-aflatoxin antibody and HRP could effectively monitor the level of Aflatoxin in food to ensure food safety. This detection method is simple and fast, and provides an important guarantee for the food industry.
In the detection of tumor markers, EIA is able to detect the levels of certain biomarkers in the blood of cancer patients. In the detection of alpha-fetoprotein (AFP), the concentration of AFP in serum can be quantitatively determined by enzyme coupled with AFP antibody, which provides a basis for the early screening of liver cancer.
Enzyme immunoassay is also used in environmental monitoring, for example to detect contaminants in water sources. Contaminant concentrations in water bodies can be detected by antibody-conjugated enzymes that bind to specific contaminants, such as polychlorinated biphenyls (PCBs). This method is not only sensitive, but also can provide scientific basis for environmental protection.