Immunoassay is an analytical method based on specific antibody-antigen interactions to detect specific molecules present in a sample. Its core advantage is the precise matching of antibodies to antigen molecules, which gives the analytical method extremely high specificity and accuracy, making antibodies an ideal tool for identifying and capturing target biomolecules, and thus occupy an important position in life science research and industrial laboratories. The technology is diversified, not only to help the assessment of diseases, protein dynamic monitoring, but also suitable for the identification of environmental pollutants.
In the face of molecular identification and separation of complex mixtures, immunoassay technology shows its unique value. Through magnetic particle mediated magnetic immunoassay, magnetite core particles coated with biocompatible materials and chemical coupling of antibodies were used to achieve efficient capture and separation of specific molecules. This process demonstrates the flexibility of the technology, but requires careful consideration before implementation to select the immunoassay model that best fits the needs of the experiment.
Immunoassays can be divided into two categories, labeled and unlabeled, depending on whether or not markers are used.
Labeled immunoassay is typically represented by enzyme immunoassay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA) and chemiluminescence immunoassay (CLIA). Each technique has its own unique subclassification and continues to evolve with the progress of science and technology. As the cornerstone, ELISA is widely used in the quantitative analysis of a variety of soluble molecules by its simple operation and specificity. Through antibody fixation, antigen capture, enzyme-labeled antibody binding and substrate color rendering, the visual determination of the target object can be realized. The color depth is proportional to the concentration of the target antigen.
RIA uses the radio-labeled antigen to competitively bind antibodies to the corresponding antigen in the sample, and inversely calculates the concentration of the target molecule through the strength of the radioactive signal. Although its sensitivity and specificity are extremely high, it must strictly abide by the radioactive safety procedures. FIA quickly and sensitively quantifies the target molecule by the fluorescence intensity generated by the binding of the fluorescently labeled antibody to the antigen. Chemiluminescence immunoassay uses light signals stimulated by chemical reactions to detect the antigen-antibody complex, showing excellent sensitivity and specificity, and is widely used in many fields from environmental monitoring to disease diagnosis, especially the magnetic bead-based CLIA technology, which further broadens the application boundary.
The emerging field of photonic biosensors is driving the development of a new generation of marker-free detection that does not rely on a central laboratory. Photonics is the science of detecting and manipulating light. By using technologies developed by the electronics industry in microprocessor production and adapting them to light, photonic biosensors combine photon sensing with biometrics to create marker-free analysis on a chip. Instead of moving around the silicon chip, light moves around the silicon chip through waveguides.
The technology allows for the development of lab-on-a-chip marker-free immunoassay (LFIA) devices. These devices are functionalized with trapping antibodies and have light resonance conditions. Due to the change in refractive index, the reaction between the trapping antibody and the target antigen will change this resonance wavelength. Measuring the offset of the resonant wavelength provides a reading of the combined event. As a result, marker-free assas can detect antigen-antibody binding without the use of additional markers, resulting in increased analytical sensitivity and reduced operating time
Chemical reagents commonly used in immunoassay experiments include antigens, antibodies, buffers, enzyme-linked reagents, substrate developing agents and washing solutions. Antigens and antibodies are the core components, which detect target molecules by specific binding; The buffer is used to maintain the pH stability of the reaction environment and ensure the consistency of experimental conditions. Enzyme-linked agents (such as horseradish peroxidase HRP) bind to antibodies or antigens to produce detectable signals by catalyzing substrate color reaction; The substrate developing agent (such as TMB/H 2 O 2) changes color under the action of enzymes to provide quantitative or qualitative analysis results; The washing solution is used to flush the reaction plate to remove substances unless specifically bound to reduce background interference and improve detection accuracy. These chemicals work together to ensure the sensitivity and specificity of the immunoassay assay. In different types of immunoassay experiments, specific chemical reagents may also be added, including radioisotopes, fluorescent dyes, chemiluminescent substrates, etc.
Immunoassay plays an important role in drug detection. Immunoassay Drug testing uses binding reactions between specific antibodies and drug molecules to achieve rapid and accurate detection of drugs and their metabolites. This method has the advantages of high sensitivity, high specificity and simple operation, and is especially suitable for rapid screening of large quantities of samples. Through the high-throughput immunoassay method, the potential bioactive compounds can be quickly screened, which provides the direction for further research. In addition, immunoassays can be used to evaluate drug safety by testing the ability of a drug to bind to a specific biomarker to assess the potential impact of a drug on an organism.
Lateral flow immunoassay is a simple and rapid immunoassay method. Its principle is to use the antibodies on the lateral flow strip to bind specifically to the antigens in the sample to achieve rapid detection of target molecules. The method has the advantages of simple operation, low cost, field detection, etc., and has a wide application prospect in environmental monitoring, food safety detection and other fields. Chemical materials such as nitrocellulose membranes, gold nanoparticles, and porous substrates are integral parts of LFIAs function. The gold nanoparticles bound to the antibody produce visible lines on the test paper when the target is detected, simplifying the interpretation of the results.
Multiple immunoassay is an immunoassay technique that can detect multiple target molecules simultaneously. It uses multi-parameter detection technology, such as multicolor flow cytometry, microarray technology, beads, etc., combined with specific chemical reagents, to achieve the simultaneous detection of multiple antigens or antibodies. Bead-based multiplexing uses microspheres coated with different trapping antibodies, each labeled with a unique fluorescent dye. Multiple immunoassay has the advantages of high throughput, high efficiency and high sensitivity, and has important application value in biomedical research and clinical diagnosis.
Hemoglobin immunoassay specifically targets the screening and diagnosis of hemoglobin molecules for various conditions such as anemia, diabetes (glycated hemoglobin), and blood disorders. These tests employ antibodies that target hemoglobin or its derivatives to ensure specificity. The use of magnetic beads combined with anti-hemoglobin antibodies can effectively capture and separate the target and improve the analytical performance of complex biological matrices.
BOC Sciences provides comprehensive immunoassay solutions, including various chemical reagents used in immunoassay, antibody modification and conjugation, and fluorescent labeling service.
Rizzo, F. Optical immunoassays methods in protein analysis: An overview. Chemosensors. 2022, 10(8): 326.
