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Fluorescence immunoassays are difficult to quantify due to the high background in general fluorescence assays. In recent years, several special fluorescent immunoassays have been developed, which, like enzyme immunoassays and radioimmunoassays, are used in clinical tests.
Basic knowledge about fluorescence:
First, the phenomenon of fluorescence
(1) Generation of fluorescence: This chemical substance can absorb and store energy (such as light energy, chemical energy, etc.) from the outside and enter the excited state. When it returns from the excited state to the ground state, the excess energy can be in the form of electromagnetic radiation. Radiation (ie, luminescence). The characteristic of fluorescence emission is that the molecules or atoms that can generate fluorescence immediately cause luminescence after receiving energy; and once the energy supply is stopped, the luminescence (fluorescence) phenomenon disappears in an instant. There are many types of energy that can cause fluorescing, and fluorescence caused by photoexcitation is called fluorescing. What is caused by chemical reaction is called chemical fluorescence, and X-ray or cathode ray is called X-ray fluorescence or cathode ray fluorescence, respectively. Fluorescent immunoassays are generally labeled with a fluorescent substance.
(b) Fluorescence efficiency Fluorescent molecules do not convert all absorbed light energy into fluorescence, and are more or less released in other forms. Fluorescence efficiency refers to the percentage of fluorescent molecules that convert the absorbed light energy into fluorescence, which is proportional to the value of the emitted fluorescent photon. Fluorescence efficiency = number of molecules that emit fluorescence (fluorescence intensity) / number of light quantum of absorbed light (excitation light intensity) The quantum number of light that emits fluorescence, that is, the fluorescence intensity, is affected by the intensity of the excitation light and also by the wavelength of the excitation light. Each fluorescent molecule has its specific absorption spectrum and emission spectrum (fluorescence spectrum), that is, a maximum absorption peak and a maximum emission peak at a specific wavelength. The wavelength of the excitation light is selected to be close to the maximum absorption peak wavelength of the fluorescent molecule, and the measured fluorescence intensity is also the largest when the measured light wave amount is close to the maximum emission light peak.
(3) The radiant ability of the fluorescent quenching fluorescent molecule is weakened or even quenched after being irradiated by the excitation light for a long time, because the electrons of the excited state molecule cannot return to the ground state, and the absorbed energy cannot be in the form of fluorescence. emission. Some compounds have a natural fluorescence quenching effect and are used as quenchers to eliminate unwanted fluorescence. Therefore, the preservation of the fluorescent substance should take care to avoid direct irradiation of light (especially ultraviolet light) and contact with other compounds. Some non-fluorescent pigment materials such as methylene blue and alkaline red complex are commonly used in fluorescent antibody technology. Evans blue or low concentrations of potassium permanganate, iodine solution, etc., are properly counterstained to attenuate the non-specific fluorescence nature, making the specific fluorescence more prominent.
Second, the fluorescent substance
(I) Fluorescent Pigments Many substances can produce fluorescence, but not all can be used as fluorescent pigments. Only organic compounds that produce significant fluorescence and can be used as dyes can be called immunofluorescent pigments or fluorescent dyes.
Commonly used fluorochromes are:
1. Fluorescein isothiocyanate (FITC) is a yellow or orange-yellow crystalline powder and is easily soluble in solvents such as water or alcohol. The molecular weight is 389.4, the maximum absorption wavelength is 490495nm, and the maximum emission wavelength is 520530nm. It exhibits bright yellow-green fluorescence. The structural formula is as follows: There are two isomeric structures, in which isomer type I is in efficiency, stability, and protein binding. It is better in terms of ability and so on. It can be stored for many years in cold and dark and dry place. It is the most widely used fluorescein.
The main advantages are: 1 human eye is more sensitive to yellow-green, 2 usually green specimen fluorescence is less than red.
　
2. Rhodamine (RIB200) is an orange-red powder that is insoluble in water and soluble in alcohol and acetone. It is stable in nature and can be stored for a long time. The structural formula is as follows: the maximum absorption light wavelength is 570 nm, and the maximum emission light wavelength is 595-600 nm, which is orange-red fluorescence.
3. The structural formula of tetramethylrhodamineisothiocyanate (TRITC) is as follows: The maximum attractive light wavelength is 550 nm, and the maximum emission light wavelength is 620 nm, which is orange-red fluorescence. Contrasting with FITC's emerald green fluorescence, it can be used for double labeling or contrast staining. Its isothiocyanato group can bind to proteins, but its fluorescence efficiency is low.
(2) Other fluorescent substances
1. Some substances that produce fluorescence after enzymatic action Some compounds have no fluorescence effect, and once they are enzymatically formed, they form a substance with strong fluorescence. For example, 4-methylumbellone-β-D-galactoside is decomposed into 4-methylumbelliferone by the action of β-galactosidase, which emits fluorescence with an excitation light wavelength of 360 nm and an emission wavelength of 450 nm. Other substrates such as alkaline acid enzyme 4-methylumbellone phosphate and horseradish peroxidase substrate p-hydroxyphenylacetic acid and the like.
2. Lanthanide chelates Some trivalent rare earth lanthanides such as europium (Eu3+), strontium (Tb3+), cerium (Ce3+) and other chelates can also emit characteristic fluorescence after excitation. Among them, Eu3+ is the most widely used. . The Eu3+ chelate has a wide range of excitation light wavelengths, a narrow wavelength range of emission light, and a long fluorescence decay time, which is most suitable for the resolution of fluorescence immunoassays. Basic knowledge of immunology: antigens, antibodies and monoclonal antibodies are well known. Humans or animals can rely on their own ability to resist the invasion of certain diseases. Some diseases can be self-healed by the body's own regulation. This is because there is an immune system inside the body. . The immune system is immunocompetent cells (B lymphocytes, plasma cells) from central immune tissues (bone marrow, thymus, digestive immune tissue) and peripheral lymphoid tissues (lymph nodes and spleen), and various lymphokines and antibodies produced by them. Composition. The process by which the immune system produces a series of reactions to foreign substances is called immunity, and the reaction itself is called an immune response. A substance capable of causing a specific immune response in the body is called an antigen, and the result of the immune response is to stimulate the body to produce a specific antibody corresponding to the antigen and cause cellular immunity. Since the immune response depends on the body at the same time, not just the substance that enters the body, the definition of the antigen is relative. Thus, according to the effect of the immune response, the antigen can be divided into a complete antigen and a hapten: a complete antigen refers to a substance capable of directly inducing a specific immune response in the body. The complete antigen has a molecular weight of more than 5,000, and its chemical components are mostly proteins or lipopolysaccharides. Others such as lipoproteins, glycoproteins, polysaccharides, polypeptides, nucleic acids and the like are also complete antigens. A hapten can specifically bind to an antibody, but it cannot stimulate the body to produce an antibody. It must bind to a protein (carrier) and enter the body to produce an effective immune response. The hapten of the macromolecule can undergo a visible reaction (agglutination, precipitation) in combination with the corresponding antibody in vitro, and the hapten of the small molecule can bind to the corresponding antibody without a visible reaction. In addition, the antigen can be classified into soluble (toxin, heterologous protein) or granular (bacterial, cell) antigen according to its own material properties, or divided into exogenous and endogenous antigens according to clinical significance. An antibody is the product of an immune response in the body stimulated by an antigen. All antibodies have the ability to specifically bind to antigen. The chemical nature of antibodies is the immunoglobulin, gamma-globulin. However, this immunoglobulin can be called an antibody only when the immunoglobulin is directed against a known antigen. Immunoglobulins are structurally strong molecular substances that can withstand large environmental changes, such as 56 ° C warming, storage at room temperature for extended periods of time, short-term high or low pH treatment, even after contact with detergent or urea. It still retains its antibody activity. Antibody activity refers to the ability to specifically bind to an antigen, ie a specific affinity between the two. The two are non-covalently bonded, and the combined forces include hydrogen bonding, electrostatic force, van der Waals force, and hydrophobic binding force. The reaction between antibody antigens is reversible and can be dissociated under the appropriate conditions without changing the properties. There are about 100 million different B lymphocytes in the body. Each independent B lymphocyte can only be stimulated by one antigen to form the ability to secrete an antibody. Therefore, specificity is a unique indicator of immune response. As common sense sees, measles patients get a lifetime immunization against measles, and this does not form immunity to smallpox. The essence of antibody antigen-specific binding is the specific adaptability of the antigenic determinant to the antibody-binding cluster. The antigenic determinant refers to the specific chemical gene on the antigen molecule, which can determine the specificity of the antibody produced by the stimulating organism. The antigenic determinant is fully compatible with its corresponding antibody binding cluster stereoconfiguration. An antigen molecule can carry multiple different determinants. The larger the molecular weight of the antigen, the greater the number of determinants. The number of determinants represents the valence of the antigen molecule. An antibody-binding cluster is a site that specifically binds to a corresponding antigenic determinant on an antibody molecule, and is located on an antigen-binding segment (V segment) of an Ig molecule, and is composed of a part of a variable region of a heavy chain and a light chain. Its specificity is determined by the amino acid arrangement order and includes about 5 to 15 amino acids. Each monomeric immunoglobulin molecule such as IgG has two binding clusters, IgA is a dimer, there are 4 binding clusters, and IgM is a pentamer so there are 10 binding clusters. (However, things are not absolute. In addition to the specific reaction of an antibody with a corresponding antigen, it is also possible to react with other antigens that are similar in chemical structure. This is known as the interaction reaction. It may be due to the fact that the two antigens have a common determinant, or because the antigenic determinants of the two are not identical, but are closely related in stereochemistry, resulting in one antigen binding to the corresponding antibody of the other antigen.) Normal In this case, after the antibody and the antigen are bound to the body, they can be phagocytized and excreted to remove the antigen. If the antigen belongs to an exogenous pathogenic antigen (bacteria, virus, toxin), it can achieve the purpose of killing or weakening the antigen. For endogenous antigens, such as abnormal cells that have been degraded, damaged or mutated, they can be eliminated in time to achieve the body's own immune regulation. Of course, in an abnormal situation, the immune complex formed by the binding of the antibody antigen will damage the tissues and cells, causing adverse effects such as allergic reactions such as pollen allergy or immune diseases. However, the so-called specific immune serum antibodies, whether obtained from humans or animals, whether acquired actively or passively, are actually mixed antibodies composed of many antibodies with different characteristics. This is because antigens entering the body often carry several antigenic determinants. In addition, different individuals respond differently to the same antigenic determinant, even if the same individual receives antigen stimulation at different times, the response is not completely consistent. Since people cannot distinguish between different B lymphocyte clones that have been stimulated by antigens in the body, even if they can be divided into single cells in vitro, they cannot continue to grow, proliferate and secrete antibodies. Therefore, the immune serum antibody prepared by the conventional immunization method can only be a mixture of a large number of monoclonal antibodies, and is now generally referred to as a polyclonal antibody (PcAb). Such polyclonal antibodies have inherent defects such as poor specificity, low potency, limited number, large individual differences between animals, and difficulty in repeated preparation, and are difficult to use in many cases. In 1975, Kohler and Milstein of the Laboratory of Molecular Biology, University of Cambridge, UK, published a famous paper entitled "Continuous Culture of Fusion Cells Secreting Specific Antibodies" (Nature, 256:495, 1975). They fuse mouse myeloma cells that have been adapted to culture in vitro with spleen cells (B lymphocytes) of sheep erythrocyte-immunized mice, and found that the hybridoma cells formed by fusion have the characteristics of amphiphilic cells: ie, like myeloma cells in vitro In culture, it can proliferate indefinitely, and can continuously secrete specific antibodies. By cloning, hybrid cells can be made into simple cell lines, and thus the monoclonal system can obtain high-purity antibodies having the same structure and various characteristics. That is, a monoclonal antibody (McAb). The above method creates a new and epoch-making technique for the production of monoclonal antibodies using B lymphocyte hybridomas. The creation and rapid spread of this technology has provided new tools and preparations for all research fields that require the preparation and use of antibodies, and has promoted the development of life sciences. The two scientists also won the 1984 Nobel Prize in Medicine and Physiology for this outstanding contribution. Aflatoxin B1 is a derivative of dihydrofuran oxaphthalene, which has a molecular weight of 312 and belongs to the hapten and cannot directly induce the body to produce an immune response. Therefore, our work firstly links aflatoxin B1 to a carrier protein and synthesizes it into a complete antigen. Later studies have basically adopted the classical method of hybridoma-monoclonal antibody technology: animal immunization → immune spleen cells and myeloma Cell fusion→cell screening→Single cells secreting specific antibodies were isolated by limiting dilution method, and then hybridoma cell lines were established by cloning and culture to prepare a large number of monoclonal antibodies, thus aflatoxin was finally established. Immunoassay method for B1 (AFB1).
Immunolabeling
(a) Fluorescence immunoassay: The principle is a sandwich method using a pair of monoclonal antibodies. The substrate was phosphate--4-methylumbelliferone, and the fluorescence emitted by the product was detected. The fluorescence intensity was proportional to the concentration of Mb, and the result was obtained within 8 min. The results are expressed as the rate of release of Mb per hour (ΔMb). The method has good repeatability, wide linear range, and is fast, sensitive and accurate. Taking the double-antibody sandwich method as an example, a specific antibody is first linked to a solid phase carrier to form a solid phase antibody. The unbound antibody is removed, and then the test sample is added to form a antigen-antibody complex with the solid phase antibody. The unbound material is removed by washing, followed by the addition of a fluorescently labeled antibody to specifically bind to the antigen to form an antibody-antigen-antibody complex. Finally, protein antigens can be quantified based on fluorescence intensity. The traditional fluorescence immunoassay is greatly interfered by the background fluorescence. The time-resolved fluorescence immunoassay is a rare earth metal with a long life span, such as strontium, as a marker. After the addition of normal liquid, the excitation assay can effectively remove the short-lived background fluorescence. Interference.
(ii) Radioimmunoassay Radioimmunoassay is an antigen that is labeled with an excess of unlabeled antigen and radioactive material, competitively bound to the antibody to form a radioactive antigen-antibody complex and a non-radioactive antigen-antibody complex, and There are excess labeled antigens and unlabeled antigens. Then, the antigen-antibody complex is separated from the free antigen by centrifugation and the like, and the radioactivity intensity is compared with a standard curve to quantify the unlabeled antigen to be tested. The RIA method has high sensitivity and specificity for the determination of serum protein, and can be accurately quantified to the ng/ml level. But the early methods were cumbersome to operate, time consuming, and radioactive. In recent years, with the application of monoclonal antibodies, the sensitivity of RIA has been greatly improved, and the operation has been greatly simplified, and commercial kits have been supplied, which is convenient to use.
(3) Enzyme-linked immunosorbent assay (ELISA) ELISA has two methods: competition method and sandwich method. The competition method is based on the principle that standard or serum Mb and coated Mb on microplates are competitively combined with monoclonal antibodies. The minimum detection limit is 10 μg/L and the linear range is 1 000 ug/L. . The sandwich ELISA method has a good correlation with EIA (r=0.92). The ELISA method has high sensitivity, strong specificity, good precision and simple operation. It is suitable for the detection of multiple specimens, and does not require special equipment and equipment, and is easy to popularize. But it is not suitable for rapid detection of emergency. Take the double-antibody method as an example. The antigen is first coated, and then the primary antibody is added to form an antigen-antibody complex with the coated antigen. The enzyme-labeled secondary antibody is then added to form an antigen-antibody-antibody complex. Finally, the substrate is added and the product is produced by the enzyme catalyzed substrate. Protein antigens can be quantified by the amount of product produced.
(4) The enzyme-free method of coupling the biotin-avidin system utilizes the characteristics that one avidin molecule can bind to four biotin molecules, so that the sensitivity of the conventional sensitivity-sensitive enzyme is obvious. Amplification.
(5) Time-Resolved Fluorescence Immunoassay (Time Resolved Fluoroimmunoassay, TRFIA) is a non-isotopic immunoassay technique that uses a lanthanide to label an antigen or antibody according to the luminescent characteristics of the lanthanide chelate. The time-resolved technique measures fluorescence and simultaneously detects the wavelength and time parameters for signal resolution, which can effectively eliminate the interference of non-specific fluorescence and greatly improve the sensitivity of analysis.
(VI) Dissociation enhanced lanthanide fluorescence immunoassay Dissociation Enhanced Lanthanide Fluoroimmunoassay DELFA is one of the time-resolved fluorescence immunoassays. It uses a chelating agent with a bifunctional group structure to link one end to Eu (Eu) and the other end to a free amino group on the antibody/antigen molecule to form an EU-labeled antibody/antigen, which is immunologically complexed to generate an immune complex. Object. Since the fluorescence intensity of this complex in water is very weak, an enhancer is added to dissociate Eu3+ from the complex, and free Eu3+ is chelated with another chelating agent in the enhancer to form a colloidal molecule. This group of molecules can emit strong fluorescence under the excitation of ultraviolet light, and the signal is enhanced by a million times. Because this analytical method uses an understanding of the enhancement step, it is called dissociation-enhanced lanthanide fluorescence immunoassay.
Introduction to fluorescent immunotechnology
Fluorescence immunoassay is one of the earliest developments in labeling immunoassays. Some scholars have long tried to combine antibody molecules with some tracer substances, and use antigen-antibody reactions to locate the antigens in tissues or cells. Coons was the first to be successfully labeled with fluorescein in 1941. This technique of labeling antibodies with fluorescent substances for antigen localization is called fluorescent antibody technology. The fluorescence immunoassay can be further divided into several types according to the reaction system and the quantitative method. Compared with radioimmunoassay, fluorescence immunoassay has no radioactive contamination, and most of them are easy to operate and easy to promote. A large number of TDM kits produced abroad are of this type, and there are also automatic analyzers for TDM fluorescence polarization immunoassay.