Fluorescent Antibody Technique

Fluorescence immunoassay technology is one of the earliest developed labeling immunoassay techniques. For a long time, some scholars have tried to combine antibody molecules with some tracer substances, and use antigen-antibody reaction to locate antigenic substances in tissues or cells. Coons was equal to the first successful use of fluorescein for labeling in 1941. This technique of labeling antibodies with fluorescent substances for antigen localization is called fluorescent antibody technique (fluorescent antibody technique).
Due to the high background in general fluorescence measurement, it is difficult to use fluorescence immunoassay technology for quantitative measurement. In recent years, several special fluorescent immunoassays have been developed, which are used in clinical tests like enzyme immunoassays and radioimmunoassays.

Basic knowledge about fluorescence

1. fluorescence phenomenon

　　 (1) Fluorescence

　　 This chemical substance can absorb and store energy from the outside world (such as light energy, chemical energy, etc.) into an excited state. When it returns from the excited state to the ground state, the excess energy can be emitted in the form of electromagnetic radiation (ie, emit light).
　　 The characteristic of fluorescence emission is that the molecules or atoms that can produce fluorescence cause luminescence immediately 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 fluorescence, and fluorescence caused by light excitation is called fluorescence. What is caused by chemical reaction is called chemical fluorescence, and what is caused by X-ray or cathode ray is called X-ray fluorescence or cathode ray fluorescence, respectively. Fluorescence immunotechnology generally uses fluorescent substances for labeling.

　　（2）Fluorescence efficiency

　　 Fluorescent molecules do not convert all the absorbed light energy into fluorescence, and they are always released in more or less 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 light quantum.
　　 Fluorescence efficiency = the number of light molecules emitting fluorescence (fluorescence intensity) / the number of photons of absorbed light (excitation light intensity)
　　The number of photons that emit fluorescence, that is, fluorescence intensity, is not only affected by the intensity of the excitation light, but also related to the wavelength of the excitation light. Each fluorescent molecule has its specific absorption spectrum and emission spectrum (fluorescence spectrum), that is, there is a maximum absorption peak and maximum emission peak at a specific wavelength. When the wavelength of the excitation light is selected to be close to the maximum absorption peak wavelength of the fluorescent molecule, and the measured light wave is close to the peak of the maximum emission light, the obtained fluorescence intensity is also maximum.

　　 (3) Quenching of fluorescence

　　The radiation ability of fluorescent molecules will be weakened or even quenched after being irradiated by the excitation light for a long time. This is because the electrons of the excited molecules cannot return to the ground state and the absorbed energy cannot be emitted in the form of fluorescence. Some compounds have natural fluorescence quenching effect and are used as quenchers to eliminate unnecessary fluorescence. Therefore, care should be taken to avoid direct exposure of light (especially ultraviolet light) and contact with other compounds in the storage of fluorescent materials. Some non-fluorescent pigment substances such as methylene blue and basic fuchsin are commonly used in fluorescent antibody technology. Evans blue or low-concentration potassium permanganate, iodine solution, etc., properly counterstain the specimen to weaken the nature of non-specific fluorescence and make specific fluorescence more prominent.

2. Fluorescent substance

Many substances can produce fluorescence, but not all can be used as fluorescent pigments. Only those organic compounds that can produce obvious fluorescence and can be used as dyes can be called immunofluorescent pigments or fluorescent dyes. Commonly used fluorescent pigments are:Fluoresceinisothiocyanate (FITC) is a yellow or orange-yellow crystalline powder, 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, showing bright yellow-green fluorescence. The structural formula is as follows:

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