At present, the measurement principle adopted by most automatic immunoassay analyzers is enzyme immunoassay. These analyzers can also be called fully automatic enzyme immunoassay analyzers. These analyzers can be divided into different types according to whether the reagents used are dedicated or not. There are two types of reagents: “restricted†and “open†reagents. There are relatively few fully automated enzyme immunoassays for reagents, such as BIO-RAD's CODA and Per-sonalLab; and reagent-defined fully automated enzyme immunoassay types. There are many, such as Roche's COBASeCOREII and ENZYMESYSTEM (ES), ABBOTT's AXSYM@ and IMX, BioMérieux's VIADS@, Johnson & Johnson's Vitros ECi@, BECKMANCOULTER's Access@ and DPC's IMMULITE42000. Both types of fully automatic enzyme immunoassay analyzers use the basic principle of ELISA, especially the open reagents, which only change the basic ELISA process, such as loading, incubation, and washing, from manual operation to machine operation. If the reaction product of the enzyme and the substrate after zui is used, the fully automatic enzyme immunoassay analyzer can be classified into three types: color development, fluorescence measurement and luminescence measurement. According to the enzyme used for labeling, it can also be classified into horseradish peroxidase (HRP) and alkaline phosphatase markers. This section introduces the basic measurement principle and mode of the fully automatic enzyme immunoassay analyzer defined by reagents according to the methods of color development, fluorescence and luminescence.
I. Colorimetric Determination Roche COBASeCOREII and ENZYMESYSTEM (ES) Automated Enzyme Immunoassay System Roche COBAS*COREII Automated Enzyme Immunoassay System uses plastic beads (0.6 cm in diameter) as a solid phase carrier with horseradish Peroxidase (HRP) is used as a labeling enzyme, and the chromogenic substrate is tetramethylbenzidine (TMB). Each time the antigen-antibody binding reaction is completed on the solid phase, a special washing device is installed in the instrument to rapidly roll the plastic beads to remove unbound antigen, antibody and enzyme conjugate. The ES type automatic enzyme immunoassay system uses a streptavidin-coated polyphenylene plastic tube as a solid phase carrier, and the pseudo-solidified antibody or antigen can be indirectly adsorbed by biotin, the antibody or antigen The aphacity technology Zui was originally patented by Boehringer-Mannheim. The enzyme used for labeling in this system is also HRP, but the substrate is different, and ABTS is used.
2. Fluorescence measurement (1) ABBOTT's IMX and AXSYM~ fully automatic enzyme immunoassay system ABBOTT's IMX automatic enzyme immunoassay system uses a solid phase of superficially porous plastic microparticles with a diameter of 0.5 tzm. The enzyme used for labeling is alkaline phosphatase, and the substrate of the enzyme is 4-methylumbelliferone phosphate (4-MUP). Since 4-MU is producing 4-methylumbelliferone under the action of alkaline phosphatase, Under illumination of 360 nm excitation light, 448 nm fluorescence is emitted, and thus Zui finally calculates the concentration of the measured substance based on the fluorescence intensity generated. It is worth mentioning that the washing and separation process of the enzyme immunoassay system, that is, after the antigen-antibody binding reaction is completed on the solid phase of the plastic microparticles, the instrument transfers the microparticles to a specific glass fiber, and adds a washing buffer without binding. The antigen or antibody is washed away, and the antigen antibody bound to the solid phase of the plastic particles remains on the glass fibers due to the irreversible binding of the particles to the glass fibers. Therefore, the effect of such washing separation is good.
ABBOTT's AXSYM* automated immunoassay system is a combination of two immunoassays, IMX and TDX (using fluorescence polarization immunoassay). Therefore, this fully automated immunoassay system can perform enzyme immunoassay in addition to IMX. In addition, fluorescence polarization immunoassay of TDX can be performed, which is more convenient for the determination of some small molecules such as drugs.
(II) VIADS@Automatic Enzyme Immunoassay System of BioMérieux The basic principle of the analysis system is similar to ABBOTT's IMX, that is, it uses alkaline phosphatase as a labeling enzyme and 4-MUP as an enzyme. The fluorescent substrate, which is detected after zui, is also fluorescent. The difference is that the reaction solid phase of the system is the inner surface of the plastic phase (SRP), which is automatically pumped by the instrument, and the antigen-antibody binding reaction, washing separation and fluorescence excitation process are completed on the inner surface of the tip.
III. Luminescence measurement class (1) Vitros ECi@ fully automatic enhanced chemiluminescence enzyme immunoassay system of Johnson & Johnson Co., Ltd. The enzyme immunoassay system uses a bullet-type small-pore tube as a solid phase carrier, HRP as a labeling enzyme, and a substrate luminol. For the luminescent substrate of HRF, the system uses a chemiluminescence enhancer 3-chloro-4-hydroxyacetanilide, which not only makes the chemiluminescence intensity stronger than that of luminol alone, but also is stable and lasts for a long time. Sensitivity. The surface of the solid phase carrier of the assay system is also pre-coated with streptavidin, and the antibody or antigen is indirectly adsorbed by biotin.
(2) Access@automatic microparticle chemiluminescence enzyme immunoassay system of BECKMANCOULTER Company This system uses paramagnetic microparticles of polyphenylene encapsulating iron oxide as the solid phase carrier, and alkaline phosphatase as the enzyme for labeling. A novel alkaline phosphatase substrate AMPPD (dioxetanes) is used as a luminescent substrate. The molecular structure of this substrate has two important parts: one is a dioxane ring connecting a benzene ring and adamantane, which can be broken and The photon is emitted; the other is a phosphate group that maintains the stability of the entire molecular structure. Under the action of alkaline phosphatase, this substrate rapidly dephosphorizes the phosphate group to form the unstable intermediate AMPD. AMPD quickly decomposes itself, returning from a high-energy excited state to a low-energy steady state, while emitting photons. This chemiluminescence is stable for up to several hours, easy to measure and easy to control. Since the solid phase of the system is paramagnetic particles, when the electromagnetic field is added, the particles will quickly sink and adsorb, and the separation of the solid phase and the liquid phase is achieved, and the washing separation is simple and rapid, which accelerates the measurement speed of the entire measurement system.
(3) DMC's IMMULITE 72000 fully automatic chemiluminescence enzyme immunoassay system. The system uses polyphenylene plastic beads as a solid phase carrier, alkaline phosphatase as a labeling enzyme, and AMPPD as a substrate. The method of washing away unbound antigen or antibody and enzyme conjugate from the solid phase is a centrifugal wash.
I. Colorimetric Determination Roche COBASeCOREII and ENZYMESYSTEM (ES) Automated Enzyme Immunoassay System Roche COBAS*COREII Automated Enzyme Immunoassay System uses plastic beads (0.6 cm in diameter) as a solid phase carrier with horseradish Peroxidase (HRP) is used as a labeling enzyme, and the chromogenic substrate is tetramethylbenzidine (TMB). Each time the antigen-antibody binding reaction is completed on the solid phase, a special washing device is installed in the instrument to rapidly roll the plastic beads to remove unbound antigen, antibody and enzyme conjugate. The ES type automatic enzyme immunoassay system uses a streptavidin-coated polyphenylene plastic tube as a solid phase carrier, and the pseudo-solidified antibody or antigen can be indirectly adsorbed by biotin, the antibody or antigen The aphacity technology Zui was originally patented by Boehringer-Mannheim. The enzyme used for labeling in this system is also HRP, but the substrate is different, and ABTS is used.
2. Fluorescence measurement (1) ABBOTT's IMX and AXSYM~ fully automatic enzyme immunoassay system ABBOTT's IMX automatic enzyme immunoassay system uses a solid phase of superficially porous plastic microparticles with a diameter of 0.5 tzm. The enzyme used for labeling is alkaline phosphatase, and the substrate of the enzyme is 4-methylumbelliferone phosphate (4-MUP). Since 4-MU is producing 4-methylumbelliferone under the action of alkaline phosphatase, Under illumination of 360 nm excitation light, 448 nm fluorescence is emitted, and thus Zui finally calculates the concentration of the measured substance based on the fluorescence intensity generated. It is worth mentioning that the washing and separation process of the enzyme immunoassay system, that is, after the antigen-antibody binding reaction is completed on the solid phase of the plastic microparticles, the instrument transfers the microparticles to a specific glass fiber, and adds a washing buffer without binding. The antigen or antibody is washed away, and the antigen antibody bound to the solid phase of the plastic particles remains on the glass fibers due to the irreversible binding of the particles to the glass fibers. Therefore, the effect of such washing separation is good.
ABBOTT's AXSYM* automated immunoassay system is a combination of two immunoassays, IMX and TDX (using fluorescence polarization immunoassay). Therefore, this fully automated immunoassay system can perform enzyme immunoassay in addition to IMX. In addition, fluorescence polarization immunoassay of TDX can be performed, which is more convenient for the determination of some small molecules such as drugs.
(II) VIADS@Automatic Enzyme Immunoassay System of BioMérieux The basic principle of the analysis system is similar to ABBOTT's IMX, that is, it uses alkaline phosphatase as a labeling enzyme and 4-MUP as an enzyme. The fluorescent substrate, which is detected after zui, is also fluorescent. The difference is that the reaction solid phase of the system is the inner surface of the plastic phase (SRP), which is automatically pumped by the instrument, and the antigen-antibody binding reaction, washing separation and fluorescence excitation process are completed on the inner surface of the tip.
III. Luminescence measurement class (1) Vitros ECi@ fully automatic enhanced chemiluminescence enzyme immunoassay system of Johnson & Johnson Co., Ltd. The enzyme immunoassay system uses a bullet-type small-pore tube as a solid phase carrier, HRP as a labeling enzyme, and a substrate luminol. For the luminescent substrate of HRF, the system uses a chemiluminescence enhancer 3-chloro-4-hydroxyacetanilide, which not only makes the chemiluminescence intensity stronger than that of luminol alone, but also is stable and lasts for a long time. Sensitivity. The surface of the solid phase carrier of the assay system is also pre-coated with streptavidin, and the antibody or antigen is indirectly adsorbed by biotin.
(2) Access@automatic microparticle chemiluminescence enzyme immunoassay system of BECKMANCOULTER Company This system uses paramagnetic microparticles of polyphenylene encapsulating iron oxide as the solid phase carrier, and alkaline phosphatase as the enzyme for labeling. A novel alkaline phosphatase substrate AMPPD (dioxetanes) is used as a luminescent substrate. The molecular structure of this substrate has two important parts: one is a dioxane ring connecting a benzene ring and adamantane, which can be broken and The photon is emitted; the other is a phosphate group that maintains the stability of the entire molecular structure. Under the action of alkaline phosphatase, this substrate rapidly dephosphorizes the phosphate group to form the unstable intermediate AMPD. AMPD quickly decomposes itself, returning from a high-energy excited state to a low-energy steady state, while emitting photons. This chemiluminescence is stable for up to several hours, easy to measure and easy to control. Since the solid phase of the system is paramagnetic particles, when the electromagnetic field is added, the particles will quickly sink and adsorb, and the separation of the solid phase and the liquid phase is achieved, and the washing separation is simple and rapid, which accelerates the measurement speed of the entire measurement system.
(3) DMC's IMMULITE 72000 fully automatic chemiluminescence enzyme immunoassay system. The system uses polyphenylene plastic beads as a solid phase carrier, alkaline phosphatase as a labeling enzyme, and AMPPD as a substrate. The method of washing away unbound antigen or antibody and enzyme conjugate from the solid phase is a centrifugal wash.
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