Chemiluminescence immunoassay and its progress

Summary:
Chemiluminescence immunoassay combines chemiluminescence with immunoassay methods, combines the high sensitivity of chemiluminescence and the high selectivity of immunoassays, and is widely used in clinical testing and drug analysis. With the development of new luminescent reagents, new solid phase materials and the application of new labeling techniques, the sensitivity and reproducibility of chemiluminescence immunoassay methods will be greatly improved.

In analytical chemistry, chemiluminescence is the light produced when the energy released in the absorption reaction of the ground state molecule transitions to the excited state, and the molecule in the excited state returns to the ground state in the form of optical radiation. The analytical method based on the relationship between the chemiluminescence intensity and the content of the measured substance is called chemiluminescence analysis, and the combination of chemiluminescence and high performance liquid chromatography, capillary electrophoresis, immunoassay, etc., has high sensitivity, wide linear range, and instrument. Simple and easy to operate, it has been widely used in environmental, clinical, food and industrial analysis. The combination of chemiluminescence and immunoassay methods combines the high sensitivity of chemiluminescence with the high selectivity of immunoassay. In recent years, it has become a research hotspot.

1 Classification of chemiluminescence immunoassays

Chemiluminescence immunoassay can be divided into immunoassays, enzyme-catalyzed chemiluminescence immunoassays and electrochemiluminescence immunoassays based on the different methods used in immunoassay.

1.1 Immunoassay of Directly Labeled Luminescent Materials <br> This method uses a chemiluminescent agent to directly label an antigen or antibody. The chemiluminescent substance commonly used for labeling is an acridinium ester compound, which is an effective luminescent label, which emits light by the action of activating luminescent reagent, and strong direct luminescence is completed in 1 S, which is a fast scintillation luminescence. Acridinium ester is used as a marker for immunoassay. Its chemical reaction is simple, rapid, and requires no catalyst. The detection of small molecule antigens is based on competition, the macromolecule antigen is sandwiched, the non-specific binding is low, and the background is low. The combination does not reduce the amount of light produced, thereby increasing sensitivity. Zhang Wei et al. used a fully automated double-anti-sandwich immunoassay method using direct chemiluminescence technology to determine serum B-HCG content in 130 patients. Detection by chemiluminescence immunoassay. HCG, easy to operate, high sensitivity, specificity, superior to the general enzyme-linked immunoassay and radioimmunoassay, suitable for clinical laboratory use.

1.2 Enzyme-catalyzed chemiluminescence immunoassay <br>From the perspective of labeled immunoassay, enzyme-catalyzed chemiluminescence immunoassay should be an enzyme immunoassay, except that the substrate of the enzymatic reaction is a luminescent agent, and the procedure is identical to the enzyme immunoassay: The enzyme-labeled biologically active substance (such as an enzyme-labeled antigen or antibody) performs an immune reaction, and the enzyme on the immune reaction complex acts on the luminescent substrate, emits light under the action of a signal reagent, and performs luminescence measurement using a luminescence signal meter. Currently used labeling enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (ALP), which have their own luminescent substrates. The common substrate for horseradish peroxidase is luminol or its derivatives such as isoluminol, the oxidation reaction of luminol in alkaline buffer, in peroxidase and reactive oxygen species (peroxidation anion) In the presence of singlet oxygen, hydroxyl radicals, hydrogen peroxide, an excited state intermediate is generated, which emits light when it returns to the ground state, and its wavelength is 425 nln. Niu Xinyi and others use two specific C. P monoclonal antibody, horseradish peroxide luminol enzymatic sensitization chemiluminescence system, detection by double antibody sandwich method. The sensitivity is 0.102 g/L; the linear range of detection is 0.102-20 L; the multi-person test confirms that the intra-assay variation of the kit is less than 10%, the inter-assay variation is less than 15%; and the 18 g/L human proinsulin There was no significant cross-reactivity of human insulin at 2000 mIU/L.

The substrate used for alkaline phosphatase is the ring 1,2. The dioxyethane derivative is used in the molecular structure designed for the chemiluminescent enzyme to analyze the substrate, and contains a stabilizing group, adamantyl group, in which the luminescent group in the molecule is an aromatic group and an enzyme-active group. , causing chemiluminescence under the action of enzymes and starting luminescent reagents. Feng Yanming et al. _3』Using a double antibody ectopic one-step sandwich method, using alpha-fetoprotein monoclonal antibody as solid phase coating, using modified sodium periodate method to prepare alpha-fetoprotein polyclonal antibody and alkaline phosphatase coupling The enzyme-labeled antibody; using the amantadine derivative CSPD as a substrate, optimizes the CSPD and Sapphire II luminescence system to calibrate the quantitative standard with the national standard, and establishes a chemiluminescence immunoassay method for human serum AFP. Using the chemiluminescent enzyme immunoassay method, alkaline phosphatase was used as a marker, and the ring 1,2-dioxyethane derivative AMPPD was used as a luminescent substrate to measure the sugar antigen 50 in the serum, and the detection limit was 1.0 umL. The linear range is 0 to 300 umL.

The intra-assay precision is <7%, and the intra-assay precision is 11%.

1.3 Electrochemiluminescence immunoassay <br> The reaction of electrochemiluminescence immunoassay was carried out on the surface of the electrode, the luminescent substrate was tripyridinium, and the other reactant tripropylamine was used to excite the photoreaction. On the surface of the anode, these two substances can simultaneously lose electrons and undergo oxidation reaction. The divalent terpyridine ruthenium on the electrode plate is rapidly oxidized to trivalent terpyridine ruthenium, while the tripropylamine is also oxidized to the tripropylamine cation radical on the electrode plate, and the tripropylamine cation free radical spontaneously releases a proton. It becomes a tripropylamine unstable molecule, and an electron is transferred to the trivalent terpyridine oxime to form an excited state of the divalent terpyridine. The excited state of the divalent triplet pyridinium emits a photon having a wavelength of 620/l/n while attenuating, and returns to the ground state divalent terpyridine. This process is repeated on the surface of the electrode, resulting in efficient, stable continuous illumination and continuous enhancement. Zhang Jidong used electrochemiluminescence immunoassay to detect hepatitis B markers and found that it was detected by electrochemical method with higher sensitivity and more specificity.

2 new developments

New advances in chemical immunoassay methods are mainly reflected in the application of new markers and labeling techniques, the application of novel solid phase carriers, and the combination of techniques.

2.1 New Markers <br> In recent years, scientists have devoted themselves to the study of chemiluminescent substances by adding chemical enhancers to the luminescence system to increase quantum yield and luminescence time. At the same time, some scholars have focused on the development of new marker illuminants. And achieved corresponding results. It has been demonstrated that the anionic isoenzyme (HRP2A) of HRP extracted from palm trees and soybeans can efficiently and frequently catalyze the luminol-H2O2 system luminescence without an enhancer, and HRP. A is more stable than HRP-C, which is very important in the storage of enzyme markers. Leukocyte Alkaline Phosphatase (LAP), like AIJP, also has catalytic properties and has been used for chemiluminescence detection. In addition, acridine ester chemiluminescent reagents were synthesized by synthetic techniques. Zhuang et al. synthesized a novel bi-acridine compound: 10,102 dimethyl 23,32 disulfo 29,9'2 dipyridinium (DMDSBA), and studied its chemiluminescent properties in detail. DMDSBA was used to label anti-CEA antibodies. The ratio of the marks is close to 1.15 to 1.32, and the average mark ratio is 1.25. The results showed that the immunoreactivity of the labeled antibody and the quantum efficiency of DMDSBA did not change significantly after attachment to the anti-CEA antibody. A new sandwich chemiluminescence immunoassay method was established to determine the linear range of CEA in human serum from 1.0 to 100 ng/mL and the detection limit of 0.53 ng/mL.

2.2 Application of new labeling technology <br> Application of solid phase labeling method to reduce free label and polymerized protein; combination of nanotechnology and biological analysis, that is, using cations such as Au and Ag to catalytically amplify chemiluminescence and improve Detection sensitivity. HonglanQ et al. use electrochemiluminescence immunoassay to apply N. Aminobutyl-N. Isoluminol (ABEI) was labeled as a luminescent labeling reagent on a gold nanoparticles modified paraffin-activated graphite electrode to determine the human body [gG. The intensity of electrochemiluminescence is greatly enhanced. The detection limit of ABEI is 2×10 ^-14 mol/L (S/N=3), and the detection limit of lgG is 1×10. g/mL (S/N=3). The linear range is from 3.0 x 10 ^-ll to 1.0 x 10 ^-9 g/mL. The RSD at a concentration of 1.0 × 10 ^-10 /mL was 3.1%. It is indicated that the catalytic amplification of gold particle chemiluminescence has great application prospects in chemiluminescence immunoassay. Quantum dots (QDs), also known as semiconductor nanocrystals, are a type II. VI family or III. A semiconductor nanoparticle composed of a group V element capable of receiving fluorescence by a laser, having a diameter of about 2 to 6 nm. Compared with CLIA commonly used labeling enzymes and fluorescein, QD is not easy to be deactivated, has little influence from the external environment, is stable in nature, has good reproducibility, and has a wide spectral range. The emission spectrum is narrow and symmetrical, and it is a rare marker.

2.3 Application of new solid phase materials
The new solid phase material is mainly embodied in (1) a functional group bound to the protein on the surface, which can sufficiently immobilize the antibody antigen; (2) the surface property is ensured not to affect the immunoreactive activity of the bound protein; (3) the specific surface area is large enough to The immobilized sufficient amount of protein (4) should be hydrophilic to avoid non-covalent binding to the analyte and the sample matrix; (5) to maintain the antibody binding activity of the surface under the impact of the carrier liquid. Currently used for 丌. The solid phase materials mainly include nylon, agarose gel, magnetic particles, and silicone materials such as Controlled Poleglass (CPG) and Glass Beads.

2.4 Used in conjunction with other technologies
At present, the most research is capillary electrophoresis-chemiluminescence immunoassay (CECLIA) combined with the efficient separation of biological samples by CE and the high sensitivity detection of CLIA. Wang Junhua et al. Detection of zmol bone morphogenetic protein in rat vascular smooth muscle cells by capillary electrophoresis with immunochemiluminescence. 2, using horseradish peroxidase (HRP) to catalyze luminol. Hydrogen peroxide, which enhances its chemiluminescence reaction with p-iodophenol, has a detection limit of 4.4 pmol/L (53 zmol), which is one of the highest sensitivity reports for detecting HRP. Hepatitis B surface antigen and antibody in human serum were determined by capillary electrophoresis with chemiluminescence immunoassay, and compared with enzyme immunoassay, the feasibility of capillary electrophoresis chemiluminescence immunoassay for clinical use was verified. Flow injection chemiluminescence immunoassay Flow injection chemiluminescence analysis technology is a high sensitivity micro and trace analysis technology combining chemiluminescence analysis and flow injection. It has fast analysis speed and simple equipment, and is currently in the field of analytical chemistry. Research hotspots. The non-ionic surfactants were analyzed by sequential flow injection technique. The linear range was 0-1000 ppb, the minimum detection limit was 10 ppm, and the analysis time of each sample was 15 min. It has been successfully used for the detection of anti-ApnEOs in river water.

3 Conclusion

As a high-selection and high-sensitivity detection method, chemiluminescence immunoassay has been widely used in clinical testing and drug analysis. With the development of new luminescent reagents, new solid phase materials and the application of new labeling techniques, the sensitivity and reproducibility of chemiluminescence immunoassay methods will be greatly improved. The emergence of various combinations of technologies, such as capillary electrophoresis and chemiluminescence immunoassay, has greatly improved the selectivity and reproducibility of chemiluminescence immunity. It is widely used in clinical testing and drug analysis, and is developing towards automation, intelligence and miniaturization.

(Changchun University of Traditional Chinese Zhai Yan, Wang Hui)