Chemiluminescence Immunoassay Imaging

This thesis consists of two parts.In the first part, a review about chemiluminescence immunoassay(CLIA), the basicprinciples and new technologies of CLIA, the types and application ofchemiluminescence immunoassay imaging were summarized. CLIA is that usingchemiluminescence(CL) species or an enzyme as a label to study the reaction of antigenand antibody, and we can determine the CL intensity by affiliating the oxidant orenzyme to the chemiluminescence reaction after immunoassay reaction. In this way, theconcentrations of samples can be gained according to the CL intensity. Due to the mainadvantages of simple, high sensitivity, wide dynamic range, long activity of the label,non-radiation and automatic control, CLIA has raised worldwide interest. Combined tomany analytical methods such as flow injection, HPLC, capillary electrophoresis, andCCD (charge-coupled device) camera imaging, the application of CLIA could obtaindevelopment greatly. The current developments of CLIA are that they should synthesizenew labels in immunoassay analysis, develop new chemiluminescent systems that havemore sensitive and higher quantum yield.Combined CCD imaging technology to CLIA, Chemiluminescent immunoassayimaging-has the disadvantages of CLIA, and also has the advantages of high throughputand simultaneity detection of imaging device. It has been paid more and more attentionworldwide.The research part includes four sections. In the first section, a sensitive, simple andhigh throughput method has been used for quantitative analysis of several availablefluorescent probes (butyl rhodamine, rhodamine B, rhodamine 6G, tetramethylrhodamine isothiocyanate, fluorescein and fluorescein isothiocyanate and so on), and anew chemiluminescent system, bis(2, 4, 6-trichlorophenyl) oxalate (TCPO)-hydrogenperoxide (H₂O₂)-glyoxaline-fluorescent substance, has been used in this research. Theresults showed that TCPO Chemiluminescence imaging analysis has the advantage ofsensitivity of chemiluminescence and high throughput of imaging. It had good linearand the detection limit was 10^-11 mol/1. In the second section, horseradish peroxidase(HRP)-catalyzed fluorescent reaction has been combined to TCPO-H₂O₂-glyoxaline-fluorescent substance chemiluminescence imaging analysis, and these enzymed-catalyzed reactions contain HRP catalyze tyramine, o-phenylenediamine(OPD), 3-(4-hydroxyphenyl)-acetic acid(PHPPA) and the reduced forms of rhodamineB. Based on the advantages of enzymed-catalyzed reaction and chemiluminescenceimaging analysis, the quantitative analysis of HRP could be achieved by determinationof chemiluminescence intensity, and it had good linear and the determination limit was10^-14 g/ml. The investigation provides a possibility of using TCPO chemiluminescencesystem for immunoassay. Based on enzymed-linked immunosorbent(ELISA), andHRP-catalyzed fluorescent reaction, TCPO chemiluminescence imaging analysis hasbeen used for determination of interferon alpha(α-IFN) and carcinoembryonic antigen(CEA) in fresh human serum samples in the third and forth section. A typical "sandwichtype" immunoassay was used. The antigen standard or samples were added into the96-well plates coated with antibody. After incubation in 37℃, washing steps followed,and the HRP-conjugated anti-α-IFN monoclonal antibody was added to react withantigen. After incubation and washing steps, the fluorogenic substrate and oxidant wereadded into the microtitre, then an incubation steps followed. After addition of mixture,TCPO chemiluminescence substrates were added to determine CL intensity. TCPOchemiluminescence imaging measurements have been successfully used for analysis ofthe content ofα-IFN and CEA in human serum samples. Under the optimum conditions,the CL intensity was proportional with the concentration ofα-IFN and CEA in the rangeof 1.3 to156.0pg/ml(R²=0.9991) and 1.0 to 128.0ng/ml(R²=0.998), respectively. Thedetection limits were 0.8pg/ml and 0.5ng/ml(3σ), respectively. The relative standarddeviation(RSD) for nine parallel measurements of 25pg/mlα-IFN and CEA 32ng/mlwere 4.7% and 4.2%, respectively. Compared with ELISA, the main advantages of thisnovel method is sensitive, simple, rapid, high throughput and simultaneous detection.The results indicate that the proposed CLIA method for the determination of somesubstance is feasible in clinical diagnoses.