Studies On Novel Immunoassay Methods For Tumor Markers And Clinical Applications

The determination of serum tumor markers (TM) plays an important role in screening and auxiliary diagnosing a disease, monitoring therapy in the tumor prevention stage, as well as in the follow-up examination during therapy for the patients with certain tumor-associated diseases. The developments of new TM and novel immunoassay methods have been the focused object in tumor diagnosis. Flow injection analysis (FIA) has been applied to many fields such as clinical, pharmaceutical, environmental and food assays due to the shortened assay time, good reproducibility and easy automation for high sample throughput in non-equilibration state. The immunosensors based on electrochemical, chemiluminescent and time-resolved fluorescent detection methods have been interestingly researched due to the high sensitivity, characterization and accuracy. The combination of the different detection techniques and immunoassay methods has been the basis for the efficient determinations for the TM. This thesis focuses on the clinical application of TM, flow injection- time resolved fluorescence and the preparation of immunosensors based on the electrochemical and chemiluminescent detection.1. Detection and clinical significance of serum progastrin-releasing peptide in patients with lung cancerProgastrin-releasing peptide (ProGRP) is one kind of brain-gut hormone, and the precursor of gastrin-releasing peptide. The enzyme immunoassay method for ProGRP has been developed and applied in the diagnosis of small cell carcinoma. The levels ofserum progastrin-releasing peptide ProGRD in 78 patients with lung cancer were detected by using ELISA and compared with healthy controls and lung benign group to evaluate and probe into their clinical significance in distinctive diagnosis of lung cancer. The serum ProGRP levels in patients with small cell carcinoma (SCLC) were obviously higher than those of controls (P<0.01). Using 46 pg/ml as cutoff value the positive rate of ProGRP for SCLC was 64.6%. There was a proportional relation between ProGRP and NSE levels with a correlation coefficient of 0.9 (P<0.01). The detection of serum ProGRP was useful in auxiliary diagnosis of small cell lung cancer.2. Flow injection immunoassay for carcinoembryonic antigen combined with time-resolved fluorometric detectionA simple, sensitive and specific method has been developed for immunoassay of serum carcinoembryonic antigen (CEA) by combining the time-resolved fluorescence with high sensitivity and flow injection analysis with high flexibility. Based on a sandwich immunoassay format, a monoclonal antibody immobilized immunoaffinity column inserted in a flow system was used for immunoreactions. The Eu-labeled antibody and analyte antigen were injected into the immunoaffinity column respectively to form the sandwich immunocomplex. The enhancement solution that was used to cleave the Eu-labels from the immunocomplex was injected into the column after the above immunoreaction. Finally, the cleaved solution was collected and then detected by time-resolved fluorescence. Serum CEA could be detected in the linear range from 2.5 to 100 ng/ml with a correlation coefficient of 0.997 and the detection limit of 1.0 ng/ml. The whole assay time for one sample was 25 min. The coefficient variations for the concentrations of 2.0, 10 and 50 ng/ml were 3.5%, 1.0% and 7.65% for 10 times detection. Twenty-four human serum samples have been detected by this method were in good agreement with the results obtained by the electrochemiluminescence immunoassay (Elecsys(?)2010). This method realized the basic requirements for rapid, low-cost, sensitive and accurate assay and could be further developed for fast practical clinical detection of serum CEA levels.3. Chemiluminescent and electrochemical immunosensors for the tumor markersA chemiluminescent immunosensor for carbohydrate antigen 19-9 (CA19-9) based on the immobilization of CA19-9 on the cross-linked chitosan membrane wasdeveloped. Based on a noncompetitive immunoassay format, after an off-line incubation of the analyte CA19-9 with ho...