| Electrochemiluminescence immunoassay (ECLJA) is a new tag immunology method. It has been won the attention because of the advantages of high sensitivity and better selectivity. Some foreign teams had been at the work on the exploiture and application of ECLIA. And some companies had made the apparatus but with high price. Few teams in our contrary have been studied in the field of ECLIA. So we investigated the status and progress of the electrochemiluminescence (ECL) and ECLIA. Then some studies in improving the ECL efficiency and making a high-sensitive ECLIA device was done. At last, we used the analyzer to detect the concentration of p53 antibodies in human sera. The results are primary but significative, suggesting that ECLIA is a feasible method for clinical diagnosis.Firstly, a high-sensitive ECLIA device was improved. In the device, a single photon multiplier rube was used to detect the weak photon signal. And a quartz fiber bundle was used between the working electrode and PMT to avoid the influence of magnet under the working electrode. Thus, make a high efficiency coupling of photons; the detection efficiency was obviously improved.To study the problems of ECL efficiency and detection efficiency, three kinds of platinum electrodes with different geometry, such as round disk, four-hole round disk and spiral thread, were designed. The relation of the ECL intensity with the geometry of these electrodes has been investigated. The optical character of these electrodes was directly studied by observation of ECL image and measurement of the ECL intensity. The results showed that the ECL not only concentrated on the edge of the electrode but also concentrated on the edge of holes, which contain in the electrode. The detection limit was improved to 10-18 mol/LRu(bpy)3C12 6H2O on four-hole round disk electrode, three times higher than that on round disk electrode. So we concluded, given an identical surface area, when the length of the edge is added, the surface area of per gram is increased; the ECL efficiency can be improved. Results showed that the dynamic linear range extended over five orders, from 10-13 mol/L to 10-8 mol/L.In the experiments of ECLIA, the ECL probe-Ru(bpy)32+-NHS ester was chemosynthesized by ourselves. From the studies of the infrared spectroscopy and ultraviolet-visible absorption spectra, we calculated the yield was 75.2%.Furthermore, we applied the ECLIA in tumor early diagnosis. We designed a new method to detect p53 antibodies in human sera. The results suggested that ECLIA is a feasible method for clinical diagnosis. We used ECLIA to detect an extremely low concentration p53 antibodies introduced in normal human sera. The calibrated assay was then used to quantitatively evaluate the presence of p53 antibodies in cancerous human sera that was determined with conventional diagnosis. The positive rate of p53 antibodies was 28.6% in lung carcinoma and 8.33% in nasopharyngeal carcinoma, respectively. P53 antibody concentration in the carcerous human sera was quantified from the calibration curve. To lung carcinoma, a trend was found that higher the p53 antibody concentration in the serum likely linked to a higher stage of the cancer.In conclusion, results of the study suggested that ECLIA is a simple, rapid, sensitive method. This method could provide either qualitative or quantitative results for analysis clinical samples. It is a feasible method for rapid and sensitive detection of p53 antibodies in human serum. ECLIA, thus, may provide a new means for quantitatively evaluated the production of p53 antibodies. |
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