Development Of Chemiluminescence Enzyme Immunoassay Methods For The Detection Of Hepatocelluar Carcinoma Tumor Markers And Their Applications

Hepatocellular carcinoma (HCC) in its early stage is asymptomatic, and consequently the diagnosis of HCC is often delayed. Thus, the mortality rate is still high for pateints with liver cancers because of poor prognosis. An early diagnosis is the key to an effective treatment of HCC and tumor markers play an important role in early clinical diagnosis of cancers. Chemiluminescence has attracted great attentions in terms of sensitivity and small footprint in bioananlytical field, and has been applied in clinical diagnosis with a great market share. In this work, several chemiluminescence enzyme immunoassay(CLEIA) methods have been developed for the detection of HCC tumor markers as well as their applications in clinics. The major works are as follows:1. The diagnostic methods for HCC and their development are reviewed. The application of chemiluminescence immunoassay for detection of tumor markers are introduced in detail. Then, we summarized whatever key works should be concerned and have a overlook about the application of CLEIA. 2. In order to improve the linear detection range and the sensitivity for the detection of AFP, magnetic particles with surface modification are employed, which can covently binding antibodies. The contents are briefly summarized as bellow,CLEIA was developed by alkaline phosphatase (ALP) labeling AFP antibodies for the detection of AFP. A sanwich immo-complex was formed by immunoreaction between FITC labled AFP monoclonal antibodies, AFP samples and ALP labled AFP antibodies. Magnetic micro-particles coated with anti-FITC antibodies were used as solid phase to separate immuno-complex from the liquid system. The linear detection range was enlarged to 1300 ng-mL-1 with a shorter immunoassay time compared with conventional CLEIA based on microplates.In order to decrease background interference and increase sensitivity, CLEIA with horseradish peroxidase labeling AFP antibodies was proposed for the detection of AFP. A one-step immunoassay was performed by reaction between magnetic micro-particles coated with AFP antibodies, AFP samples and HRP labled AFP antibodies to form sanwich immo-complex. Additionaly, polystyrene StartubesTM were used as solid phase for AFP antibodies coating in parallel with magnetic micro-particles. Our results indicated that the magnetic micro-particles based CLEIA showed better performance with less consumption of immunoreagents, larger linear detection range (2000 ng-mL-1) and shorter immunoassay time by comparing with StartubesTM based CLEIA. 3. AFP used in early diagnosis of HCC show low diagnosis positive rate, therefor more sensitive and specific tumor markers should be exlored in combination with or instead of AFP for the diagnosis of HCC. Glypican-3 (GPC3) highly expressed in liver tissues of HCC pateins, is a promising tumor marker for HCC, and its detection was often reported by molecular biochemistry. A serological immunoassay method was proposed in our work for detection of GPC3, and its application in clinical diagnosis was studied.Firstly, N-terminal and C-terminal GPC3 circulated in blood were simultaneously determinaed by competitive radioimmunoassay between 125I labeled GPC3, GPC3 samples and goat anti-human GPC3 polyclonal antibodies. The levels of GPC3 were evaluated in normal liver, hepatitis B, hepatitis C, hepatocirrhosis and HCC by our proposed method. We got that the levels of GPC3 were obviously higher in pateints with HCC than other benigh liver diseases. Addtionaly, the diagnostic sensitivity and specificity of GPC3 were also much higher compared with AFP and AFP/CA19-9 combination detection.Sencondly, in order to save assay time, avoid radio-pollution and increase sensitivity, magnetic nano-particles based CLEIA was proposed for detection of serological GPC3, and the performance of the method in terms of sensitivity and specificity were evaluated. The GPC3 levels in HCC after transarterial chemoembolization(TACE)treatment indicated that GPC3 was an effective tumor marker for prognosis of HCC patients. Additionally, the influence of nano-particles in immunoassay was compared with micro-particles. The results revealed that nano-particles based CLEIA exhibted better performance in terms of higher bioactivity, less consumption of immunoreagents and shorter immunoassay time than micro-particles based CLEIA.4. Simultaneous detection of HCC tumor markers was developed by CLEIA on microfluidics device. Glass substrate was processed with three-dimensional modification to improve antibodies coating efficiency. AFP and Ferri were simultaneously determined on microfluidic chip with high performance. No cross-reaction occurred and high specificity was obtained. The linear detection range for AFP and Ferri were 500 ng-mL-1 and 800 ng-mL-1, respectively.5. Capturing and detecting liver cancer cells circulating in peripheral blood were performed on a microfluidic chip by chemiluminescence. In order to realize early diagnosis and prognosis of HCC by counting liver cancer cells, immunocytochemistry, chemiluminescence and microfluidic chip were integrated together in this work with advantages of high specificity, sensitivity and time-saving.