The Preparation Of Immunomagnetic Microspheres And Application In Breast Cancer Diagnose Research

At present, cancer is increasingly becoming one of the most troubling diseases of human beings. Now, the morbidity of cancer is about 22%. So, it is an urgent task for people to find out some effective measures for treatment of cancers. However, most of the cancers have gone into their late stage when have been found out because of being not easy to be diagnosed in their early stage by the current medical method. In the late stage, cancer cells have basically diffused and hard to be killed completely. The early the cancer is diagnosed, the higher the possibility of it to be cured is. So finding out cancer cell earlier become very important for the treatment of it. At the first stage of cancer occurrence, the disordered growth of the tumor cells makes them lost the ability to adhere with other cells. The loose tumor cells can go into the blood vessel and lymphatic vessel and enter the blood circulation or lymphatic circulation. The cells transferred by blood are called circulating tumor cells; CTCs. At the early stage of the tumor transferring, circulating tumor cells can go along the circulation system to destroy the distant organs, and become the metastasis gradually. When we use current, conventional and empiric diagnostic tools, we can't see this transferring process. So, it is very important to find a new method to measure transferring tumor cells. Many researches proved that assaying circulating tumor cells exist in a small amount of blood has been an obvious improvement to evaluate the response to the treatment accurately and efficiently for each independent patient after cured. Obviously, single cell level assay of peripheral blood's circulating tumor cells is more sensitive than imaging analysis. Recently, specific antibody which is coated on the microspheres for combining with circulating tumor cells is used in some advanced technology. Breast cancer cell existed in human breast in vivo is relatively much easier to come off and become the circulating tumor cells under the external mechanical force than other cancer cell to do so. Therefore, using specific immunomagnetic microspheres to capture the circulating tumor cells of the breast cancer in the earlier period for early detection is more convenient and accurate than that of other cancer cells. In this research, we will study the synthesis of micron immunomagnetic microspheres and its functionalization. The focus of functionalization of micron immunomagnetic microspheres is to obtain the most appropriate synthetic condition, high promiscuous cells'removal rate, high circulating tumor cells capturing rate and so on. Firstly, the most appropriate microspheres for making micron immunomagnetic microspheres will be get by analyzing microspheres'particle size, magnetic response, surface carboxyl content etc. Then immunomagnetic microspheres complex will be synthesized by covalent coupling method. Finally, this complex will be used to capture target cells, and to remove cells and enrich circulating tumor cells. Specific research methods and results are summarized as follows:Transmission electron microscope and scanning electron microscope were used to determine microspheres'particle size and observe their morphology. Conductivity of the micron immunomagnetic microspheres represents its surface carboxyl content. The suitable magnetic microsphere will be picked out by the average size of about 10μm, better ball formation ability and about 0.1~0.5mmol/g surface carboxyl content. To make micron immunomagnetic microspheres complex, certain oscillation conditions and the covalent coupling method will be used. Spectrophotometer will be used to measure the binding rate of neutravidin. The study showed that the suitable condition of the synthesis of neutravidin-magnetic polymer microspheres was at 4℃of the synthetic reaction temperature, 0.2mg/ml density of neutravidin, 2h reaction in pH 5.8 of PBS and its capacity of neutravidin was 25~55mg/g. For making biotinylated antibody, the highest binding rate was get when BNHS and antibody mass ratio was at 1:7 and the highest coupling ratio was get when neutravidin and biotin molar ratio was at 1:3. Anti-CD45 immune magnetic polymer microspheres'leukocyte removal was up to 78%, and microspheres'recovery rate was 98%.