Preparation Of Specific Antibodies Against GPC3 And Establishment Of Hepatocellular Carcinoma Ctc Detection Assay

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide.Its incidence is still increasing. Early diagnosis of HCC is very essential for HCC. Recently, it has been reported that GPC3 is overexpressed in hepatocellular carcinoma,but not expressed in normal liver and benign liver lesions. It indicates that GPC3 may be a novel marker for early detection of HCC. Liver cancer cells have the characteristics of invading into blood circulation easily, which maybe the main reason leads to matastasis, recurrence and poor prognosis of liver cance after HCC resection or liver transplantation. Therefore, CTC (Circulating Tumor Cell) detection in peripheral blood of HCC patients has great significance in diagnosis, stage judgement, treatment and prognosis of liver cancer. In this study, we prepared the specific polyclonal antibodies against GPC-3 and identified a monoclonal antibody against GPC3.We also Established a novel assay for hepatocellular carcinoma CTC Detection.1. Preparation and Identification of Specific Antibodies against GPC-3â‘ . GPC3-N polyclonal antibody preparation and identification: GPC3-N terminal truncated protein was expressed by gene technology. Total RNA was extracted in liver cancer cell line HepG2, and then GPC3-N terminal sequence was amplified by RT-PCR. Truncated GPC3-N terminal sequence (32-249aa) was subcloned into pQE30 vector and GPC3-N truncated protein were then expressed and purified in E.coli. Purified GPC3-N protein was used to immunize rabbit to obtain anti-GPC3 polyclonal antibody. Its titer could reach higher than 1:200,000 detected by ELISA. Rabbit polyclonal antibody was purified by ammonium sulfate precipitation method. Then its immunological character was identified by WB and Immunofluorescence technique. Results showed: the polyclonal antibody we prepared was antibody against GPC3-N recombinant protein with high specificity.Immunodominant epitopes of GPC3 protein were analyzed by biological analysis software. Two peptides (named LW1 and LW2) were synthesized according to the results and then were used to immunize rabbits to prepare polyclonal antibody. Titer of the antibody against LW1 could reach more then 1:200,000, higher then those against LW2 detected by ELISA. Anti-LW1 polyclonal antibody was purified by ammonium sulfate precipitation method. Immunofluorescence assay showed that the anti-LW1 polyclonal antibody could react with GPC3 protein specifically.â‘¡. Preparation and Identification of Monoclonal Antibody against GPC3: BALB /c mice were immunized by synthetic peptide LW1. After the third immunization, serum antibody titers reached 1:200000 detected by ELISA. Immunized mice with the highest titer were selected to produce monoclonal antibodies by spleen cell- Sp20 fusion technology. After scanning and identification ten cell line producing monoclonal antibodies were successfully obtained. Anti-LW1 monoclonal antibody was purified by using Protein G column. Western blot was used to detect the expression of GPC3 in liver cancer cell line HepG2 and Huh7 and normal human liver cell line L02 with purified monoclonal antibody as primary antibody. Results showed that anti LW1 monoclonal antibody can specifically recognize GPC3 protein expressed in liver cancer cell line HepG2 and Huh7 cells but not normal human liver cell line L02. Immunofluorescence assay showed that GPC3 was located in cell membrane and cytoplasm and only specifically expressed in liver cancer cell line HepG2, but not Human cervical carcinoma cell line Hela and normal liver cell line L02. The monoclonal antibody was also used in circulating tumor cells (CTC) detection and results showed it could be able to recognize CTC as well. In summary, anti-LW1 monoclonal antibody we prepared has very good specificity and provides a fundamental basis for further study on the biological function of GPC3 protein and its clinical application.2. Establishment of Hepatocellular Carcinoma CTC Detection AssayCirculating tumor cells (CTC) means the tumor cells which invade into blood circulation autonomously or caused by operation of diagnosis and treatment. Tumor cell mark referred to those membrane proteins that expressed abnormally when tumor cell grow or invade into blood circulation. CTC detection has great significance in tumor stage determination, metastasis and recurrence. In this section, we use immunomagnetic separation technique to remove the vast majority of white cells of cancer cell patients in peripheral blood circulation of patients so that remaining rare cells, including tumor cell, were enriched and then immunofluorescence techniques were used to identify tumor cells. We used cancer cells enrichment test to testify if the CTC collection method was effective. Results showed the rate of liver cancer cells enrichment from blood sample was 99% which indicates that our method was very effective to remove white cells in blood sample and enrich liver cancer cells exactly and stably. Blood sample of HCC patients from Youan Hospital were analyzed by the CTC detection method established by us. Results showed, positive rate of CTC was 26.7% in 30 cases of HCC patients in early stage; 94.12% in 17 cases of HCC patients in advanced stage who did not took chemotherapy within six months; but 16.67% in 6 cases of HCC patients in advanced stage who took chemotherapy within six month. It indicated that CTC detection was more sensitive in HCC patients in advanced stage. CTC detection showed positive in eight cases of HCC patients before taken liver transplantation surgery, which indicated that the possibility of recurrence after surgery might be increased. Only one case in 6 cases of HCC patients in advanced stage who took chemotherapy within six month showed positive in CTC detection and its CTC number was very low- just one. The other negative cases were samples collected from patients who took chemotherapy just one week ago. It showed that the change of CTC numbers may reflect the sensitivity of tumor against chemotherapy and our CTC detection method could reflect the effect of clinical treatment of HCC patients. In summary, our results showed CTC detection in HCC patients has great significance in diagnosis, evaluation of treatment effect, prognosis and prediction of recurrence.