Amplification Of Hepatocellular Carcinoma Associated Antigen And Liver Cancer-related Protein Antibody Preparation

Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors and is the second most common cause of cancer death in China. The incidence and mortality of HCC is found increasing year by year. Because of late diagnosis, the prognosis of HCC remains poor. The important measures to improve the survival rate of HCC are early detection, early diagnosis and early treatment.At present, there has the research on immune diagnosis and immune therapy of HCC, 11 kinds of cDNA encoding HCC-related antigens were successfully cloned from several different cancer cell lines, including MAGEA1, MAGEA2, MAGEA6, MAGEA10, GPC3, GP73, HCA519, HCA661, CTp11, TPTE and PTEN. The positive detection of MAGEA1 and MAGEA10 in HCC was 66% and 36.7% respectively, they are the promising candidate molecules for therapeutic vaccine against HCC. GPC3 and GP73 are the most promising candidate biomarkers for HCC diagnosis. Because their expression has no relation with AFP, combined detection of GPC3, GP73 and AFP could improve the diagnosis of HCC. HCA519 and HCA661 are two HCC associated antigen cloned by recombinant cDNA expression library serological analysis (SEREX) technique. HCA519 is a nuclear proliferation-related protein, which only exists in cells during cell cycle. HCA519 is highly expressed in hepatoma cells, and also expressed in prostate, lung and pancreas, but not expressed in normal tissues; HCA661 is a cancer-testis antigen, it is mainly expressed in testis, weakly expressed in normal pancreas, but not expressed in other normal tissues, it may be a vaccine candidate molecule. Because of the weak expression of HCA661 in normal pancreas, the pancreatic toxicity should be focused on when the therapeutic vaccine is designed based on HCA661. CTp11 was first discovered in melanoma cells, it was also found expressed in 62.9% of HCC. CTp11 has a potential HLA-A2 restricted CTL epitope and more than 50% of Asians express HLA-A2, therefore, CTp11 is candidate molecule for immune therapy for HCC. TPTE1 was found in 39% of HCC patients, while the anti-TPTE1 antibody in serum was found in more than 25% of HCC patients. TPTE1 might be used for HCC screening. As a tumor suppressor gene, the expression of PTEN was found decreased in HCC patients. PTEN may play an important role in preventing tumor invasion and the migration. PTEN can be used for gene therapy for HCC and other tumors.GPC3 is a new marker for HCC diagnosis and its expression has no correlation with AFP. For carrying out the study of immune diagnosis of HCC, the human serum soluble GPC3 (sGPC3) was expressed and purified on the basis of cloning of GPC3 cDNA. sGPC3 is the NH2-terminal portion of GPC3 cleaved between Arg358 and Ser359, and could be detected in the sera of patients with HCC. In order to make monoclonal antibody(McAb) to sGPC3, mice were immunized with purified sGPC3. When the serum antibody titers reached 1:106, the splenocytes from the immunized mice were fused with SP2/0 cells by PEG. The positive clones were selected by indirect ELISA. Two positive hybidoma cell lines (D1 and G6) stably secreting antibodies against sGPC3 were obtained after successive limiting dilutions. The McAb D1 and G6 were found to be of immunoglobulin IgG1 subclass, and had high specificity for sGPC3. The McAb D1 had higher affinity than G6, it could detect 10 ng/mL of sGPC3 while G6 could only detected 10μg/mL of sGPC3. The results of the competition ELISA indicated that D1 and G6 recognized different epitopes of GPC3, this meant that D1 and G6 could be paired for the use in sandwich ELISA for detection of sGPC3 in serum. In addition, D1 and G6 could also recognize the GPC3 proteins on cell surface.HCCR is another good marker for HCC diagnosis, which was first cloned from human cervical cancer by South Korea's Ko using differential display RT-PCR. Compared to normal people or patients with benign liver diseases, the level of HCCR protein was found increased significantly in the serum of HCC patients. Because HCCR cDNA was not successfully cloned, another strategy for generation antibody directed to HCCR was adopted. In general, the C-terminal peptide of protein has good accessibility, it is often good B cell epitope candidate for the corresponding protein antigen. The C-terminal peptide of HCCR (TNYLGTRR, 37c) also has good accessibility and hydrophilicity, and its sequence is unique analyzed by blasting in human protein library. This means that the C-terminal peptide of HCCR can be used as a specific tag for HCCR. Based on above analysis, the peptide was displayed on the surface of T7 phage, each T7 phage displayed 415 copies of the peptide. This recombinant T7 phage was used as the alternative antigen of HCCR to immunize two rabbits to make antiserum. After four times of immunization, the serum antibody titer reached 1:105. Serum from rabbit 1 had good specificity, it not only specifically recognized the C-terminal peptide of HCCR, but also specifically recognized HCCR167-360 protein, whereas the serum from rabbit 2 showed poor specificity. This may be related to the individual differences of animals.Hepatopoietin Cn (HPPCn) is a novel liver stimulating factor isolated from hepatic stimulatory substance (HSS), it belongs to LANP (leucine-rich acidic nuclear protein) family. HPPCn plays an important role in liver regeneration. It could activate the hepatocyte proliferation related signal pathway, and could alleviate CCl4–induced liver damage. For further study of the biological functions of HPPCn and analysis of its dynamic changes in liver diseases, it necessary to make McAb against HPPCn. The recombinant HPPCn was used to immunize female BALB/c mice. The hybridoma cell lines were established by cell fusion. After screening, a stable positive cell line W2-D5 was obtained. McAb W2-D5 possessed good specificity and high affinity. Its detection limit of HPPCn is 1 ng/mL. Using phage peptide library technology, the binding epitope of McAb W2-D5 was mapped to HPPCn7-13(IHLELRN).In summary, 11 kinds of cDNA encoding HCC-related antigen were successfully cloned. Of them, GPC3 was expressed and purified, and two specific, high affinity McAbs to GPC3 were generated, they could be paired for sandwich ELISA. Using the alternative strategy, the specific antiserum against HCCR was produced, it could be paired with mouse McAbs for sandwich ELISA for detection of serum HCCR. In addition, McAb W2-D5 to HPPCn was developed, it could specifically recognize HPPCn protein, its binding epitope was also confirmed.