Liver Cancer Candidate Biomarkers The HCCR Monoclonal Antibody Preparation And Preliminary Applications

Hepatocellular carcinoma(HCC) is one of the most common tumors in the world. 500 000 new patients with HCC were diagnosed annually. The incidence of HCC in developing countries(>20 per 100 000) is higher than that in developed areas(<20 per 100 000), and it is increasing year by year. In early stage of HCC, no obvious symptom can be found, HCC is usually discovered too late to treat.To date, serum AFP assay is the first choice for HCC screening, but the diagnostic sensitivity is low. In small HCC(<3cm), the sensitivity of AFP ranged 26. 9 %?39. 3 %, while in smaller HCC(<2cm), it varied from 17%?42%. So it is necessary to explore new biomarkers for HCC diagnosis to improve the early detection of HCC.The rising incidence of HCC worldwide has sparked a renewed interest in HCC serum markers. Human Cervical Cancer Oncogene (HCCR), a new candidate bio-marker for HCC, was first reported by Ko and his colleagues in 2003. HCCR has two subclasses,HCCR-1 and HCCR-2. They comprise a consensus sequence except HCCR-2 lacks exon 1 of HCCR-1. Now, HCCR was found not only expressed in cervical cancer but also in many other different tumors. This hinted that HCCR is associated with the genesis and development of many tumors. Yoon reported that HCCR was over-expressed in HCC, moderately-expressed in cirrhosis, but not detected in normal liver tissues. The diagnostic accuracy of HCCR for HCC was higher than that of AFP, its sensitivity and specificity were up to 78.2% and 95.7%, respectively. Its positive rate in small HCC of <2cm was up to 69. 2 %,significantly higher than 46. 2 % of AFP。In this paper, we hope to make the anti-HCCR antibodies with high specificity and sensitivity, and to establish a new serum diagnostic method for HCC to improve the diagnostic sensitivity and accuracy for HCC.HCCR-1 is a typeⅡtransmenbrane protein, the segment of 167th-360th amino acids (HCCR-1167-360) exposes outside of cell membrane, it could be released to blood when liver lesions occurred. In this paper, HCCR-1167-360 was used to be the antigen to make antibody. Firstly, the coding sequence of HCCR-1167-360 was cloned from HepG2 cells, and was inserted in pET-28a vector to construct the expression vector. After induction, HCCR-1167-360 was highly expressed as inclusion bodies, and could not be refolded. For obtaining of the immunogen to make antibody, HCCR-1167-360 was separated by SDS-PAGE, and the gel containing HCCR-1167-360 proteins was cutting and collected to use to immunize Balb/c mouse. After three times of immunization, the serum antibody titers had reached 1:104. The results indicated that the renatured recombinant proteins still were good immunogens for antibody generation.Another recombinant protein Ep-HCCR, which displayed an HCCR epitope(YLGTRR, the 355th-360th aa of HCCR-1) was constructed and purified for the uses of detection of serum antibody titers and screening of the positive hybridomas.The immunized mouse splenocytes and SP2/0 cells were fused by PEG-1500. The positive clones were screened by ELISA, and subcloned by dilution methods. Finally, one hybridoma clone, which secreted anti-HCCR mAb, was obtained. The anti-HCCR antidody is IgG1 subclass identified by Sigma IsoQ5 test . The anti-HCCR antibody was characterized by ELISA, Western blot, immunofluorescence and immunohistochemistry.The ELISA analysis indicated that the anti-HCCR antibody specifically recognized Ep-HCCR protein, and showed no cross-reaction with other proteins. The detection limit of the antibody reached 10ng/ml of Ep-HCCR protein, and the antibody titer of ascites reached 1:104.The affinity constant of the monoclonal antibody(McAb) was 5.4×106 L/mol, analyzed by indirect ELISA according to the formula Kaff=(n-1)/2(n[Ab?]t?[Ab]t).The HCCR expression in HCC cell lines and other human tumor cells was detected by Western blot. HCCR was found high-expressed in HCC cell lines including HepG2, BEL-7402, 7721 and BEL-7402, while no expression was detected in normal hepatic cell line L02. HCCR was also found differently expressed in colon cancer cell line Lovo, breast cancer cell line MCF-7, lymphoma cell line Raji and cervical cancer cell line Hela, but no expression was found in ovarian cancer cell line OV3. These results implied that HCCR was not specific for HCC, it was probably involved in genesis and development of many tumors. Immunofluorescence results showed that the McAb could recognize HCCR protein expressed in plasma membrane and cytoplasm of HepG2 cells. These were consistent with those reported by others. These results indicated that the McAb could recognized the expressed HCCR proteins in cells.The McAb could also react with the expressed HCCR protein in HCC tissues, while no expression was detected in normal liver tissues.The ELISA method for detection of HCCR protein in serum was established. Using this method, the serum HCCR statuses of 121 cases of HCC patients and 20 cases of healthy individuals were analyzed. The results showed that positive rate of HCCR in HCC patients reached 71.07% (86/121), which was higher than 59.50% (72/121) of AFP. The positive rate of union detection of HCCR and AFP reached 70.97%(44/62). In 62 HCC patients with HBV/HCV infection, the positive rate of HCCR was 70.97% (44/62), in contrast, in patients without HBV/HCV infection, the positive rate was 71.17%. They were no different. This meant that HCCR expression was not related to HBV/HCV infection. The positive rates of HCCR in HCC patients inⅠ,Ⅱstage andⅢ,Ⅳstage were 71.08% and 71.05%, respectively, this hinted that HCCR expression was not associated with tumor clinical stage.The serums from HCC patients were stored in 4℃and room temperature for 48 hours, and then detected their HCCR proteins. The results were not significantly different. This indicated that HCCR protein was relatively stable in short storage period. In addition, the serum GP73 of HCC patients was also analyzed. Its positive rate reached 80.99%. The positive rate of union detection of HCCR, AFP and GP73 reached 97.51%. This demonstrated that HCCR had good complementarity with AFP and GP73 for the detection of HCC.In conclusion, one hybridoma cell line secreting McAb against HCCR protein was obtained by immunizing mouse with the gel containing HCCR-1355-360 protein. The McAb could be used in ELISA, Western blot, immunofluorescence and immunohistochemistry. It could effectively recognized HCCR protein in cells and tissues. The positive rate of HCCR in HCC patients was higher than that of AFP, and HCCR showed good complementarity with AFP and GP73, this laid the foundation for future development of good methods for HCC diagnosis.