Preparation And Identification Of Monoclonal Antibody Against Human Growth Differentiation Factor 15

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; especially the early diagnosis.At present, HCC conventional screening and diagnosis methods include physical examinations, imaging technologies such as ultrasonography and blood serum Alpha fetoprotein (AFP) examination. AFP is a clinically useful tumor marker for many years, However, it has been recognized that AFP has poor sensitivity and specificity in the detection of HCC.therefore,it is essential to develop new HCC diagnose markes.In our early research ,we found that there were 12 genes include gdf15(growth differentiation factor 15) and ctgf(connective tissue growth factor) expressed abnormally in rat early HCC by gene chips. Subsequently, we have confirmed that GDF15 were over-expressed at the transcript and protein level both in rats and humans.The results suggest that GDF15 may become a new potential HCC diagnosis marker.OBJECTIVE:To prepare the monoclonal antibody (mAb) against GDF15 and identify the biological characteristics of the mAb.This research will provide the basis for further developing HCC diagnose kit.METHODS:1. RT-PCR technology was used to amplify the sequence of gdf15 .The expression vector pGEX-4T-2-gdf15 was constructed and transformed into E.coli Top10F'for expression by IPTG. The fusion protein was identification by Western blot.Then; positive colnes were transformed into LB for massive expression. Affinity chromatography method was used to purify the GST-GDF15 fusion protein.2. The purified GST-GDF15 fusion protein was used to immunize the BALB/c mice. Then, the mouse myeloma cells (Sp2/0) were fused with spleen cells from BALB/c immunized by the purified protein. Subsequently, three times limited dilution method was used to screen hybridoma cell lines.3. The titer of the mAb was detected by ELISA and its specificity was analyzed by Western blot. Isotypes and subclasses were identified by co-immunoprecipitation(IP), mass spectrometric analysis and immunohistochemistry (IH) were detected whether the mAb can combine with human native GDF15 protein. Affinity chromatography method was used to purify the mAb.RESULTS:1. The expression vector pGEX-4T-2-gdf15 was constructed successfully.2. The GST-GDF15 fusion protein was successfully expressed and purified. The concentration and purity of the protein is 2.1mg/ml and more than 80% respectively.3. One hybridoma cell line designated 9G3 against GDF15 was obtained. And the anti-GDF15 mAb 9G3 belonged to IgG1 subclass. IP and mass spectrometric analysis revealed that the mAb could combine with GDF15 in human serum with high specificity. Futher more, the mAb can be used for IH. And the staining of GDF15 was mainly located in the cytoplasma of HCC. The concentration and purity of the 9G3 mAb is 5mg/ml and more than 85% respectively. CONCLUSION:1. The expression vector pGEX-4T-2-gdf15 was constructed and GST-GDF15 fusion protein was expressed and purified successfully.2. GST-GDF15 fusion protein was used as antigen and we successfully prepare a anti-GDF15 mAb which could be appllied to Western blot and IH.The mAb preparation provides the basis for further developing HCC diagnose kit.