| Primary carcinoma of the liver(PCL)is a kind of malignant tumor which is very common in clinic and has a very high mortality rate,which seriously threatens the health of modern people.Hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma and hepatosarcoma are divided into hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma and hepatosarcoma,among which hepatocellular carcinoma is the most common in clinical practice with a high incidence.It accounts for more than 90%of liver cancer incidence.HCC has a very high fatality rate among malignant tumors,ranking the fourth among cancers.Its early onset symptoms are not obvious,and most patients have advanced disease when they are diagnosed.Therefore,early diagnosis and treatment of HCC are crucial for postoperative rehabilitation and survival time of patients.Serological examination is the main means of early screening and diagnosis of liver cancer due to its low damage and low price.At present,alphafetoprotein(AFP)is one of the main markers in the serological diagnosis of HCC,but its accuracy and sensitivity are relatively low.It is of great significance to search for independent markers that can be combined with AFP in the early diagnosis of HCC.Vasorin(VASN)protein is involved in the repair of blood vessels in human body and the occurrence of tumors,and also plays an important role in the development of tumors.VASN protein is higher in smooth muscle cells in aortic blood,only in liver,placenta,and higher in intracellular cells and serum specimens in hepatocellular carcinoma,and may be used as a potential candidate marker for HCC.In this study,the company synthesized pc MV3-VASN recombinant plasmid was identified by PCR,transferred to DH5αreceptor cells for plasmid amplification,then transiently transfected into HEK-293F cells,purified by magnetic bead method to obtain VASN protein,and validated by Western blot analysis of VASN protein.The target protein was used to immunize mice,and hybridoma cells that stably secreted antibody were screened by PEG-4000-induced cellular chemical fusion.The prepared anti-human VASN monoclonal antibody was subjected to potency,subclass,affinity assay and western blot identification analysis.The four VASN monoclonal antibodies were labeled with horseradish peroxidase(HRP),and the antibody with higher enzyme potency was paired with the unlabeled monoclonal antibody to screen the antibody pairing combination with the best detection effect,determine the optimal working concentration of the captured antibody,the incubation time of the antigen and enzyme-labeled antibody,and finally perform sensitivity testing to establish the VASN protein double antibody sandwich ELISA assay.The main experimental results were as follows:(1)The eukaryotic pc MV3-VASN expression plasmid was successfully constructed and transformed into DH5αEscherichia coli receptive cells for expression.After transient transfection into HEK-293F cells and purification,VASN protein can be 95.4%pure.(2)(2)Six hybridoma cell lines with stable secretion of anti-human VASN antibody were screened by PEG-4000 induced cell fusion and named as 4E3,4F7,4G10,1H12,2F9 and 4H6,respectively.The most effective antibody price can reach 1:25600.The results showed that 4E3and 4G10 were Ig G2a.The subclasses of 4H6 and 2F9 antibodies were Ig G3 type.The subclass of 1H12 antibody was Ig G2b.The subclass of 4F7 antibody was Ig G1 type.The affinity constants of 2F9,4G10,1H12,4F7,4E3 and 4H6 were 1.2×10~6L/mol,4.9×10~6L/mol,1.6×10~6L/mol,1.5×10~6L/mol,5.2×10~6L/mol and 5.1×10~6L/mol,respectively.The highest affinity constant of 4E3 is 5.2×10~6L/mol.(3)Four monoclonal antibodies were labeled with HRP,namely 4E3,2F9,4F7 and 4G10,of which the enzyme-labeled monoclonal antibody 2F9 had the highest potency of 1:25600,and the antibody pairing of which two pairs were successfully paired,namely 4F7 and 4E3,2F9 and4E3,of which 2F9 and 4E3 were paired with the best detection effect.The optimal working concentration of VASN monoclonal antibody 2F9,antigen VASN protein and enzyme-labeled antibody 4E3 incubation time were 4μg/m L,1 h and 45 min,respectively,and the optimal dilution of enzyme-labeled antibody 4E3 was determined by tessellation titration method to be1:6400 times,and the antibody sensitivity was detected by preliminary establishment of the double antibody sandwich ELISA assay using its method,and its linear regression equation was y=0.6962x-0.2195,R~2=0.9909,and the method was used to detect the VASN standard protein with a minimum detection concentration of 4 ng/m L.In conclusion,this study prepared monoclonal antibodies against human VASN with high accuracy and strong specificity,which laid a foundation for the establishment of a new method for the early diagnosis of HCC. |
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