Establishment Of A Nanobody RT-PCR Based On Ultra-sensitive Assay For Detection Of AFP

There is a heavy chain antibody with lack of natural light chain in camel source.Variable domain of heavy chain of heavy-chain antibody is consist of the heavy chain variable region(variable region) genetic engineering antibody, which is called nanobody because of its small weight. Compared with ordinary antibodies and recombination single antibody, nanobody has many advantages such as small molecular weight, water-soluble high, stable strong, easy to express etc. Therefore, it is applied in many fields.A fetoprotein(AFP) is mainly composed in the fetal liver that molecular weight is 69 KDa. AFP in healthy adult serum is below 30 ng/m L. However, AFP level apparently increase in serum when hepatocyte undergoes a cancer change. Since AFP is a specific clinical diagnosis of primary liver cancer. In this research,we construct sandwich Enzyme-linked immunosorbent assay to detect AFP by using nanobody that specifically bind to AFP. The main research contents are as follows:The establishment of the AFP immune library After Alpacas immunized four times with AFP, alpacas’ s serum was collected two weeks later and isolated hemameba.m RNA was extracted from hemameba. c DNA library was constructed by reverse PCR.We obtained VHH genes by nest-PCR. Using restriction enzyme NotⅠand SfiⅠ,VHH genes were inserted into p HENⅠ Vector. The recombination plasmids were transferred into E. coli TG1 by electrotransformation.Biopanning from immune Library Phages that specifically bind to AFP were obtained by Solid-phase biopanning, reducing AFP concentration in each round with alternate using BSA and OVA blocking agent. After three rounds selection, 95 clones were picked to bind AFP. Finally, P-A-18 /P-A-65/P-A-80 and P-A-83 were adapted with Anti-AFP Mc Ab to establish a sensitivity assay for detection AFP by Sandwich ELISA. The limit of detection is 0.45 ng/m L and the linear rang is 1-100 ng/m L.VHH expression pHEN-VHH plasmids that were extracted from P-A-18/P-A-65/P-A-80 and P-A-83 were inserted into p ET25b(+) vector by restriction enzyme NotⅠand NocⅠ. The recombination plasmids were transferred into E.coli Rosetta and induced expression by 0.05 m M IPTG in 30℃ for 11 h. N-A-18 andN-A-65 nanobodies were purified by Ni-NTA Resin and construct sandwich enzyme-linked immune assay with Anti-AFP Mc Ab to detect AFP antigen. The limit of detection is 0.65 ng/m L and the line range is 1-100 ng/m L.Establishment a double-nanobody assay to detect AFP by IPCR. N-A-18 as coated antibody was adapted with P-A-5 which is detecting antibody to establish a sensitive assay for detection AFP by sandwich Immunofluorescence polymerase chain reaction.The limit of detection is 5 pg/m L and liner range is 0.01-10000 ng/m L. The linear correlation coefficient is 0.9855. RT-PCR is applied in early cancer diagnose based double- nanobody.