Directed Biotinylation Of Nanobodies And Its Application In Detection Of Carcinoembryonic Antigen

Carcinoembryonic antigen?CEA?,as a biomarker of cancer,is of great significance in preoperative diagnosis and post-treatment monitoring of cancer patients.The general method for detecting carcinoembryonic antigen is to use a traditional monoclonal antibody?McAb?,which has high cost of clinical detection due to its high cost of preparation,transportation and preservation.Secondly,the traditional antibody labeling efficiency is unstable and the operation is cumbersome.Nanobody?Nb?is a small antibody that is gradually used in the diagnosis and treatment of diseases due to its small molecular weight,low immunogenicity,high solubility and easy modification.In this paper,nano-antibodies are used to detect CEA in a double-anti-sandwich system,in order to reduce costs,simplify the detection steps,and improve detection sensitivity.In the early stage of the laboratory,two nano-antibodies?Nb1 and Nb2?targeting different epitopes of CEA have been successfully screened by phage display technology,and their affinity reaches pM level,with good thermal stability and high specificity.The biotinylated tag?Avi?was added to the C-terminus of Nb1 by gene engineering,and cloned into three different expression vectors pMES4,pET47b and pGEX-6p-1 for expression,purification and identification.Finally,Nb1-AVI-pET47b strain was screened,which had good solubility and high expression,and the affinity parameter value was 1.706×10^-10,which was used for biotinylation.At the same time,the BirA enzyme gene was cloned by PCR,and then inserted into the pGEX-6p-1vector for expression and purification.A commercial enzyme?AVIDITY?was used as a control to detect the activity of the self-made enzyme before and after the GST tag was cleaved.Biotinylation results showed that the enzyme activity was comparable before and after excision of the GST tag,and not lower than the commercial enzyme.Targeted biotin catalysis of Nb1-AVI using a specific enzymatic reaction of the BirA enzyme in vitro and in vivo,both the HABA assay and the biotin-streptavidin system assay demonstrated that Nb1-AVI can be biotinylated.Further,a new generation of fluorescent quantum dots was used to couple Nb2,and a one-step method for detecting CEA double-antibody sandwich ELISA was established.The double-nanobody sandwich ELISA method developed in this study has a linear detection range of about 1⁸0 ng/mL for the standard in the immunoassay,and the lowest detection limit is 0.805 ng/mL.The linear curve shows an acceptable correlation coefficient R²=0.9937,the recovery rate of carcinoembryonic antigen in human serum samples was 96.6%¹03.7%.These results indicate that the biotinylated Nanobody can detect carcinoembryonic antigen in a biotin-streptavidin-based double-nanobody sandwich ELISA reaction,which is simple in operation,high in sensitivity,and more stable in optical properties.It lays the foundation for rapid and low-level detection of carcinoembryonic antigen.