Technological Improvement And Application Of Nanobody Library Construction

Since the unique functional heavy chain antibody(HCAbs)was discovered in the serum of camelids in 1993,the variable domain of heavy chain antibody(VHH),also know as nanobody,showes a great promise in a number of applications.How to increase the success rate of generation of the VHHs for small molecules is a challenge task.For this purpose,most efforts were focused on the following three aspects:hapten design and synthesis,different nanobody display technologies,novel biopanning strategy.However,most of previous studies for generation of high-affinity nanobodies were just based on optimizing extracellular experimental operations,such as improving the immune response,increasing the yield of expression in vitro and reducing the loss rate of biopanning.The success rate of generation VHHs specific for small compounds is still very low.Based on some successful experience and deficiencies from previous studies,an innovative strategy was designed to improve the success rate technology system for VHHs generation.(1)The distribution and transcript abundance of different allotypes in three kinds of camelus were studied.The distribution of different allotypes in camel,llama and alpaca were supplemented.The transcript abundances of HCAbs were higher than the protein abundances(62.9-97%vs 10-80%).However,camel possesses all seven allotypes and the highest transcript abundances of HCAbs.Therefore,camel is postulated to be a better source for the generation of nanobodies than other camelids.(2)A series of novel reverse primers were designed to enhance the diversity and quality of the VHH library.At the same time,a series of polymerases with different functions were evaluated to increase the yield of VHH gene amplifications.The importance of primer design and polymerase choice had been demonstrated.(3)Ultimately,based on the modified technology system of VHHs generation,several VHHs specific for triazophos and fipronil were isolated from the library derived from an immunized Bactrian camel.Enzyme linked immunosorbent assays(ELISAs)based on VHHs were developed for the detection of fipronil and triazophos.The half maximum inhibition concentration(IC50)of fipronil and triazophos for VHH F1 and VHH T1 were 3.68 and 6.6 ng/mL,respectively.The introduction of new genes to the VHH library was crucial since over 60%of identified positive clones were derived from allotype IgG3a.Therefore,it is possible that IgG3a is the most important for the VHH generation among all the allotypes.In addition,a new nucleotide sequence of hinge region was observed.This result may guide us to generate VHHs for small molecules with higher efficiency.(4)ELISAs based on the VHH F1/FLP2-BSA and VHH F2/FLP4-CON were developed to detect fipronil and fipronil-sulfone,respectively.The IC50 values of fipronil and fipronil-sulfone were 3.68 ng/mL and 31.84 ng/mL,respectively.The equivalent concentration of fipronil and fipronil-soufone from the serums of rat and prairie dog determined by the VHH ELISA correlated well with those from a HPLC-MS method,indicating the developed ELISA is suitable for the detection of fipronil and its metabolites in the sera...