Generation, Characterization And Applications Of Nanobodies Specifically Against Human Serum Albumin (HSA)

Human serum albumin (HSA) plays role as vital biomarkers for disease-re la ted diagnosis, and its depletion approaches in plasma proteomic analysis and other biomarker discovery also have received much attention. Therefore, the importance of the methodological study of the HSA detection has been highlighted and effective depletion method for further study supposed to be developed.We constructed an immune phage display naobody library after immunization of a camels with HSA. By taking advantage of phage display technology, PE-ELISA, and sequencing, HSA-specific Nbs can be obtained. After expression and characterizing by a series of analysis, detection method would be developed. And for removal of HSA from serum, we would use these Nbs combined with NHS-Activated Sefinose Resin to assemble a spin tube filter.The immune phage display libraries were successfully constructed and four HSA-specific Nbs were obtained. After performing further characterization, these Nbs demonstrated good quality:high purity, good yield, strong specificity and high affinity. As for detection of HSA, the absorbance at 450 nm displayed a linearity with the concentrations of HSA ranging from 10 to 1000 ng·mL-1 and the minimum detection limit was calculated as 21.2 ng·mL-1. Finally, HSA in the blood serum sample was successfully depleted by means of nanobody-assembled resins.In conclusion, we successfully generated and characterized a number of nanobodies against HSA. And a directional, rapid and sensitive immunoassay for HSA detection was developed. Furthermore, an effective method for removal of HSA from serum have been established mainly based on Nbs and NHS-Activated Sefinose Resin. Our work indicated a promising future in further antigen detection and diagnostic applications.