title: Development of nanobody-based flow injection chemiluminescence immunoassay for sensitive detection of human prealbumin.
abstract: Nanobodies, derived from camelid heavy-chain antibodies, have novel and impactful applications in clinical diagnostics. Our objective is to develop a nanobody-based chemiluminescence immunoassay for sensitive detection of human prealbumin (PA). In this context, a phage display nanobody library is constructed via immunizing dromedary camel with human prealbumin. Three nanobodies have been identified by five successive bio-panning steps. Based on their high expression level and good affinity, two out of three are chosen for further study. Magnetic beads (MBs) were functionalized with PEI by acylamide bond formed between the carboxyl group on the surface of the MB. Then, an anti-PA nanobody (Nb1) can be effectively immobilized onto the surface of the functionalized MB using glutaradehyde as the link. The modified MBs with Nb1 can specifically capture the target PA and reacted with silica nanoparticles with co-immobilized HRP and anti-PA nanobody (Nb2). The concentration of PA was detected by flow injection chemiluminescence. When using MB/PEI as the carrier of anti-PA Nb1, the CL signal significantly increased to 4-fold compared with the signal using MB without PEI modification. The CL signal was further amplified to 5-fold when Si/Nb2 was used as the signal probe. Under optimized conditions, the present immunoassay exhibited a wide quantitative range from 0.05 to 1000 μg L(-1) with a detection limit of 0.01 μg L(-1). The sensitivity of the proposed immunoassay offers great promises in providing a sensitive, specific, time saving, and potential method for detecting PA in clinical settings.
authors.affiliation: Key Laboratory of Child Development and Learning Science (Ministry of Education), Research Center for Learning Science, Southeast University, Nanjing 210096, P.R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Ma
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, P.R. China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Key Laboratory of Child Development and Learning Science (Ministry of Education), Research Center for Learning Science, Southeast University, Nanjing 210096, P.R. China. Electronic address: xjkang64@163.com.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Kang
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, P.R. China; Jiangsu Nanobody Engineering and Research Center, Nantong 226010, P.R. China. Electronic address: ywan@seu.edu.cn.
authors.forename: Yakun
authors.initials: Y
authors.lastname: Wan
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation; D002162:Camelus; D017022:Flow Injection Analysis / Q000295:instrumentation*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation*; D057230:Limit of Detection; D008163:Luminescent Measurements / Q000295:instrumentation*; D019151:Peptide Library; D011228:Prealbumin / Q000032:analysis*; D061905:Single-Domain Antibodies / Q000737:chemistry*
chemical_list: D019151:Peptide Library; D011228:Prealbumin; D061905:Single-Domain Antibodies; D006735:Horseradish Peroxidase
keywords: Flow injection chemiluminescence immunoassay; Human prealbumin; Nanobodies