title: Human alpha-fetal protein immunoassay using fluorescence suppression with fluorescent-bead/antibody conjugate and enzymatic reaction.
abstract: The aim of the study was to develop a simple and rapid immunoassay using fluorescent microbeads and enzyme-substrate reactions to measure alpha-fetal protein (AFP) concentrations. We demonstrated the functionality of the fluorescent immunosensor using antibody-conjugated fluorescent latex beads (AB-FLBs) and horseradish peroxidase (HRP) to catalyze a reaction, where the products would precipitate and suppress the fluorescence of AB-FLBs. First, the AB-FLBs were incubated with antigen, biotinylated antibodies (bABs), and streptavidin-HRP (SAv-HRP) to form a sandwich-type immunoreaction. The mixture was then filtered through a membrane to concentrate the beads on a small area. After washing to remove unbound bABs and SAv-HRP, a chromogenic HRP substrate and H2O2 were added to form precipitates on the FLB surface. The suppression of the fluorescence was measured with a fluorescent image analyzer system. Under optimized conditions, AFP could be measured at concentrations as low as 1 pg mL(-1) with a dynamic range up to 100 ng mL(-1).
authors.affiliation: Department of Nano Manufacturing Technology, Nano Convergence Mechanical Systems Research Division, Korea Institute of Machinery and Materials (KIMM), Yuseng-Gu, Daejeon 305-343, Republic of Korea. Electronic address: ajh@kimm.re.kr.
authors.forename: Junhyoung
authors.initials: J
authors.lastname: Ahn
authors.affiliation: Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-Gu, Daejeon 305-806, Republic of Korea.
authors.forename: Yong-Beom
authors.initials: YB
authors.lastname: Shin
authors.affiliation: Department of Nano Manufacturing Technology, Nano Convergence Mechanical Systems Research Division, Korea Institute of Machinery and Materials (KIMM), Yuseng-Gu, Daejeon 305-343, Republic of Korea. Electronic address: jjlee@kimm.re.kr.
authors.forename: JaeJong
authors.initials: J
authors.lastname: Lee
authors.affiliation: Biosensors and Bio-Photonics Laboratory, Department of Chemistry, Gwangju Institute of Science and Technology (GIST), Buk-Gu, Gwangju 500-712, Republic of Korea. Electronic address: mkim@gist.ac.kr.
authors.forename: Min-Gon
authors.initials: MG
authors.lastname: Kim
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D011232:Chemical Precipitation; D004867:Equipment Design; D005453:Fluorescence; D005455:Fluorescent Antibody Technique / Q000295:instrumentation / Q000379:methods*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D018796:Immunoconjugates / Q000737:chemistry*; D057230:Limit of Detection; D000509:alpha-Fetoproteins / Q000032:analysis*
chemical_list: D018796:Immunoconjugates; D000509:alpha-Fetoproteins; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase
keywords: Alpha-fetal protein (AFP); Enzyme-catalyzed precipitation; Fluorescence suppression; Fluorescent immunoassay