title: A human kringle domain-based fluorescence-linked immunosorbent assay system.
abstract: As a non-immunoglobulin protein scaffold, human kringle domain (KD) has attractive properties such as high specificity, stability, and production in bacterial hosts. Here, we developed a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) system using a fluorescent kringle domain (fluoKD), a fusion protein of a green fluorescent protein (GFP), and a kringle domain variant (KD548). Two kinds of fluoKDs in which KD was fused to the N terminus of GFP (N-fluoKD) or the C terminus of GFP (C-fluoKD) were constructed and characterized. In Escherichia coli host, both fluoKDs were produced in high yield and solubility and were successfully purified by a simple procedure. The purified fluoKDs exhibited strong fluorescent activities and high affinities to the target antigen. Furthermore, it was successfully demonstrated that the FLISA with purified fluoKDs allowed for more rapid detection of target antigens with higher sensitivity compared with conventional enzyme-linked immunosorbent assay (ELISA), indicating that a simple, rapid, and sensitive immunoassay system could be developed by using KD instead of antibody or antibody fragments.
authors: Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors: Gu Min
authors: GM
authors: Jeong
authors: Department of Molecular Science and Technology, Ajou University, Yeongtong-gu, Suwon 443-749, Republic of Korea.
authors: Yong Sung
authors: YS
authors: Kim
authors: Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Yuseong-gu, Daejeon 305-701, Republic of Korea; Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: kjjeong@kaist.ac.kr.
authors: Ki Jun
authors: KJ
authors: Jeong
mesh_terms: D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018082:Kringles; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
chemical_list: D000906:Antibodies; D000941:Antigens; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: FLISA; GFP; Immunoassay; Kringle domain