Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody.
Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, an anti-BAFF single-chain antibody fragment (scFv) was genetically linked to the C terminus of the enhanced green fluorescent protein (EGFP) to generate an EGFP/scFv fusion protein. The EGFP/scFv fusion protein had an apparent molecular weight of 52 kDa and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. After being purified by immobilized metal affinity chromatography, the fusion protein exhibited similar fluorescence spectra with native EGFP. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorting showed the EGFP/scFv could bind to human soluble BAFF and BAFF positive cell lines in vitro. The binding of EGFP/scFv can also be visualized under laser scanning confocal microscopy. Furthermore, the results of the competition assay indicated its antigen binding specificity. Therefore, the fusion protein EGFP/scFv has several characteristics including high sensitivity, stability and convenience for manipulation, and can be a powerful tool for the study of the underlying pathology of BAFF relevant to autoimmune diseases.
Cao
Meng
M
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, Jiangsu, People's Republic of China.
Cao
Peng
P
Yan
Huaijiang
H
Ren
Fang
F
Lu
Wuguang
W
Hu
Yunlong
Y
Zhang
Shuangquan
S
D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D053264:B-Cell Activating Factor / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000906:Antibodies; D000941:Antigens; D053264:B-Cell Activating Factor; D011993:Recombinant Fusion Proteins; C505719:TNFSF13B protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins