期刊
BIOSENSORS & BIOELECTRONICS
卷 17, 期 4, 页码 305-313出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/S0956-5663(01)00292-5
关键词
F-ab; single-chain F-v; immobilized metal affinity chromatography; s-triazine; immunoassay; antibody stability
Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected singic-chain F-v (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy, was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F-ab fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F ab fragment in a high cell density fermenter was induced by addition of anhydrotetracycline, The F-ab fragment was subsequently purified from the periplasmic extract in a singe stop by immobilized metal affinity chromatography (IMAC). A yield of 100 mug/l x OD550 purified F-ab fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F-ab was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F-ab fragment exhibited significantly improved long-term stability. (C) 2002 Eisevier Science B.V. All rights reserved.
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