Expression and purification of an anti-clenbuterol single chain Fv antibody in Escherichia coli.
Protein Expr Purif
; 72(1): 26-31, 2010 Jul.
Article
em En
| MEDLINE
| ID: mdl-20206697
ABSTRACT
Recombinant antibodies with desirable characteristics that can replace polyclonal or monoclonal antibodies are important for enzyme-linked immunosorbent assay (ELISA) of residues of clenbuterol (CBL), an illicit veterinary drug. Here, we report our work on expression and purification of a mouse-derived anti-CBL single chain Fv (scFv) antibody in Escherichia coli (E. coli). An expression plasmid pBV220-CBL was constructed and transformed into E. coli BL21 (DH3) strain cells. After induction by temperature, the 6x His-tagged anti-CBL scFv antibodies were expressed with the yield of 31%. The solubilized inclusion bodies were extracted, denatured and then purified by Ni-NTA column chromatography. The purified recombinant target protein was analyzed by high performance liquid chromatography, SDS-PAGE and Western blotting, respectively. The results showed the prepared anti-CBL scFv antibodies posed HRP-anti-His-tag antibody-recognized activity and their purity was up to 96%. Moreover, an indirect competitive ELISA based on the anti-CBL scFv antibodies revealed that the limit of detection for CBL was 0.5 ng/ml and the linear range was 1.5-10.6 ng/ml. Taken together, these findings suggest that the prepared recombinant antibody can be used for future immunoassay detection for CBL.
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Base de dados:
MEDLINE
Assunto principal:
Região Variável de Imunoglobulina
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Clembuterol
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Agonistas Adrenérgicos beta
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Escherichia coli
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Anticorpos de Cadeia Única
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article