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Wien Kusharyoto
Research Centre for Biotechnology, Indonesian Institute of Sciences (LIPI)
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Immunology & microbiology Medicine & Pharmacology

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Sensitivity Improvement of A Direct Competitive Elisa for Atrazine by Exploiting Low Cross-Reactivity of An Atrazine-Specific Recombinant Antibody Fab-Fragment Wien Kusharyoto; Rolf D. Schmid
ANNALES BOGORIENSES Vol 9, No 1 (2004): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (3557.578 KB) | DOI: 10.14203/ann.bogor.2004.v9.n1.1-8

Abstract

The hapten-binding site of the antibody Fab-fragment K411 B specific towards the herbicide atrazine (2-cllloro-4-(ethylamino)-6-(isopropyJamjno)- 1,3,5-triazine) was modified by means of structural modeling and site-directed mutagenesis. A triple mutant (GlnL89GlulValH3711e/GluL3Val) of the Fab-fragment showed an increased affinity towards the hapten HlCVC6 (4-arnino-6-chloro-l ,3,5-triazine-2-(6-aminohexanecarboxylic acid» compared to the affinity ofthe wild-type Fab-fragment lowards the same hapten. However, the mutant exhibited substantially lower affinity towards the hapten H/CVC6 Ihan towards atrazine and the hapten iPr/ CVC6 (4-chloro-6-(isopropylamino)-1,3,5-triazioe-2-(6-aminohexanecarboxylic acid», which is usual ly used in the synthesis of enzyme tracers in ELISA for atrazine. Advantage was taken of the low cross-reactivity and increased affinity of the mutant Fab fragment towards H/CVC6 to improve the sensitivity of a direct-competitive ELISA tor atrazine. HlCIlC6 was covalently conjugated with horseradish peroxidase (HRP), and the conjugate H/CI/C6-HRPwas used as enzyme tracer in the ELISA for atrazine. An eight-fold improvement in sensitivity ofa direct-competitive ELISA for atrazine could be achieved using the tracer H/CVC6-HRP compared to the sensitivity of ELISA using the tracer iPr/CVC6-HRP. The detection limit for atrazine was as low as 0.01 i g/l. 
Subcloning, Expression and Characterisation of A Recombinant Antibody Fab-Fragment Specific Towards 2,4-D Wien Kusharyoto
ANNALES BOGORIENSES Vol 10, No 1 (2005): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (3633.744 KB) | DOI: 10.14203/ann.bogor.2005.v10.n1.23-30

Abstract

A generic strategy was established for subcloning the VH and VL gene of antibody variable domains into the plasmid pASK85 for the expression of Fab antibody fragments. pASK85 bears coding sequences for murine constant domains including a His6-tag at the carboxy-terminal end of the constant heavy-chain domain. The VH and YL gene derived from the monoclonal antibody E2/B5 specific towards 2,4-dichloropbcnoxyacetic acid (2,4- D) were used in this study. Eschericia coli was used as host cells for the biosynthesis of the Fab-fragment. The Fab­ fragment wa subsequently purified from the periplasmic extract in a single step by immobilised metal-ion affinity chromatography (IMAC). The production level obtained were 0.5-0.8 mg purified Fab-fragments per liter E. coli culture. The sensitivity and cross-reactivity of the Fab-fragment determined by direct competitive ELISA were similar to those of the parental monoclonal antibody E2/B5 .  
Co-Authors Rolf D. Schmid