Molecular engineering of biotin–glutamic acid decarboxylase 65 fusion protein (Biotin–GAD65) for non-radioactive GAD65 antibody assay

We constructed biotinylated fusion proteins that linked to three detection tags to GAD65 at the N-terminus, and expressed them in an E. coli expression system. The Biotin14–GAD65 protein exhibited the strongest binding to both the GAD65 antibody and the streptavidin among the three constructs. We de...

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Veröffentlicht in:	Journal of biotechnology 2003-08, Vol.103 (3), p.249-255
Hauptverfasser:	Luo, Dong, Rogers, Chad N., Hampe, Christiane S., Lernmark, Åke
Format:	Artikel
Sprache:	eng
Schlagworte:	Antibodies - analysis Antibodies - immunology Antigen-Antibody Complex - analysis Binding assays Biological and medical sciences Biosensing Techniques - methods Biotin Biotin - chemistry Biotin - genetics Biotin - metabolism Enzyme-Linked Immunosorbent Assay - methods Expression Fundamental and applied biological sciences. Psychology GAD65 Glutamate Decarboxylase - chemistry Glutamate Decarboxylase - genetics Glutamate Decarboxylase - metabolism Isoenzymes - chemistry Isoenzymes - genetics Isoenzymes - metabolism Protein Binding Protein Engineering - methods Radioimmunoassay Recombinant Recombinant Fusion Proteins Reproducibility of Results Sensitivity and Specificity
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Zusammenfassung:	We constructed biotinylated fusion proteins that linked to three detection tags to GAD65 at the N-terminus, and expressed them in an E. coli expression system. The Biotin14–GAD65 protein exhibited the strongest binding to both the GAD65 antibody and the streptavidin among the three constructs. We describe the optimal conditions using a Biotin14–GAD65-based immunoassay for the detection of GAD65 antibody.
ISSN:	0168-1656 1873-4863
DOI:	10.1016/S0168-1656(03)00125-1