An enzyme-linked immunosorbent assay for detecting proteolytic activity of hepatitis C virus proteinase.
An ELISA method for the quantitation in vitro of HCV serine proteinase activity was developed. A peptide substrate, Ac-Gly-Glu-Ala-Gly-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Tyr-Thr-Trp-Thr-L ys (biotin) -OH (Sub-1), was hydrolyzed by a recombinant NS3 proteinase fused with maltose binding protein (MBP-NS3) into a product with a free amino moiety at the N-terminus. The product was immobilized, and the amino moiety was analyzed by digoxigenin labeling followed by immunological reaction with anti-digoxigenin-alkaline phosphatase conjugate and then the colorimeteric reaction. This method is suited for the high throughput screening of inhibitors, and the screening can be accelerated by automatic operation.
Takeshita
N
N
HQL Research Laboratories, Sumitomo Metal Industries Ltd., Kyoto, Japan.
Kakiuchi
N
N
Kanazawa
T
T
Komoda
Y
Y
Nishizawa
M
M
Tani
T
T
Shimotohno
K
K
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005069:Evaluation Studies as Topic; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012697:Serine Endopeptidases / Q000032:analysis* / Q000235:genetics; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases