Generation and characterization of chimeric antibodies against NS3, NS4, NS5, and core antigens of hepatitis C virus.
Mouse-human chimeric antibodies (cAbs) against hepatitis C virus (HCV) core, NS3 (nonstructural), NS4, and NS5 antigens were developed as quality control (QC) reagents to replace the use of human sera/plasma for Abbott HCV immunoassays. The cAb retains the mouse monoclonal antibody (MAb) specificity and affinity but still reacts in the existing HCV assay format, which measures human anti-HCV immunoglobulin. Mouse heavy-chain (V(H)) and light-chain (V(L)) variable regions of anti-HCV core, NS3, NS4, and NS5 antigens were PCR amplified from hybridoma lines and then cloned with human IgG1 heavy-chain (C(H)) and light-chain (C(L)) constant regions, respectively. A single mammalian expression plasmid containing both heavy-chain and light-chain immunoglobulin genes was constructed and transfected into dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cells. The transfected CHO cells were selected using hypoxanthine- and thymidine-free medium and screened by an enzyme immunoassay (EIA). The clone secreting the highest level of antibody was isolated from the CHO transfectants and further subcloned. Each cAb-expressing CHO cell line was weaned into serum-free medium, and the cAb was purified by protein A affinity chromatography. The levels of cAb production for the various CHO cell lines varied from 10 to 20 mg/liter. Purified anti-HCV cAbs were tested with Abbott HCV immunoassays and showed reactivity. Moreover, yeast surface display combined with alanine-scanning mutagenesis was used to map the epitope at the individual amino acid level. Our results suggest that these HCV cAbs are ideal controls, calibrators, and/or QC reagents for HCV assay standardization.
Tu
Bailin
B
Department of Antibody Research, Abbott Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA. bailin.tu@abbott.com
Ziemann
Robert N
RN
Tieman
Bryan C
BC
Hawksworth
David J
DJ
Tyner
Joan
J
Scheffel
James
J
Pinkus
Mary S
MS
Brophy
Susan E
SE
Werneke
Jeffrey M
JM
Gutierrez
Robin
R
White
Michael
M
D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D016174:Hepacivirus / Q000276:immunology; D018937:Hepatitis C Antibodies / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D018936:Hepatitis C Antigens / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D014758:Viral Core Proteins / Q000276:immunology*; D017361:Viral Nonstructural Proteins / Q000276:immunology*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D058846:Antibodies, Monoclonal, Murine-Derived; D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase