"Affinity maturation" of ligands for HCV-specific serum antibodies.
We have previously screened a phage-displayed random peptide library using sera from patients and identified ligands binding to antibodies specifically associated with the hepatitis C virus infection. The ability of these peptides to detect HCV-specific antibodies was improved through an in vitro procedure which mimics the natural process of antibody affinity maturation operating in secondary immune response. Libraries were generated by mutating the sequence of the original peptide through a protocol that efficiently introduced substitution, insertion and deletion mutations on a single or population of clones. Screening these libraries isolated mutants that displayed increased specific reactivity with a broader range of sera from HCV-infected patients. Several variants of the original peptide were identified which discriminate between the various components of the specific polyclonal response. This methodology to select artificial ligands from RPL using sera and to enhance their diagnostic properties by affinity maturation makes the development of a diagnostic assay to detect disease-associated antibodies feasible, without requiring the natural antigen.
Urbanelli
L
L
Istituto di Ricerche di Biologia Molecolare P. Angeletti, Roma, Italy.
Fortugno
P
P
Bartoli
F
F
Nuzzo
M
M
De Tomassi
A
A
Felici
F
F
Monaci
P
P
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D019020:Directed Molecular Evolution; D004797:Enzyme-Linked Immunosorbent Assay; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology; D006526:Hepatitis C / Q000276:immunology; D018937:Hepatitis C Antibodies / Q000097:blood*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library
D000956:Antigens, Viral; D017931:DNA Primers; D018937:Hepatitis C Antibodies; D008024:Ligands; D019151:Peptide Library