Development of single-chain Fv fragments from a human anti-double-stranded DNA antibody to study the influence of somatic mutations on antigen binding.
Exp Clin Immunogenet
; 18(2): 96-9, 2001.
Article
em En
| MEDLINE
| ID: mdl-11340298
ABSTRACT
OBJECTIVE:
The monoclonal IgG anti-double-stranded (ds) DNA antibody 32B9, obtained from a patient with systemic lupus erythematosus, was found to be encoded by somatically mutated immunoglobulin genes. We examined the input of several somatic mutations into antibody specificity and affinity.METHODS:
Five single-chain (sc) Fv fragments [variable domain of the heavy chain (V(H))-linker-variable domain of the light chain (V(L))] derived from 32B9 were constructed and expressed in Escherichia coli. These scFv fragments contained V(H) or V(L) fragments, differing in the somatic mutation pattern. The antigen binding features of the 32B9 IgG were compared with the corresponding scFv fragments, and the binding to DNA of all fragments was analyzed by ELISA. Binding constants to dsDNA were determined by surface plasmon resonance and ELISA.RESULTS:
The scFv 32B9 reflected the binding features of the 32B9 IgG. Independently of the somatic mutations, all scFv fragments bound to dsDNA in ELISA. The affinity data indicated that the mutations studied had only a marginal effect on affinity maturation of the 32B9.DISCUSSION:
We discuss the approach to constructing scFv fragments as a tool to study autoantibody maturation.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Região Variável de Imunoglobulina
/
Fragmentos de Imunoglobulinas
/
Anticorpos Antinucleares
Limite:
Humans
Idioma:
En
Revista:
Exp Clin Immunogenet
Assunto da revista:
ALERGIA E IMUNOLOGIA
/
GENETICA
Ano de publicação:
2001
Tipo de documento:
Article
País de afiliação:
Alemanha