Development of single-chain Fv fragments from a human anti-double-stranded DNA antibody to study the influence of somatic mutations on antigen binding.
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.


CONCLUSIONS
We discuss the approach to constructing scFv fragments as a tool to study autoantibody maturation.
Kersten
B
B
Department of Dermatology, Medical Faculty (Charité), Humboldt-University Berlin, Berlin, Germany. Kersten@molgen.mpg.de
Niemann
B
B
Jahn
S
S
D000974:Antibodies, Antinuclear / Q000235:genetics / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D004247:DNA / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000974:Antibodies, Antinuclear; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D004247:DNA