Interaction of the octapeptide angiotensin II with a high-affinity single-chain Fv and with peptides derived from the antibody paratope.
The amino-acid sequence of the very high-affinity anti-angiotensin II monoclonal antibody 4D8 was predicted from the nucleotide sequence of the heavy and light chain variable genes. The single-chain variable fragment (scFv) was constructed and expressed in Escherichia coli as a soluble protein and at the surface of the filamentous M13 phage and was compared with the full-length antibody (Ab). The scFv showed the same specificity profile and affinity constant as the intact antibody (5.0x10(10) and 8.0x10(10) M(-1), respectively, by Scatchard analysis). Several peptides from the set of overlapping dodecapeptides covering the variable domains of 4D8 mAb were found to specifically bind biotinylated angiotensin II: peptides from the L1, L2, L3 and H1 regions had the strongest capacity to bind the antigen.
Cohen
P
P
Department of Immunology/Oncology, Sanofi-Synthelabo, 371 rue du Professeur Blayac, 34184 Montpellier Cedex 4, France.
Laune
D
D
Teulon
I
I
Combes
T
T
Pugnière
M
M
Badouaille
G
G
Granier
C
C
Mani
J C
JC
Simon
D
D
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D016415:Sequence Alignment; D012995:Solubility
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009842:Oligopeptides; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D000804:Angiotensin II