Engineered recombinant single-chain fragment variable antibody for immunosensors.
A recombinant single-chain fragment variable (scFv) antibody (designated A10B) was engineered to contain two histidines within the linker peptide used to join the scFv heavy and light chains. A piezoimmunosensor using the scFv was successfully developed. A10B scFv bound to the gold piezoimmunosensor surface were correctly oriented, retained antigen-binding activity, and coupled at high surface concentration. These results, and results obtained from an earlier study using an scFv containing a linker cysteine, suggest that the location on the linker sequence in which the amino acids were incorporated was well tolerated by the scFv and did not interfere with scFv antigen-binding activity. The scFv-modified QCM sensor was thoroughly characterized and used to specifically detect antigen in crude serum sample and had a sensitivity of 2.3 +/- 0.15 nM (n = 4) with a linear range over 2.3 x 10(-9) - 3.3 x 10(-(8) M. The piezoimmunosensor was also used to study the kinetics and thermodynamics of antigen/scFv antibody binding.
Shen
Zhihong
Z
Department of Chemistry, Oakland University, Rochester, Michigan 48309, USA.
Mernaugh
Raymond L
RL
Yan
Heping
H
Yu
Lei
L
Zhang
Ying
Y
Zeng
Xiangqun
X
D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D006046:Gold / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000032:analysis; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity
D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D006639:Histidine; D006046:Gold