Abstract
In antigen-antibody interactions, the high avidity of antibodies for their specific antigens can be achieved both by exploiting the high affinity of each binding site and by multivalence of antibodies. In this study, we developed artificial antibodies with multiple valency. The Fv fragments of an antibody fused with one and with two domains of the Fc-binding protein A from Staphylococcus aureus (designated Fv-P and Fv-PP, respectively) were expressed in secreted forms in Escherichia coli. Their physical characteristics were examined. The Fv portions of Fv-P and Fv-PP had virtually the same affinity for the antigen as that of the original Fv molecules, and each protein A-derived domain could be bound to IgG. When Fv-P was mixed with IgG, the complex formed was composed of two Fv-P molecules and one IgG molecule. In the case of Fv-PP, a complex composed of three Fv-PP molecules and two IgG molecules was preferentially formed. Analysis of surface plasmon resonance in a BIAcore system indicated multivalency of these complexes for antigens. There was 3.5-fold decrease in the dissociation constant of the complex between Fv-PP and the antigen upon the addition of IgG. The use of these complexes as reagents in enzyme-linked immunosorbent assay and Western blotting gave much stronger signals than Fv, Fv-P, and Fv-PP alone.
Highlights
Since the forward reaction, which is ruled by kon[Ag][Ab],is dependent on the rateof diffusion and on the probability that a collision will result inbinding, the value of kondoes not vary over a wide range [8].since the backward reaction, and two IgG molecules was preferentiallyformed. which is ruled by k,ff[Ag.Ab], is directly determined by the Analysis of surface plasmon resonance in a BIAcore strength of the bonds that are formed at thebinding sites, the system indicated multivalency of these complexes for value of koffvaries considerably among combinations of antiantigens
The reason that immunoblotting still works as a useful procedure seems to be that antibodies have An antibody can bind to its respective antigen. multiple binding contacts, they are anchored at more The antigen-binding siteis localized in thecomplementarity- than two sites on an antigen-linked membrane.In this study, determining regions of the variable domains of the antibody. we devised a new type of gene construct that encodes an Fv Progress in protein engineering has made it possible to con- fragment fused with a fragment derived from protein A of struct libraries of antibodies expressed in Escherichia coli (1, Staphylococcus aureus [10]
Expression of the Fu Fragment Fused with a Fragment of Protein A in E. coli-It has been shown that theFv fragment of D1.3 that has antigen binding activity can be expressed in a large quantities in E. coli [18,20]
Summary
We devised a new type of gene construct that encodes an Fv Progress in protein engineering has made it possible to con- fragment fused with a fragment derived from protein A of struct libraries of antibodies expressed in Escherichia coli 20 p1 of 100 nM, 50 nM, and 25 nM solutions of antibody (Fv, Fv-P, and Fv-PP), with or without human IgG (at a molar ratio of 2:1), wereinjected into the bio-sensor chip with subsequent washing with HBS, and the chips were regenerated with 100 mM HCl. The mixing conditions were the same as those for the analysis by HPLC except that HBS was used as the dilution buffer. Fv-P or Fv-PP was mixed with human IgG as thesame method as thatof ELISA These mixtures were used as thefirst antibody after 1,000-folddilution with phosphate-buffered saline containing 0.05% Tween 20. Horseradish peroxidase-conjugated antibodies raised in goat against the Fc fragment of human IgG were used as thesecond antibody for detection
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