Antibody affinity maturation in vitro using unconjugated peptide antigen

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Antibody library selection typically uses immobilized antigens on solid phases.
• Selecting small molecule binders often requires antigen conjugation, leading to issues like non-specific binding and reduced specificity.

Purpose of the Study:

• To develop a method for improving anti-small molecule antibody affinity without antigen derivatization.
• To enhance the sensitivity and specificity of antibody selection for small molecules.

Main Methods:

• Utilized the open-sandwich (OS) principle, leveraging antigen-dependent stabilization of the antibody variable domain (Fv).
• Employed PCR-randomized V(H)-displaying phages selected for binding to immobilized V(L) fragments in the presence of limited antigen peptide.
• Applied saturation mutagenesis to further refine antibody mutants.

Main Results:

• Obtained V(H) mutants with superior detection sensitivity in OS ELISA.
• Characterized mutants to confirm improved antigen-binding affinity.
• Demonstrated further sensitivity enhancement through saturation mutagenesis.

Conclusions:

• The 'OS-selection' method enables the selection of anti-small molecule antibodies without using conjugated antigens.
• This approach is particularly beneficial for affinity maturation of antibodies with inherently unstable Fv domains in the absence of antigen.