Antibody Affinity Maturation Technique

Affinity maturation is the process to improve antibody affinity for an antigen.

In vivo, natural affinity maturation by the immune system takes place by
somatic hypermutation and clonal selection. In vitro, in the laboratory affinity
maturation, can be obtained by mutation and selection.
Creative Biolabs has gained extensive experience in antibody affinity
maturation. We usually take scFv as the antibody format in affinity
maturation. Also, a monovalent display phagemid system is used to reduce
the avidity effects during antigen-binding screening. We also provide affinity
maturation services for single domain antibodies. Two methods, untargeted
mutagenesis and oligonucleotide-directed mutagenesis, are employed to
construct random or defined sub-libraries to introduce a large number of
mutants of the original antibody. Antibody binders of higher affinity are then
selected by increasing the screening stringency. By constructing a series of
sub-libraries of a scFv/Fab antibody, our proprietary protocol allows increase
of the affinity of the scFv antibodies from 10 -9 to 10 -10. We have
successfully obtained a scFv antibody that has an extremely high affinity of 10
-12, whose binding to the antigen is essentially irreversible.
Antibody Affinity Measurement
We offer Biacore Analysis services for binding kinetic analyses of antibodies.
We typically capture the antibody on the chip and run antigen on top of the
captured antibody. The antigen will be ran at 6 different concentrations for
each antibody and chi-square analysis will be performed on the binding
constants we obtain from each antigen concentration. The documentation
package will include a real time on-rate (Ka), off rate (Kd), an affinity constant
(KD), chi square value and a graph of real-time binding kinetics. We would like
to obtain ~50 uL of 1 mg/mL antigen and antibody solutions. We will need
~100 ug of antigen and ~50ug for each antibody. We would need MW
information for the antigen as well. It may require special considerations for
antigens with repeated or multiple epitopes for affinity determination.>>
Learn more about Antibody Affinity Measurement Services
Peptide Affinity Maturation
Alanine scanning mutagenesis is our favorite method in affinity maturation of
peptide binders. In this method, each single AA of a selected binding peptide
will be replaced with an alanine, and then the binding of the modified
peptides to the target protein will be assayed using Biacore technology. The
non-essential AAs will be specifically identified. After that, we will create a
directed/constrained peptide sub-library that contains random sequences on
the non-essential AA positions. Here, again, we frequently randomize the nonessential residues using "NNK" or "trimer codon" strategy. Mutants with
increased binding affinity are identified by enhancing the screening
stringency, followed by phage ELISA.