Antibody Affinity Maturation

Alpha Lifetech Inc. can offer the antibody affinity maturation service by using mutation and selection technologies. We usually use scFv as the antibody format in the processes of affinity maturation service and a monovalent display phagemid system which has been used to reduce the avidity effects during antigen-binding screening.  Our professional team can provide affinity maturation services for single-domain antibodies as well.

Alpha Lifetech Inc. is proud to offer comprehensive services to meet the diverse needs of global clients. Our aim is to understand and meet the demands of different customers and to assist with any upcoming and emerging problems in research work.

What Is Affinity Maturation?

Affinity maturation is the process by which antibodies undergo repeated mutations and selection to enhance their affinity for specific antigens. Mainly occurring in the germinal center of secondary lymphoid organs, B cells undergo somatic hypermutation and selection under the influence of T cells and follicular dendritic cells.
During the affinity maturation process, B cells with low-affinity surface immunoglobulin receptors are activated by antigen encounters and undergo proliferation and differentiation. Through somatic hypermutation affinity maturation, random nucleotide substitutions occur in the variable region of immunoglobulin genes, leading to the production of various antibody variants. After somatic hypermutation, antigen-driven selection is performed on B cells with mutated immunoglobulin receptors. B cells that produce antibodies with increased antigenic affinity are preferentially retained and allowed to proliferate, while B cells with decreased or unchanged affinity undergo apoptosis. This iterative process of mutation and selection leads to the production of B cell clones, producing antibodies with gradually higher affinity for the antigen.

Antibody Affinity Maturation Service

The chief advantages of PCR-based methods are that mutations are precisely targeted to the amplified fragment; the error rate is easy to control and the method is quick and easy to set up and does not use hazardous chemicals. It is well known that the Taq DNA polymerase duplicates DNA with low fidelity, substantially because of the absence of 3' to 5' proofreading activity. Alpha Lifetech's antibody engineering platform applies an error-prone PCR approach to mutate mainly CDR regions during sub-library construction. And often we create mutations at completely random positions across the entire VH and VL fragments, in order to increase the genetic diversity of the sub-library. After the construction of a mutated antibody gene library by error-prone PCR, the selection of high-affinity variants (affinity of the scFv antibodies can reach 10 -8 - 10 -10 M) is either performed by panning in solution or on immobilized antigen with washing conditions optimized for off-rate-dependent selection.
  
Scientists in Alpha Lifetech Inc. use Escherichia coli mutator strains mutAD5TM as one of several mutation strategies to introduce random mutations, and thereby modify the affinity and expression of recombinant antibody fragments. Selection conditions can be modified for antibody fragments with increased production levels. Growth conditions in Escherichia coli mutator cells can be adjusted to introduce a single random point mutation per kilobase of DNA, nearly equivalent to one codon change per scFv fragment. After several cycles of mutation, display, and selection, we can improve the affinity constant of from 10-5 - 10-6 M to 10 -8 - 10-10 M.

Improve Affinity Maturation Strategies

Certain positions of antibodies can be randomized at a defined diversity (such as full randomization with all 20 amino acids or biased randomization with selected amino acids at fixed percentages) to improve the affinity. We have two strategies to improve the antibody affinity: first, mutations are introduced at restricted positions in the complementarity-determining regions (CDRs) by site-directed mutagenesis; second, mutations are introduced into the entire V-coding regions by random mutagenesis. By using these two methods, the conserved amino-acid sequences or the whole framework region of the antibody will be substituted by other amino acids, which combined with our peptide library scanning technologies, could extremely increase the affinity of antibodies of interest to a very high level.

Phage Display-Antibody Affinity

Once the scFv mutant library has been constructed, two screening strategies are available: biopanning and solid-antigen sorting. In the former, a subtractive concentration of substrate is used to isolate the high-affinity antibody, as the low-affinity mutants would be washed out, leaving the high-avidity phage particles. The second strategy uses a labeled antigen in solution, selection based on the equilibrium constant (Kd), and selection based on binding kinetics, this selection approach singles out antibody variants with improved Kd.

If you have any questions, please feel free to contact us at any time.