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.
Antibody Affinity Maturation Service - Affinity Maturation by Error-Prone PCR
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 Inc.'s antibody discovery 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, 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.
Antibody Affinity Maturation Service - Affinity Maturation by E. coli Mutator Cells
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.
E. coli mutAD5TM strain introduces random, predominantly point mutations into DNA, a function of a defective dnaQ gene, which results in proofreading errors. The rate and specificity of mutation are governed by the growth conditions: mutation in rich media is increased by up to 5-fold compared to the rate in minimal media; the ratio of transitions is also affected; the mutation rate is highest when the cells are in exponential growth and decreases as the cells approach stationary phase. 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.
Antibody Affinity Maturation Service - Affinity Maturation by Targeting Random Mutations
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 combines with our peptide library scanning technologies, could extremely increase the affinity of antibodies of interest to a very high level.
Antibody Affinity Maturation Service - Affinity Maturation by Antibody Library Screening
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 is used, selection based on the equilibrium constant (Kd) and selection based on binding kinetics, this selection approach singles out antibody variants with improved Kd.
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.
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