1. What is Antibody Affinity?

Antibody affinity refers to the binding strength between an antibody and an antigen epitope or antigenic determinant. Its essence is a non-covalent force that includes the binding force between amino acids, such as hydrogen bonds and hydrophobic forces. The strength of antibody affinity depends on the degree of coordination between the antibody and the antigen epitope used. Its determining factors include the size of the contact area, the degree of affinity, and the distribution of charged groups and hydrophobic groups. Affinity is an important indicator for evaluating antibody quality, which can reflect the binding ability of antibody molecules and antigen molecules or haptens. The higher the degree of binding between the antibody and the corresponding antigen, the stronger the affinity.

The equilibrium dissociation constant KD ((mass molar concentration/liter) can evaluate the affinity of antibodies, and its calculation formula is: KD= ([Ab][H])/[Ab*H], where [] represents molar concentration, Ab represents antibody, H represents antigen/hapten, and Ab*H represents the conjugate of antibody and antigen/hapten. The lower the equilibrium dissociation constant KD value, the higher the ability of the antibody to bind to the antigen/hapten. Of course, there is also a practice of defining the reciprocal of the equilibrium dissociation constant as the affinity constant, and using the affinity constant to directly characterize the affinity.

In a shorter period of time, antibodies with high affinity are more likely to bind to antigens than antibodies with low affinity. Because of the high activity of high-affinity antibodies and the stability of the complex, high-affinity antibodies are better used in immunochemical techniques - for example, the dissociation time of low-affinity antibodies binding to small molecule protein antigens is only a few minutes, while above the dissociation half-life of high-affinity antibodies is 30 minutes.

2. What is Antibody Affinity Maturation?

Antibody affinity maturation refers to a state of immune function. In humoral immunity, the phenomenon that the antibodies produced by the secondary reaction have a higher average affinity than the primary immune reaction is called antibody affinity maturation. Specifically, antibodies are produced by B cells, but only a few B cells can produce high-affinity antibodies; they occur in lymphoid tissues called "germinal centers" and are selected from a large number of B cells with different affinities through a Darwinian evolutionary screening process, and then differentiate into plasma cells (B cells that continuously secrete antibodies). This screening process is called affinity maturation. In this process, the decisive factor that B cells with different affinities have to compete for is the help signal of T cells. T cells transmit these help signals that allow B cells to survive and clone and proliferate through direct contact with B cells. B cells with higher affinity are also more likely to obtain help signals, have better survival ability, stronger reproduction ability, and are more likely to differentiate into plasma cells.

The differentiation of B cells into plasma cells is determined by two interrelated processes. The first is the molecular basis of antibody-specific immune response - the gene fragments of the bone marrow antibody light and heavy chain germline produce more than 10^6 combinations through random rearrangement of V-(D)-J; the second is the antibody binding characteristics. After being stimulated by antigens, the antibody genes, especially the complementary determining regions (CDRs), will undergo high-frequency mutations in the germinal centers of secondary lymphoid organs, and the antibody binding characteristics will change. For high-frequency mutations, studies have shown that cytosine nucleoside deaminase (AID) can deaminize cytosine to uracil, causing base mismatch and base shearing, leading to DNA repair. Incorrect DNA repair will further aggravate the mutation, thereby producing high-affinity antibodies.

Because only B cells with the highest antigen affinity will further differentiate into plasma cells and memory cells after dendritic cells present antigens, the selection of mutation regions and how to introduce mutations during in vitro antibody affinity maturation are important issues.

In vitro antibody affinity maturation is to obtain the mutated antibody sequence by using 3D structure simulation and introducing appropriate mutant amino acids in the antibody V region, combined with in vitro panning technology.

Current mutation strategies can be mainly divided into two categories: random mutation and directed mutation.

3. What Services can Tekbiotech Provide?

TekBiotech can provide customers with multi-species antibody affinity maturation services based on its own phage antibody library technology. We have a skilled antibody team and have accumulated rich experience in antibody affinity maturation, which can improve the affinity of monoclonal antibodies provided by customers by 2 to 3 orders of magnitude, reaching the level of 10^-10-10^-12.

Figure 1 Antibody affinity maturation technology process

Based on phage display technology, TekBiotech focuses on providing one-stop scientific research services, including phage antibody library construction and screening, antibody humanization, human Fab antibody library construction, human scFV antibody library construction, phosphorylated antibody customization and other technical services, and can customize suitable experimental plans for customers.