NP [KLH]

The structural and functional analysis of antibodies in the affinity maturation pathway contributes to our understanding of the molecular mechanisms underlying protein recognition. The use of one of the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP), allows for the production of a diverse range of monoclonal antibodies at early or late stages of immunization. Affinity maturation improves the binding affinity and specificity of an antibody to its immunogen through somatic hypermutation. Catalysts such as NP and choline phosphate as well as 2-phenyloxazolone have been used to analyze the structural basis of affinity maturation. The equilibrium binding constants of germline-type antibodies to hapten are as low as 10⁵ -10⁶ M^-1, whereas affinity-matured antibodies have equilibrium binding constants up to 10⁹ M^-1. Some investigators analyzing anti-NP antibodies during affinity maturation have found that the Trp to Leu mutation at VH33, which occurs via somatic hypermutation, is essential for maturation. It has also been found that the Tyr to Gly mutation on VH95 can be classified as a mature antibody in the later stages of immunization.

During affinity maturation, antigen binding affinity and specificity are usually enhanced. Many somatic mutant antibodies have been generated and screened, some of which are expressed from plasma cells. A Trp-to-Leu mutation on the anti-NP antibody VH33 increases its affinity for NP approximately 10-fold. Including the Tyr to Gly mutation on VH95, the affinity of the antibody for NP was increased 10-10³-fold. Most Leu33^H/Tyr95^H-type antibodies appear within 2-3 weeks after immunization, while Trp33^H/Gly95^H-type antibodies appear later. During affinity maturation, both the binding rate constant and the dissociation rate constant contribute to the antigen-binding affinity. Thermodynamic parameters such as enthalpy change (ΔH) and entropy change (ΔS) of NP binding to anti-NP monoclonal antibody and single chain Fv (scFv) antibody were obtained by isothermal titration calorimetry. Binding kinetic parameters can be obtained using a surface plasmon resonance biosensor. Antibodies bind to NP with a favorable ΔH, partially compensated by an unfavorable ΔS. In summary, during antibody evolution, antigen-antibody interactions will shift from an "induced-fit" type to a "locked-bonded" type.

Figure 1. Thermodynamic parameters for NP binding to anti-NP antibody at 25°C
(Source: Oda M. 2022)

Investigators used anti-NP scFv to analyze the thermal stability of antibodies during affinity maturation in the presence or absence of antigen. One of the advantages of using scFv is that it consists of the smallest antibody unit to which the antigen binds and provides information about its structure and function without interfering with the other binding domains. The thermal stability of scFvs in the antigen-bound state is higher than in the unbound state. Notably, germline-type antibodies were more stable in the antigen-unbound state than affinity-matured antibodies. Upon binding to antigen, mature antibodies are more stable than germline antibodies. The increase in stability is closely related to antigen binding affinity.

(4-hydroxy-3-nitrophenyl)acetyl [KLH]