Antibody
Antibody antigen interaction and dynamic measurement can be carried out in different liquids, including complex liquids such as serum or saliva.
Characterization of antibodies by SPR
SPR is a sensitive platform to determine the interaction between antibody or fragment and antigen, and directly measure the affinity and kinetics of the interaction. SPR instrument can also directly measure target molecules such as liposomes and living cells, and reflect their natural interaction. The interaction can be measured in different liquids, including 100% serum, plasma or cell growth medium. SPR provides some information about the conformation and direction of the antibody.
Advantages of selecting SPR for antibody characterization:
1. Label-free affinity and kinetics test
2. Low cost
3. Conformational changes of antibody
4. Natural samples (100% serum)
5. Biopharmaceuticals, even drugs from cell mediums
6. Easy ex-situ preparation of own sensor surfaces
Key questions about antibodies that SPR can answer:
1. What is antibody affinity and kinetics to antigen?
2. What is the best antibody for immunoassay?
3. What is the binding of antibody on receptor in lipid environment?
4. Which cell signaling cascade is activated by antibody in living cells?
5. How fast is molecule X association and dissociation kinetics to molecule Y?
6. Which monoclonal antibody has highest affinity?
Antibody antigen interaction measured by SPR
Protein-antibody interaction is an important research field in pharmaceutical industry and protein research field. Antibody based biotech drugs have been shown to be effective in the treatment of several intractable diseases. SPR is a sensitive tool for determining molecular interactions. Real time and label free measurement not only provides information about affinity, but also provides information for studying system dynamics. A variety of fixed protocols can be used to bond molecules on the sensor surface, so the method can be applied to a wide range of molecules. Human serum albumin (HSA) was immobilized on the surface of carboxymethyl dextran (CMD) sensor and reacted with amine. Human serum albumin antibody (anti-HSA) interaction with immobilized human serum albumin (HSA) was measured using Surface Plasmon Resonance (SPR). Steady state binding as well as kinetic of the binding was calculated for the interaction with four different concentration. Commercial anti-HSA binds strongly to the HSA and dissociates extremely slowly from the HSA.
Antibody binding kinetics
PKC (protein kinase C) is associated with cancer, type II diabetes and Alzheimer's disease. Therefore, inhibitors and activators of PKC are expected to be used in research and drug development. Llama single chain Fv (VHHs) is a new monoclonal antibody, which can specifically activate or inhibit human PKC. On standard gold sensors, carboxymethyldextran hydrogels are self synthesized. The dynamics was measured by SPR, and the SPR was tracked and analyzed. The measurement was carried out in a fixed angle mode. In this process, the first-order or second-order Langmuir binding model is used, and the correlation and dissociation constants are determined. The activation and inhibition kinetics of PKC kinase determined by SPR were well correlated with the translocation of PKCa HeLa cells in vivo. Therefore, in SPR measurement, it is important to combine VHHs with PKC conformation for protein localization.
