Method for determining the affinity of monoclonal antibody using non-competitive ELISA: a computer program

Area of Science:

• Biochemistry
• Immunology
• Analytical Chemistry

Background:

• Determining monoclonal antibody (mAb) affinity is crucial for therapeutic and diagnostic applications.
• Existing methods for affinity calculation can be complex or indirect.
• A simplified, reliable method is needed for routine assessment of mAb-antigen interactions.

Purpose of the Study:

• To describe a simple and reliable method for calculating monoclonal antibody affinity using non-competitive ELISA.
• To validate the method based on the law of mass action.
• To develop a computational tool for facilitating affinity calculations.

Main Methods:

• Utilized non-competitive ELISA with serial dilutions of both antigen (Ag) and antibody (Ab).
• Measured antigen-antibody binding via optical density (OD).
• Plotted OD against antibody concentration to obtain a hyperbolic curve and derived affinity constant (K(aff)) using a formula based on the law of mass action.

Main Results:

• A hyperbolic curve was obtained when plotting OD against antibody concentration.
• The method allows for the calculation of the affinity constant (K(aff)) using the derived formula: K(aff) = (n - 1)/2(n[Ab'] - [Ab]), where n = [Ag]/[Ag'].
• A computer program was developed to automate the affinity calculation process.

Conclusions:

• The described ELISA-based method provides a simple and reliable approach for calculating monoclonal antibody affinity.
• The method leverages the law of mass action for accurate determination of antibody-antigen binding.
• The developed software facilitates the practical application of this affinity calculation technique.