Determining Binding Affinity (KD) of Radiolabeled Antibodies to Immobilized Antigens

Summary

Here, a method is described to determine the binding affinity (K_D) of radiolabeled antibodies to immobilized antigens. K_D is the equilibrium dissociation constant that can be determined from a saturation binding experiment by measuring the total, specific, and nonspecific binding of a radiolabeled antibody at various concentrations to its antigen.

Abstract

Determining binding affinity (K_D) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is represented by the equilibrium dissociation constant, K_D, and can be calculated as the concentration of antibody at which half the antibody binding sites are occupied at equilibrium. This method can be generalized to any radiolabeled antibody or other protein and peptide scaffolds. In contrast to cell-based methods, the choice of immobilized antigens is particularly useful for validating binding affinities after long-term storage of antibodies, distinguishing binding affinities of fragment antigen-binding region (Fab) arms in bispecific antibody constructs, and determining if there is variability in antigen expression between different cell lines. This method involves immobilizing a fixed amount of antigen to specified wells on a breakable 96-well plate. Then, nonspecific binding was blocked in all wells with bovine serum albumin (BSA). Subsequently, the rAb was added in a concentration gradient to all wells. A range of concentrations was chosen to allow the rAb to reach saturation, i.e., a concentration of antibody at which all antigens are continuously bound by the rAb. In designated wells without immobilized antigen, nonspecific binding of the rAb can be determined. By subtracting nonspecific binding from total binding in the wells with immobilized antigen, specific binding of the rAb to the antigen can be determined. The K_D of the rAb was calculated from the resulting saturation binding curve. As an example, binding affinity was determined using radiolabeled amivantamab, a bispecific antibody for epidermal growth factor receptor (EGFR) and cytoplasmic mesenchymal-epithelial transition (cMET) proteins.

Introduction

Radiolabeled antibodies (rAb) have a variety of uses in medicine. While the majority are utilized in oncology as imaging and therapeutic agents, there are imaging applications for rheumatology-related inflammation, cardiology, and neurology¹. Imaging rAbs has high sensitivity to detect lesions and has the potential to aid in patient selection for treatment^2,3,4,5. They are also used for therapy because of their specificity for their respective antigens. In a strategy known as theranostics, the same rAb is used for bot....

Protocol

NOTE: Refer to Figure 2 for a graphical representation of the protocol.

Figure 2: Schematic of the protocol. Row and column labels are indicated as a guide for setting up the breakable 96-well plate. Anticipated binding is shown in an example well for the antigen and the BSA. The curved .......

Discussion

As part of the development of rAbs, it is important to ensure a rAb binds specifically to its target with high binding affinity. Determining binding affinity can inform if the immunoreactivity of the rAb is affected by radioconjugation through the radioligand saturation assay using immobilized antigen. Determining rAb binding to BSA can be used to quantify nonspecific binding to measure specific binding to the immobilized antigen more accurately. This method tests the binding of different concentrations of rAb to generat.......

Materials

Name	Company	Catalog Number	Comments
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A9647
Gamma Counter	Hidex	Hidex Automatic Gamma Counter
GraphPad Prism Software	GraphPad		version 9.2; used for statistical analyses in this study
Immuno Breakable MaxiSorp 96-well plates	Thermo Scientific	473768
Microplate Sealing Tape	Corning	4612
Microsoft Excel	Microsoft
Phosphate Buffered Saline (PBS)	Gibco	14190144
Sodium Bicarbonate	JT Baker	3506-01
Sodium Carbonate	Sigma-Aldrich	S7795
Tween-20	Sigma-Aldrich	P7949