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

This method can determine the binding affinity of a radiolabeled antibody for its antigen. It will also measure the specificity of binding. The main advantage of this simple method is the specific characterization of binding affinity of only the radiolabeled antibody without interference from the unconjugated parental antibody.

This technique quantitatively validates the ability of a radiolabeled antibody to bind to its target. This is important for applications in nuclear imaging and in nuclear therapy in a variety of diseases. Demonstrating the procedure will be Erika Belitzky, a postgraduate associate from my laboratory.

To begin, prepare an immobilization buffer by adding 191 milligrams of sodium bicarbonate and 23.9 milligrams of sodium carbonate to a 50-milliliter conical tube. Then, add 40 milliliters of 18 megaohm water and dissolve by vortexing. Now, adjust the pH to 9.0 before bringing the total volume to 50 milliliters.

Next, prepare 200 milliliters of washing buffer by adding 200 milliliters of PBS and 100 microliters of Tween 20 to a 250-milliliter bottle. Also prepare 50 milliliters of binding buffer by adding 50 milligrams of BSA and 25 microliters of Tween 20 to 50 milliliters of PBS and mix by vortexing. Add 1.5 grams of BSA to 50 milliliters of PBS to prepare the blocking buffer and vortex gently to mix.

Dilute the antigen in immobilization buffer. Then, take a breakable, 96-well, flat-bottom plate and add 100 microliters of antigen to the bottom of each well of 24 wells in an eight-by-three array. Cover the plate with sealing tape and incubate at four degrees Celsius overnight.

The next day, wash the plate thrice with washing buffer. Invert the plate briskly in the sink to dispose of the liquid and tap the plate on a pile of paper towels to remove the excess liquid. Then, to the wells containing the antigen, add 300 microliters of washing buffer per well using a multichannel pipette.

Add 300 microliters of blocking buffer per well to both 24-antigen-coated wells and 24 empty wells of the 96-well plate. Then, incubate the plate for one hour at ambient temperature and wash each well of the plate thrice with 300 microliters of washing buffer. Make three-fold serial dilutions of the radiolabeled antibody in the binding buffer for eight rows designated as A to H on the plate.

First, calculate the volume of stock radiolabeled antibody required to make a 1.2-milliliter solution of the first concentration. Then, add 800 microliters of binding buffer to microcentrifuge tubes labeled B to H and add the required volume of binding buffer to a microcentrifuge tube labeled A.Add the calculated volume of stock radiolabeled antibody to tube A and vortex gently. Then, spin down using a mini-microcentrifuge to collect all liquid at the bottom of the tube.

Next, add 400 microliters of liquid from two A to tube B.After vortexing, spin it down using a mini-microcentrifuge. Similarly, add solution from tube B to tube C, C to D, up to tube H.Add 100 microliters of each dilution per well to three wells immobilized with antigen and three wells blocked with BSA only. Now, add 100 microliters of each dilution to the microcentrifuge tubes labeled A standard to H standard and save these tubes as radiolabeled antibody standards to be assayed in the gamma counter.

Incubate the plate for one hour at 37 degrees Celsius with gentle rocking. Label microcentrifuge tubes for each well as described in the manuscript. Aspirate the radiolabeled antibody from the wells of the 96-well plate using a vacuum aspirator.

Now, using a multichannel pipette, add 300 microliters of washing buffer to each well. Aspirate the washing buffer and repeat washing four more times. Break apart the wells into the appropriate microcentrifuge tubes.

Count the radioactivity in the tubes with the antigen marked as H1, H2, H3, H3 to A1, A2, A3, and then with BSA only marked as H4, H5, H6, to A4, A5, A6 using a gamma counter. Radiolabeled amivantamab showed specific binding for EGFR and cMET proteins. Similarly binding affinities were also retained by single-arm radiolabeled fabs bound to EGFR and cMET proteins.

The saturation binding plots showed that when the concentrations of radiolabeled antibodies were too low, saturation was not attained, as indicated by the linear curve. Concentrations of radiolabeled antibodies that were too high led to saturation without distinct logarithmic growth, as shown by the horizontal plateau. Also, high nonspecific binding was seen due to high concentrations of radiolabeled antibodies.

This procedure may take multiple attempts to optimize the concentration range for each specific radiolabeled antibody. Use data from negative results to help design future experiments. Once binding affinity is known, it can be used as a benchmark for other in vitro assays as part of the characterization of a radiolabeled antibody.