Name: determining binding affinity (kd) of radiolabeled antibodies to immobilized antigens
Uploaded: 2022-06-23T13:40:00
Description: Watch this Scientific Journal Video about Determining Binding Affinity KD of Radiolabeled Antibodies to Immobilized Antigens at JoVE.com

The authors thank 3D Imaging for the production of [89Zr]Zr-oxalate and Dr. Sheri Moores at Janssen Pharmaceuticals for providing antibodies.

NOTE: Refer to Figure 2 for a graphical representation of the protocol.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63927/63927fig02.jpg" /> 
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 ...

<ol>
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(96), e52521 (2015).</li><li>Wei, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImmunoPET:+concept,+design,+and+applications.">ImmunoPET: concept, design, and applications.</a> Chemical Reviews. 120 (8), 3787-3851 (2020).</li><li>Tavar&#233;, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+effective+immuno-PET+imaging+method+to+monitor+CD8-dependent+responses+to+immunotherapy.">An effective immuno-PET imaging method to monitor CD8-dependent responses to immunotherapy.</a> Cancer Research. 76 (1), 73-82 (2016).</li><li>Tavar&#233;, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+antibody+fragments+for+immuno-PET+imaging+of+endogenous+CD8++T+cells+in+vivo.">Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo.</a> Proceedings of the National Academy of Sciences. 111 (3), 1108-1113 (2014).</li><li>Zeglis, B. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specifically+labeled+89Zr-DFO-trastuzumab+improves+immuno-reactivity+and+tumor+uptake+for+immuno-PET+in+a+subcutaneous+HER2-positive+xenograft+mouse+model.">Site-specifically labeled 89Zr-DFO-trastuzumab improves immuno-reactivity and tumor uptake for immuno-PET in a subcutaneous HER2-positive xenograft mouse model.</a> Theranostics. 9 (15), 4409-4420 (2019).</li><li>Maguire, J. J., Kuc, R. E., Davenport, A. P., Davenport, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Radioligand binding assays and their analysis. in Receptor Binding Techniques. , 31-77 (2012).</li><li>Davenport, A. P., Russell, F. D., Mather, S. 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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+PET+imaging+of+glycoprotein+non-metastatic+melanoma+B+in+triple+negative+breast+cancer:+feasibility+of+an+antibody-based+companion+diagnostic+agent.">Preclinical PET imaging of glycoprotein non-metastatic melanoma B in triple negative breast cancer: feasibility of an antibody-based companion diagnostic agent.</a> Oncotarget. 8 (61), 104303-104314 (2017).</li><li>Ghai, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+[(89)Zr]DFO-elotuzumab+for+immunoPET+imaging+of+CS1+in+multiple+myeloma.">Development of [(89)Zr]DFO-elotuzumab for immunoPET imaging of CS1 in multiple myeloma.</a> European Journal of Nuclear Medicine and Molecular Imaging. 48 (5), 1302-1311 (2021).</li><li>McKnight, B. 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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...

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 treatment2,3,4,5. 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 both imaging and treatment6.
Ideally, the antibody chosen for radiolabeling is one already proven to have high binding affinity and specificity using non-radiolabeled methods. Radiolabeling of antibodies can be achieved via direct chemical modification of antibodies with a radionuclide that forms stable covalent bonds (eg. radioiodine), or indirectly via conjugation with chelators that subsequently coordinate to radiometals7,8. Direct radiolabeling such as with radioiodine specifically modifies tyrosine and histidine residues on the antibody. If these residues are important for antigen binding, then this radioconjugation would alter the binding affinity. Conversely, there are multiple established protocols for the conjugation and indirect radiolabeling of antibodies. For example, a common chelator used to bind zirconium-89 (89Zr) for PET imaging of antibodies is the p-isothiocyanatobenzyl-desferrioxamine (DFO), which is conjugated randomly to lysine residues of the antibody9,10. If there are lysine residues at the antigen-binding region, conjugation at these sites could sterically hinder antigen binding and therefore compromise the antibody-antigen binding. Thus, the different radioconjugation methods used for indirect or direct radiolabeling of antibodies can potentially affect immunoreactivity, defined as the ability of the antibody radioconjugate to bind to its antigen7,11. Site-specific conjugation methods can circumvent this limitation, but these techniques require antibody engineering to incorporate additional cysteine residues or expertise in enzymatic reactions on carbohydrate residues12,13,14,15,16. Once an antibody is radiolabeled, it is important to test if immunoreactivity is retained as part of the characterization of the rAb. One way to measure immunoreactivity is to determine the rAb&#39;s binding affinity.
The purpose of this protocol is to describe a process for determining the binding affinity for rAbs using an established radioligand saturation assay to quantify rAb-antigen binding. The binding trend is outlined in Figure 1. The amount of antigen bound will increase as more rAb is added to a fixed amount of immobilized antigen. Once all antigen-binding sites are saturated, a plateau will be reached, and adding more rAbs will have no effect on the amount of bound antigen. In this model, the equilibrium dissociation constant (KD)&#160;is the concentration of antibody that occupies half of the antigen receptors17. The KD represents how well an antibody binds to its target with a lower KD corresponding to a higher binding affinity. It was previously reported that an ideal rAb should have a KD of 1 nanomolar or less18. However, more recent rAbs have been developed with KD in the low nanomolar range, and are considered suitable for noninvasive imaging applications19,20,21,22. Another parameter that can be determined in the radioligand saturation assay of rAbs is Bmax,&#160;which corresponds to the maximum amount of antigen-binding. Bmax can be used to calculate the number of antigen molecules if needed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63927/63927fig01.jpg" /> 
Figure 1: Representative saturation binding curve. The percentage of antigen bound is plotted against increasing concentrations of antibodies added to a fixed amount of antigen. Pop-outs demonstrate binding at various points. The concentration and binding corresponding to KD and Bmax, respectively, are shown. This figure was created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/63927/63927fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This assay is particularly important for radiolabeled bispecific antibody constructs to determine the KD for each fragment antigen-binding region (Fab) arm of the radiolabeled bispecific antibody binding with their respective antigens. This protocol can be used to determine the KD of each Fab arm separately on immobilized antigens to independently characterize whether the binding affinity of each Fab arm for its respective antigen was affected after radioconjugation. This protocol is demonstrated by the use of radiolabeled amivantamab, a bispecific antibody for epidermal growth factor receptor (EGFR) and cytoplasmic mesenchymal-epithelial transition (cMET) proteins19. Radiolabeled single-arm antibodies, where one Fab arm binds to EGFR (&#945;-EGFR) or to cMET (&#945;-cMET) and the other Fab arm is an isotype control, were also used as examples19. This protocol is also appropriate for any radiolabeled antibody with a known antigen that can be immobilized. In this protocol, a dilution series of the rAb is added to a fixed amount of immobilized antigen in designated wells specific for each Fab arm of the rAb. The rAb is also added to wells that have only been blocked with bovine serum albumin (BSA), without antigen, to determine nonspecific binding. To determine specific binding, nonspecific binding to immobilized antigen is subtracted from the total rAb binding. The resulting saturation binding curve is then used to determine KD, as described above.
One advantage of this method is higher reproducibility when using purified antigens compared with using cell lines as the source of antigens, given that antigen expression levels could be affected during cell culture and that different cell lines have variable levels of antigen expression. In the case of radiolabeled bispecific antibodies, cell lines that only express one of the antigens without the other may not be available, which would make characterizing the binding affinity of the individual Fab arms very challenging. Notably, the key advantage of the radioligand saturation assay method over non-radiolabeled methods is the specific characterization of the binding affinity of the rAb without the contribution of the rAb&#39;s unconjugated fraction. To the best of the authors&#39; knowledge, there are currently no purification techniques to separate the rAb from its parent unconjugated antibody. Given the relatively small size of the chelator and radionuclide, their contribution to the overall molecular weight of the rAb is insignificant in size exclusion chromatography. Thus, the product generated from any radiolabeling technique is almost always a mixture of the rAb and its parent unconjugated antibody. Characterizing binding affinity using the radiolabeled saturation assay ensures that the product being tested is solely the rAb.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma Counter</td>
			<td>Hidex</td>
			<td>Hidex Automatic Gamma Counter</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism Software</td>
			<td>GraphPad</td>
			<td></td>
			<td>version 9.2; used for statistical analyses in this study</td>
		</tr>
		<tr>
			<td>Immuno Breakable MaxiSorp 96-well plates</td>
			<td>Thermo Scientific</td>
			<td>473768</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Sealing Tape</td>
			<td>Corning</td>
			<td>4612</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>JT Baker</td>
			<td>3506-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining binding affinity (kd) of radiolabeled antibodies to immobilized antigens

Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is represented by the equilibrium dissociation constant, KD, 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 KD 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.

This method calculates binding affinity (KD) for a rAb based on the saturation binding assay where different concentrations of rAb were added to a fixed amount of immobilized antigen. The binding curve should follow logarithmic growth where it is initially steep and then plateaus as the antigen is saturated. To ensure the determined KD is accurate, the concentrations of rAb must be high enough to reach saturation. For this assay, radiolabeled antibodies were conjugated to DFO and radiolabeled with <...

Watch this Scientific Journal Video about Determining Binding Affinity KD of Radiolabeled Antibodies to Immobilized Antigens at JoVE.com

Determining Binding Affinity KD of Radiolabeled Antibodies to Immobilized Antigens

Here, a method is described to determine the binding affinity (KD) of radiolabeled antibodies to immobilized antigens. KD 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.

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

Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is ...

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.

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Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the ...

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...