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>
	<li>Krecisz, P., Czarnecka, K., Krolicki, L., Mikiciuk-Olasik, E., Szymanski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiolabeled+Peptides+and+Antibodies+in+Medicine.">Radiolabeled Peptides and Antibodies in Medicine.</a> Bioconjugate Chemistry. 32 (1), 25-42 (2021).</li><li>Dun, Y., Huang, G., Liu, J., Wei, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImmunoPET+imaging+of+hematological+malignancies:+From+preclinical+promise+to+clinical+reality.">ImmunoPET imaging of hematological malignancies: From preclinical promise to clinical reality.</a> Drug Discovery Today. 27 (4), 1196-1203 (2022).</li><li>Lohrmann, C., 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=Retooling+a+Blood-Based+Biomarker:+Phase+I+assessment+of+the+high-affinity+CA19-9+antibody+HuMab-5B1+for+immuno-pet+imaging+of+pancreatic+cancer.">Retooling a Blood-Based Biomarker: Phase I assessment of the high-affinity CA19-9 antibody HuMab-5B1 for immuno-pet imaging of pancreatic cancer.</a> Clinical Cancer Research. 25 (23), 7014-7023 (2019).</li><li>Pandit-Taskar, N., 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=A+phase+I/II+study+for+analytic+validation+of+89Zr-J591+immunoPET+as+a+molecular+imaging+agent+for+metastatic+prostate+cancer.">A phase I/II study for analytic validation of 89Zr-J591 immunoPET as a molecular imaging agent for metastatic prostate cancer.</a> Clinical Cancer Research. 21 (23), 5277-5285 (2015).</li><li>Rousseau, C., 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=Initial+clinical+results+of+a+novel+immuno-PET+theranostic+probe+in+human+epidermal+growth+factor+receptor+2-negative+breast+cancer.">Initial clinical results of a novel immuno-PET theranostic probe in human epidermal growth factor receptor 2-negative breast cancer.</a> Journal of Nuclear Medicine. 61 (8), 1205-1211 (2020).</li><li>Moek, K. L., 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=Theranostics+using+antibodies+and+antibody-related+therapeutics.">Theranostics using antibodies and antibody-related therapeutics.</a> Journal of Nuclear Medicine. 58 (2), 83-90 (2017).</li><li>Chomet, M., van Dongen, G., Vugts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art+in+radiolabeling+of+antibodies+with+common+and+uncommon+radiometals+for+preclinical+and+clinical+immuno-PET.">State of the art in radiolabeling of antibodies with common and uncommon radiometals for preclinical and clinical immuno-PET.</a> Bioconjugate Chemistry. 32 (7), 1315-1330 (2021).</li><li>Kumar, K., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiochemistry,+production+processes,+labeling+methods,+and+immunoPET+imaging+pharmaceuticals+of+Iodine-124.">Radiochemistry, production processes, labeling methods, and immunoPET imaging pharmaceuticals of Iodine-124.</a> Molecules. 26 (2), 414 (2021).</li><li>Vosjan, M. J., 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=Conjugation+and+radiolabeling+of+monoclonal+antibodies+with+zirconium-89+for+PET+imaging+using+the+bifunctional+chelate+p-isothiocyanatobenzyl-desferrioxamine.">Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine.</a> Nature Protocols. 5 (4), 739-743 (2010).</li><li>Zeglis, B. M., Lewis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bioconjugation+and+radiosynthesis+of+89Zr-DFO-labeled+antibodies.">The bioconjugation and radiosynthesis of 89Zr-DFO-labeled antibodies.</a> Journal of Visualized Experiments: JoVE. (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. M., 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=Chemoenzymatic+strategy+for+the+synthesis+of+site-specifically+labeled+immunoconjugates+for+multimodal+PET+and+optical+imaging.">Chemoenzymatic strategy for the synthesis of site-specifically labeled immunoconjugates for multimodal PET and optical imaging.</a> Bioconjugate Chemistry. 25 (12), 2123-2128 (2014).</li><li>Zeglis, B. M., 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=Enzyme-mediated+methodology+for+the+site-specific+radiolabeling+of+antibodies+based+on+catalyst-free+click+chemistry.">Enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry.</a> Bioconjugate Chemistry. 24 (6), 1057-1067 (2013).</li><li>Kristensen, L. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioligand+bindsing+assays:+theory+and+practice.">Radioligand bindsing assays: theory and practice.</a> Current Directions in Radiopharmaceutical Research and Development. , 169-179 (1996).</li><li>Cavaliere, 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+[89Zr]ZrDFO-amivantamab+bispecific+to+EGFR+and+c-MET+for+PET+imaging+of+triple+negative+breast+cancer.">Development of [89Zr]ZrDFO-amivantamab bispecific to EGFR and c-MET for PET imaging of triple negative breast cancer.</a> European Journal of Nuclear Medicine and Molecular Imaging. 48 (2), 383-394 (2021).</li><li>Marquez, B. 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=Evaluation+of+(89)Zr-pertuzumab+in+breast+cancer+xenografts.">Evaluation of (89)Zr-pertuzumab in breast cancer xenografts.</a> Molecular Pharmaceutics. 11 (11), 3988-3995 (2014).</li><li>Marquez-Nostra, B. 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. N., 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=Imaging+EGFR+and+HER3+through+(89)Zr-labeled+MEHD7945A+(Duligotuzumab).">Imaging EGFR and HER3 through (89)Zr-labeled MEHD7945A (Duligotuzumab).</a> Scientific Reports. 8 (1), 1-13 (2018).</li></ol>

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.

determining-binding-affinity-kd-radiolabeled-antibodies-to

Research

JoVE Journal

Medicine

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Determining The Electromyographic Fatigue Threshold Following a Single Visit Exercise Test

Theoretically, the electromyographic (EMG) fatigue threshold is the exercise intensity an individual can maintain indefinitely without the need to recruit more motor units which is associated with an increase in the EMG amplitude. Although different protocols have been used to estimate the EMG fatigue threshold they require multiple visits which are impractical for a clinical setting. Here, we present a protocol for estimating the EMG fatigue threshold for cycle ergometry which requires a single visit. This protocol is simple, convenient, and completed within 15-20 min, therefore, has the potential to be translated into a tool that clinicians can use in exercise prescription.

This protocol describes the electromyographic fatigue threshold which demarcates between nonfatiguing and fatiguing exercise workloads. This information could be used to develop a more individualized training program.

Theoretically, the electromyographic (EMG) fatigue threshold is the exercise intensity an individual can maintain indefinitely without the need to recruit ...

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based ...

Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which has led to improved research techniques becoming available over the last decades. To increase the understanding of wheeled mobility performance, monitoring, training, skill acquisition, and optimization of the wheelchair-user interface in rehabilitation, daily life, and sports, further standardization of measurement set-ups and analyses is required. A crucial stepping-stone is the accurate measurement and standardization of external power output (measured in Watts), which is pivotal for the interpretation and comparison of experiments aiming to improve rehabilitation practice, activities of daily living, and adaptive sports. The different methodologies and advantages of accurate power output determination during overground, treadmill, and ergometer-based testing are presented and discussed in detail. Overground propulsion provides the most externally valid mode for testing, but standardization can be troublesome. Treadmill propulsion is mechanically similar to overground propulsion, but turning and accelerating is not possible. An ergometer is the most constrained and standardization is relatively easy. The goal is to stimulate good practice and standardization to facilitate the further development of theory and its application among research facilities and applied clinical and sports sciences around the world.

Accurate and standardized assessment of external power output is crucial in the evaluation of physiological, biomechanical, and perceived stress, strain, and capacity in manual wheelchair propulsion. The current article presents various methods to determine and control power output during wheelchair propulsion studies in the laboratory and beyond.

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which ...

Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The pharmacological efficacy of these immunotherapies is long lasting, extending even beyond the discontinuation of their injections, due to persistent blood concentrations. Here we developed a simple flow cytometry assay to evaluate the T cell binding status of the PD-1&#8211;blocking antibodies nivolumab and pembrolizumab. Like a glucose test, this assay requires just a single drop of peripheral blood. Visualizing antibody binding on T cells is more reliable than measuring antibody blood concentrations. In addition, if necessary, we can potentially analyze many distinctive immune-related markers on T cells bound to PD-1&#8211;blocking antibodies. Thus, this is a simple and minimally invasive strategy to analyze the pharmacological effect of PD-1&#8211;blocking antibodies in cancer patients.

We developed a simple flow cytometry assay for evaluating the binding of PD-1&#8211;blocking antibodies to T cells, requiring only a drop of peripheral blood from cancer patients.

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The ...

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular matrix (ECM), the protein meshwork that binds cells and tissues together. How the elastic properties of an ECM network relate to its composition, and whether the relaxation properties of the ECM play a physiological role, are questions that have yet to be fully addressed. Part of the challenge lies in the complex architecture of most ECM systems and the difficulty in isolating ECM components without compromising their structure. One exception is the zonule, an ECM system found in the eye of vertebrates. The zonule comprises fibers hundreds to thousands of micrometers in length that span the cell-free space between the lens and the eyewall. In this report, we describe a mechanical technique that takes advantage of the highly organized structure of the zonule to quantify its viscoelastic properties and to determine the contribution of individual protein components. The method involves dissection of a fixed eye to expose the lens and the zonule and employs a pull-up technique that stretches the zonular fibers equally while their tension is monitored. The technique is relatively inexpensive yet sensitive enough to detect alterations in viscoelastic properties of zonular fibers in mice lacking specific zonular proteins or with aging. Although the method presented here is designed primarily for studying ocular development and disease, it could also serve as an experimental model for exploring broader questions regarding the viscoelastic properties of elastic ECM&#39;s and the role of external factors such as ionic concentration, temperature, and interactions with signaling molecules.

The protocol describes a method for the study of extracellular matrix viscoelasticity and its dependence on protein composition or environmental factors. The matrix system targeted is the mouse zonule. The performance of the method is demonstrated by comparing the viscoelastic behavior of wild-type zonular fibers with those lacking microfibril-associated glycoprotein-1.

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular ...

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 regulatory mechanism of RDN on hypertension remains elusive. Thus, it&#39;s essential to establish a simpler RDN model in mice. In this study, osmotic mini pumps filled with Angiotensin II were implanted in 14-week-old C57BL/6 mice. One week after the implantation of the mini-osmotic pump, a modified RDN procedure was performed on bilateral renal arteries of the mice using phenol. Age-sex-matched mice were given saline and served as sham group. Blood pressure was measured at baseline and every week subsequently for 21 days. Then, renal artery, abdominal aorta and heart were collected for histological examination using H&#38;E and Masson staining. In this study, we present a simple, practical, repeatable, and standardized RDN model, which can control hypertension and alleviate cardiac hypertrophy. The technique can denervate peripheral renal sympathetic nerves without renal artery damage. Compared to previous models, the modified RDN facilitates the study of the pathobiology and pathophysiology of hypertension.

Here, we present a protocol for renal sympathetic denervation (RDN) in mice with hypertension induced by Angiotensin II infusion. The procedure is repeatable, convenient and allows to study the regulatory mechanisms of RDN on hypertension and cardiac hypertrophy.

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

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and experimental verification. The effective components and potential targets of Trichosanthis and Fritillaria thunbergii were collected by high-throughput experiment and reference-guided (HERB) database of traditional Chinese medicine and a similarity ensemble approach (SEA) database, and the LUAD-related targets were queried by the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. A drug-component-disease-target network was constructed by Cytoscape software. Protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted to obtain core targets and key pathways. An aqueous extract of Trichosanthes-Fritillaria thunbergii and A549 cells were used for the subsequent experimental validation. Through the HERB database and literature search, 31 effective compounds and 157 potential target genes of Trichosanthes-Fritillaria thunbergii were screened, of which 144 were regulatory targets of Trichosanthes-Fritillaria thunbergii in the treatment of lung adenocarcinoma. The GO functional enrichment analysis showed that the mechanism of action of Trichosanthes-Fritillaria thunbergii against lung adenocarcinoma is mainly protein phosphorylation. The KEGG pathway enrichment analysis suggested that the treatment of lung adenocarcinoma by Trichosanthes-Fritillaria thunbergii mainly involves the PI3K/AKT signaling pathway. The experimental validation showed that an aqueous extract of Trichosanthes-Fritillaria thunbergii could inhibit the proliferation of A549 cells and the phosphorylation of AKT. Through network pharmacology and experimental validation, it was verified that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

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

This study reveals the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma based on network pharmacology and experimental verification. The study also demonstrates that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating 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

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

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