Name: determining binding affinity (kd) of radiolabeled antibodies to immobilized antigens
Uploaded: 2022-06-23T13:40:00.000+00:00
Duration: 7 min 39 s
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="ELISA diagram showing antigen-antibody interaction on a microplate for concentration analysis." class="xfigimg" src="/files/ftp_upload/63927/63927fig02.jpg" data-altupdatedat="1747736610000" data-alt="Figure 2"> 
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 arrow designates the rAb that is expected to be washed out of wells with BSA only. This figure was created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/63927/63927fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Buffer preparation
<ol>
	<li>Prepare 50 mL of immobilization buffer (aqueous solution of 50 mM Na2CO3; pH = 9.0).
	<ol>
		<li>Weigh 191 mg of NaHCO3 and 23.9 mg of Na2CO3 on weighing paper and transfer to a 50 mL conical tube. Add 40 mL of 18 MΩ water and vortex to dissolve. Adjust the pH to 9.0 if needed before bringing the total volume to 50 mL with 18 MΩ water.</li>
	</ol>
	</li>
	<li>Prepare approximately 200 mL of washing buffer (phosphate-buffered saline (PBS) containing 0.05% Tween-20) by adding 200 mL of PBS and then 100 µL of Tween-20 to a 250 mL bottle.</li>
	<li>Prepare 50 mL of binding buffer (PBS containing 0.05% Tween-20, and 0.1% bovine serum albumin (BSA)).
	<ol>
		<li>Weigh 50 mg of BSA on weighing paper and transfer to a 50 mL conical tube. Add 50 mL of PBS and then 25 µL of Tween-20 to the tube. Vortex gently to mix.</li>
	</ol>
	</li>
	<li>Prepare 50 mL of blocking buffer (3% BSA in PBS).
	<ol>
		<li>Weigh 1.5 g of BSA on weighing paper and transfer to a 50 mL conical tube. Add 50 mL of PBS, and vortex gently to mix. 
		NOTE: All buffers are recommended to be stored for up to 1 week at 4 °C for best results.</li>
	</ol>
	</li>
</ol>
2. Antigen immobilization
<ol>
	<li>Dilute the antigen in immobilization buffer to reach a concentration of 5 µg/mL.</li>
	<li>Add 100 µL per well of antigen to the bottom of 24 wells of a breakable 96-well, flat-bottom plate in an 8 x 3 array (columns 1-3 for rows A-H). Cover the plate with sealing tape. 
	​NOTE: Ensure that the surface of the well-plate had been treated to maximize adsorption of mixed hydrophobic and hydrophilic domains. These pretreated plates are commercially available.</li>
	<li>Incubate at 4 °C overnight.</li>
	<li>The following day, wash the plate 3x with washing buffer.
	<ol>
		<li>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.</li>
		<li>Using a multichannel pipette, add 300 µL per well of washing buffer to wells containing the antigen. Remove the liquid as described in 2.4.1. Repeat the washing step a total of three times.</li>
	</ol>
	</li>
</ol>
3. Blocking of nonspecific sites with BSA
<ol>
	<li>Using a multichannel pipette, add 300 µL per well of blocking buffer to the 24 antigen-coated wells and 24 empty wells of the 96-well plate (columns 1-6 for rows A-H).</li>
	<li>Incubate the plate for 1 h at ambient temperature.</li>
	<li>Wash the plate with 300 µL per well of washing buffer a total of three times. Refer to step 2.4 for a detailed description of washing the plate. 
	 </li>
</ol>
4. Serial dilutions and addition of the rAb solution
CAUTION: The following steps involve radioactivity. Steps should only be performed by those with radiation safety training. Researchers should double glove and perform steps with adequate shielding.
<ol>
	<li>Synthesize the rAb under investigation using the method of choice. The rAbs used as an example were synthesized as described previously19. 
	NOTE: This protocol focuses on the characterization of a rAb once radiolabeled.</li>
	<li>Make 8 x 3-fold serial dilutions (designated for rows A-H on the plate) of the rAb in the binding buffer. 
	NOTE: The concentrations of the serial dilutions will vary for each rAb. Details are discussed in the Discussion section. If the dilution factor changes, the volume needed for each dilution should be recalculated to ensure enough volume for 1) binding of the rAb in each well, 2) seeding the following dilution, and 3) aliquoting a rAb standard solution for gamma counting to measure the radioactivity of the total rAb added to each well.
	<ol>
		<li>Calculate the volume of stock rAb needed to make a 1.2 mL solution of the first concentration (label as A).</li>
		<li>Add 800 µL of binding buffer to microcentrifuge tubes labeled B, C, D, ... to H. Add 1.2 mL minus the volume calculated in step 4.2.1 of binding buffer to a microcentrifuge tube labeled A.</li>
		<li>Add the volume of stock rAb calculated in 4.2.1 to tube A. Vortex gently to mix and then spin down using a mini microcentrifuge to collect all liquid at the bottom of the tube.</li>
		<li>Add 400 µL from tube A to tube B. Vortex to mix and then spin down using a mini microcentrifuge. Repeat adding from B to C, C to D, ..., G to H.</li>
	</ol>
	</li>
	<li>Add 100 µL per well of each dilution to three wells immobilized with antigen and three wells blocked with BSA only. For example, add dilution A to wells A1-A3 (antigen) and A4-A6 (BSA).</li>
	<li>Add 100 µL of each dilution to microcentrifuge tubes labeled A std - H std. Save these tubes as rAb standards to be assayed in the gamma counter.</li>
	<li>Incubate the plate for 1 h at 37 °C with gentle rocking.</li>
</ol>
5. Wash plates and assay radioactivity
<ol>
	<li>Label microcentrifuge tubes for each well (A1 to A6, B1 to B6 ... through H1-H6). Use two different colored markers to color-code samples if desired-one for wells coated with antigen and one for wells with BSA only.</li>
	<li>Aspirate the rAb from each well using a vacuum aspirator.</li>
	<li>Using a multichannel pipette, add 300 µL of washing buffer to each well. Aspirate the washing buffer. Repeat the wash a total of five times.</li>
	<li>Break apart the wells into the appropriate microcentrifuge tubes.</li>
	<li>Count the radioactivity in the tubes using a gamma counter. Count first the tubes with the antigen (H1, H2, H3 to A1, A2, A3), and then those with BSA only (H4, H5, H6 to A4, A5, A6). To minimize interference, separately count the standards for each dilution (H std to A std) at a different time.</li>
</ol>
6. Data analysis
NOTE: The supplemental files contain corresponding spreadsheet and statistical analysis templates for analyzing and plotting the data.
<ol>
	<li>In a spreadsheet, calculate the total, specific, and nonspecific binding for each sample (see the spreadsheet template attached as a supplemental file).
	<ol>
		<li>Calculate "Bound Activity" as the counts per minute (CPM) of the sample (obtained from the gamma counter) divided by the CPM of the appropriate standard. Calculate "% Bound" as Bound Activity times 100.</li>
		<li>Calculate "Total Bound, mol/L" by multiplying "% Bound" with the concentration (mol/L) of the rAb added. Calculate "Total Bound, mol" by multiplying "Total Bound, mol/L" with the volume of rAb added in liters (0.0001 L).</li>
		<li>Calculate "Specific Binding, mol" by subtracting "Total Bound, mol" of the BSA dilutions from the antigen dilutions such that A1 pairs with A4, A2 with A5, A3 with A6, B1 with B4, etc.</li>
		<li>Calculate "Nonspecific Binding, mol" by subtracting "Specific Binding, mol" from "Total Binding, mol" for each well.</li>
	</ol>
	</li>
	<li>In the statistical analysis plotting software, plot the concentration of rAb added (nmol/L) on the x-axis versus binding (mol) on the y-axis. Create separate groups to plot in triplicate total binding, specific binding, and nonspecific binding. Perform a nonlinear fit analysis by selecting the following parameters in the software used (Table of Materials; see the statistical analysis template attached as a supplemental file).
	<ol>
		<li>Select New Analysis. Under XY analyses, select Nonlinear regression (curve fit). Make sure all the data is selected under Analyze which data sets? and then select Ok.</li>
		<li>On the Model tab, under Binding - Saturation, select One site - Specific binding. On the Confidence tab, select Identify 'ambiguous' fits. Leave all other parameters as default and select Ok. 
		NOTE: This will calculate the KD and Bmax for the total, specific, and nonspecific binding. The KD of the specific binding is the KD in nmol/L of the rAb bound to the antigen.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest.

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'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) 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, which corresponds to the maximum amount of antigen-binding. Bmax can be used to calculate the number of antigen molecules if needed.
<img alt="Binding affinity graph with radiolabeled antibody concentration, showing % bound, \( K_D \) analysis." class="xfigimg" src="/files/ftp_upload/63927/63927fig01.jpg" data-altupdatedat="1747736610000" data-alt="Figure 1"> 
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 (α-EGFR) or to cMET (α-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's unconjugated fraction. To the best of the authors' 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><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 <...

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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 ...

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Research

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Medicine

6.0K Views.  Yale University. 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.

Video: Determining Binding Affinity KD of Radiolabeled Antibodies to Immobilized Antigens

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Biochemistry

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

Chemistry

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

Cancer Research

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Bioengineering

Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ failure, which is associated with high morbidity and mortality. Collagen is a key driver of fibrosis, with type I and type III collagen being the primary types involved in many fibrotic diseases. Unlike conventional protocols used to immobilize other proteins (e.g., elastin, albumin, fibronectin, etc.), comprehensive protocols to reproducibly immobilize different types of collagens in order to produce stable coatings are not readily available. Immobilizing collagen is surprisingly challenging because multiple experimental conditions may affect the efficiency of immobilization, including the type of collagen, the pH, the temperature, and the type of microplate used. Here, a detailed protocol to reproducibly immobilize and quantify type I and III collagens resulting in&#160;stable and reproducible gels/films is provided. Furthermore, this work demonstrates how to perform, analyze, and interpret in vitro time-resolved fluorescence binding studies to investigate the interactions between collagens and candidate collagen-binding compounds (e.g., a peptide conjugated to a metal chelate carrying, for example, europium [Eu(III)]). Such an approach can be universally applied to various biomedical applications, including&#160;the field of molecular imaging to develop targeted imaging probes, drug development, cell toxicity studies, cell proliferation studies, and immunoassays.

This work presents an optimized protocol to reproducibly immobilize and quantify type I and III collagen onto microplates, followed by an improved in vitro binding assay protocol to study collagen-compound interactions using a time-resolved fluorescence method. The subsequent step-by-step data analysis and data interpretation are provided.

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Immunology and Infection

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

Neuroscience

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

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

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