The authors have no acknowledgments.

Human matrices were purchased from commercial sources with approval from their Institutional Review Board (IRB) but should be treated as potentially infectious. Ensure that the laboratory environment used maintains a culture of safety12.
1. Before Starting the Assay
<ol>
	<li>Prepare ERT-conjugated streptavidin magnetic beads according to the manufacturer&#8217;s instructions13.</li>
	<li>Prepare quality control samples (QCs): spike a positive control antibody (PC) (e.g., an NAb) in the species-specific matrix (e.g., pooled CSF or serum). 
	NOTE: FDA and EMA guidance recommends preparing a high and a low QC for assay validation and routine testing1,14.
	<ol>
		<li>For example, to obtain a QC at 10 &#181;g/mL, add 10 &#181;L of 1 mg/mL stock positive control into 990 &#181;L of the relevant matrix (e.g., CSF) for a total volume of 1,000 &#181;L.</li>
		<li>Aliquot the QC samples in a volume suitable for a single use (e.g., 50 &#181;L) and freeze the aliquots at -60 to -80 &#176;C1.</li>
		<li>Aliquot a cut-point-control (CC), species-specific matrix not treated with the test article (e.g., pooled CSF or serum) for a single use (e.g., 50 &#181;L) and freeze the aliquots at -60 to -80 &#176;C1.</li>
	</ol>
	</li>
	<li>Perform a conjugation reaction between the fluorophore and the ERT using a protein-labeling kit according to the manufacturer&#8217;s protocol15. Aliquot the sample in a volume suitable for a single use (e.g., 50 &#181;L) and freeze the aliquots at -60 to -80 &#176;C.</li>
</ol>
2. Day 1: Cell Plating and Sample Preparation
<ol>
	<li>Cell plate preparation
	<ol>
		<li>Use a tissue culture hood and maintain an aseptic environment for the following steps. Spray each object that will be placed in the hood with 70% ethanol and maintain a clean environment16,17,18.</li>
		<li>Warm the cell growth medium (e.g., RPMI-1640 with 10% fetal bovine serum and 1% penicillin-streptomycin) in a water or bead bath at 37 &#176;C19.</li>
		<li>Count the human T lymphocytes Jurkat cells and plate 100 &#181;L per well at 7.5 x 105 cells/mL in a 96-well round-bottom cell culture plate appropriate for use on a flow cytometer equipped with a plate loader.
		<ol>
			<li>For example, use a hemocytometer or an automated cell counter to count the cells20,21.</li>
			<li>Ensure that the cells have at least 70% viability before proceeding with the experiment (e.g., assess the viability with trypan blue staining)17.</li>
		</ol>
		</li>
		<li>Incubate the cells in a 37 &#176;C incubator with 5% CO2 and 95% humidity overnight for 14&#8211;20 h.</li>
	</ol>
	</li>
	<li>Screening sample preparation 
	<ol>
		<li>Dilute the subject or assay control samples using serum-free media (e.g., RPMI-1640). In the example method here, dilute samples 1:2.5 in RPMI-1640 by adding 60 &#181;L of the sample to 90 &#181;L of serum-free media. (e.g., 8-strip tubes). Proceed to step 3.1 for the assay procedure to prepare a fluorophore-labeled ERT. 
		Note: The dilution of 1:2.5 was experimentally determined and is optimal for the method presented here. Optimal sample dilutions should be determined for each method that is developed.</li>
	</ol>
	</li>
	<li>Confirmatory bead and sample preparation
	<ol>
		<li>Calculate the number of ERT-conjugated beads needed for confirmatory samples.
		<ol>
			<li>For example, for 10 samples, use 10 samples x 100 &#181;L of ERT-conjugated beads per sample + 30% extra to compensate for any losses during the washing step = 1,300 &#181;L of ERT-conjugated beads.</li>
		</ol>
		</li>
		<li>Vortex the beads thoroughly. Add the calculated number of beads to a 15 mL conical tube for washing.</li>
		<li>Wash the ERT-conjugated magnetic beads by adding 1,300 &#181;L of coupling buffer or as suggested by the manufacturer (e.g., with phosphate-buffered saline with 0.1% polysorbate 20) following the steps below13.</li>
		<li>Vortex the tube thoroughly. Place the tube in a magnetic tube rack for 2 min. Without disturbing the beads, carefully aspirate and discard the supernatant. 
		NOTE: The solution will turn from dark brown to clear when beads are pulled to the magnet.</li>
		<li>Resuspend the beads with 1,300 &#181;L of coupling buffer. Repeat the wash steps for a total of four washes.</li>
		<li>After the final wash, suspend the beads in 1,300 &#181;L of coupling buffer (previously calculated in step 2.3.1.1). Add 100 &#181;L per well to a 96-well, white, round-bottom, non-binding polypropylene plate according to the plate map (e.g., one confirmatory well per sample or QC; the sample will be split into duplicates when incubated with the fluorophore-labeled ERT).</li>
		<li>Place the plate on a 96-well side-skirted magnet and allow the beads to form a pellet. Carefully aspirate the clear supernatant and discard it. 
		NOTE: The solution will turn from dark brown to clear after approximately 1&#8211;2 min on the side-skirted magnet. The obtained beads are called the &#8220;dry&#8221; beads.</li>
		<li>If the samples screened positive and are being confirmed, add 100 &#181;L of each sample with and without ERT-conjugated beads into the appropriate/assigned well. Seal the plate with a plastic film and shake it for at least 60 min on a rotary shaker at approximately 800 rpm at room temperature (RT). 
		NOTE: Previously prepared and frozen positive and negative QCs and the CC should be included on every plate.</li>
		<li>After the incubation, place the confirmatory plate on the 96-well side-skirted magnet and allow the beads to form a pellet.</li>
	</ol>
	</li>
	<li>Titer dilution series preparation
	<ol>
		<li>To prepare a titer series, serially dilute the sample in the pooled matrix a sufficient number of times to cross the predetermined titer cut point1.</li>
		<li>For example, mix 30 &#181;L of the sample to 60 &#181;L of pooled matrix in a 1:3 dilution series for a total of 8 dilutions (e.g., 8-strip tubes). Proceed to step 3.1 for the assay procedure to prepare fluorophore-labeled ERT.</li>
	</ol>
	</li>
</ol>
3. Day 1: Assay Procedure
<ol>
	<li>Prepare the fluorophore-labeled ERT in the serum-free medium (e.g., 60 &#181;L per well, or approximately 6 mL/plate).
	<ol>
		<li>For example, to prepare 10 mL of 1 &#181;g/mL fluorophore-labeled ERT, add 10 &#181;L of stock 1 mg/mL fluorophore-labeled ERT to 9.99 mL of serum-free media (e.g., RPMI-1640).</li>
	</ol>
	</li>
	<li>In a new incubation plate (e.g., 96-well, white, round-bottom, non-binding polypropylene plate), add the prepared samples from step 2.2, 2.3, or 2.4 and a fluorophore-labeled ERT in a 1:1 mixture in duplicate wells (e.g., transfer 60 &#181;L of each prepared sample and add 60 &#181;L of the fluorophore-labeled ERT per well). 
	NOTE: An example experiment plate map is provided in Figure 1.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57777/57777fig1.jpg" /> 
Figure 1: Example of an experiment plate layout. Samples and a fluorophore-labeled ERT were incubated overnight at 2&#8211;8 &#176;C. Controls such as high-quality control (HQC), low-quality control (LQC), and negative quality control (NQC) were screened and confirmed on opposite corners of the plate to assess plate uniformity. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57777/57777fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Mix the samples and the fluorophore-labeled ERT by pipetting up and down several times with a multichannel pipette. Wrap the plates in the foil and incubate them overnight at 2&#8211;8 &#176;C for 14&#8211;20 h.</li>
</ol>
4. Day 2: Adding the Prepared Samples to the Cells
<ol>
	<li>Remove the sample incubation plate(s) from the incubation at 2&#8211;8 &#176;C and warm the plates by placing them in a bead bath at 37 &#176;C for 10&#8211;15 min.</li>
	<li>Remove the cells from the CO2 incubator. Perform a visual check of the plated cells using an inverted microscope to ensure that the cells appear healthy and evenly distributed across the wells17,19.</li>
	<li>Add 100 &#181;L of the previously mixed samples and fluorophore-labeled ERT to a cell plate according to the experimental plate map.</li>
	<li>Place the cell plate with the added samples back into the CO2 incubator at 37 &#176;C. Incubate the plate for 3 h and &#177; 15 min. 
	NOTE: This time was established in the lab as optimal for the signal-to-noise response in the assay.</li>
	<li>Centrifuge the cell plate for 6 min at 320 x g in a tabletop centrifuge at 14&#8211;18 &#176;C. Confirm the presence of a cell pellet at the bottom of the plate wells.</li>
	<li>Hold the plate at a 30&#8211;45&#176; angle. Carefully remove the supernatant from each well without disturbing the cell pellet. Add 200 &#181;L of 1x DPBS onto the cell pellet in each well to resuspend the cells.</li>
	<li>Repeat the cell washing steps for a total of 3 washes. Proceed immediately to step 5 for the cell viability staining.</li>
</ol>
5. Day 2: Cell Viability Staining
<ol>
	<li>Using a multichannel pipette, add 100 &#181;L of a working solution of 1x Live/Dead stain to each well, selected for an emission wavelength different from that of the fluorophore-labeled ERT and prepared according to the manufacturer&#8217;s instructions22.</li>
	<li>Incubate the plate for 15 min at RT in the dark. Centrifuge the plate for 6 min at 320 x g in a tabletop centrifuge at 14&#8211;18 &#176;C.</li>
	<li>Carefully remove the supernatant from each well without disturbing the cell pellet. Wash the cells 1x with 1x DPBS.</li>
</ol>
6. Day 2: Fixing the Cells with 1% Paraformaldehyde
<ol>
	<li>Using a multichannel pipette, dispense 100 &#181;L of chilled (at 2&#8211;8 &#176;C) 1% paraformaldehyde (PFA) into each well.</li>
	<li>Seal the plates and gently pulse vortex to mix. Wrap the plate in the foil and place it at 2&#8211;8 &#176;C for at least 10 min to allow for fixation. 
	NOTE: Be careful to avoid splashing onto the plate seal.</li>
	<li>Add 50 &#181;L of 1x DPBS into each well using a multichannel pipette up and down prior to the analysis.</li>
</ol>
7. Flow Cytometry
<ol>
	<li>Place the plates on the flow cytometer with the plate loader and run them.</li>
	<li>Acquire and record the measured median fluorescence intensity (MFI) using the flow cytometry software (see Table of Materials). 
	NOTE: See Figure 2 as an example for the loader settings. The sample flow rate, sample volume, mixing volume, mixing speeds, and number of mixes are optimized for this assay. These criteria may be adjusted using the &#34;arrows up&#34; or&#160;&#34;down&#34; buttons next to the numbers for each criterion, depending on the flow cytometry acquisition software23.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57777/57777fig2.jpg" /> 
Figure 2: Example of the flow cytometer loader settings. The loader settings should be optimized for each assay that is developed. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57777/57777fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Create a cytometer analysis/gates/plots as shown in Figure 3. 
	NOTE: The gate creation and application may differ for each flow cytometry software23.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57777/57777fig3.jpg" /> 
Figure 3: Flow cytometry gating strategy. Jurkat cells were separated from the total events and collected by plotting the forward-scatter (FSC) vs. side-scatter (SSC) channels and drawing a gate around the target population. Singlets (single cells) were separated from doublets or larger cell aggregates using the FSC area (FSC-A) and FSC height (FSC-H) channels. Live cells were chosen by gating on singlet cells negative for a viability stain. The median fluorescence intensity (MFI) was measured in single, live Jurkat cells and is plotted as a histogram. The MFI values of cells with the drug uptake blocked (e.g., HQC) and of cells with control amounts of uptake are shown (red and blue, respectively). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57777/57777fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Exclude dead cells from the analysis and record approximately 10,000 events23.</li>
</ol>
8. Data Analysis
<ol>
	<li>Export the data (raw MFI) to the analysis software (see Table of Materials). 
	NOTE: Depending on the analysis needed, the following data analysis may be performed using analysis software.</li>
	<li>Calculate the mean MFI of duplicate wells (QCs and samples) and the coefficient of variation (%CV) to evaluate the spread variability of the duplicates from the mean. 
	<img alt="Equation 1" src="/files/ftp_upload/57777/57777eq1v2.jpg" /> 
	<img alt="Equation 2" src="/files/ftp_upload/57777/57777eq2v2.jpg" /> 
	<img alt="Equation 3" src="/files/ftp_upload/57777/57777eq3v2.jpg" /></li>
	<li>For samples and QCs that were screened in the assay, calculate the percent signal inhibition (%SI) relative to the mean MFI of the for the QCs pooled matrix CC. 
	<img alt="Equation 4" src="/files/ftp_upload/57777/57777eq4v2.jpg" /></li>
	<li>If needed, calculate the recovery ratios (RRs) for the confirmed QCs and samples relative to the corresponding screen values. 
	<img alt="Equation 5" src="/files/ftp_upload/57777/57777eq5v2.jpg" /></li>
</ol>

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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=HTS+Assay+Validation.">HTS Assay Validation.</a> Assay Guidance Manual. , 1-30 (2004).</li><li>Thermo Fisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tech Tip #34. Binding characteristics of antibody-binding proteins: Protein A, Protein G, Protein A/G and Protein L. , (2013).</li><li>Long, B., 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=Long-term+Immunogenicity+of+Elosulfase+Alfa+in+the+Treatment+of+Morquio+A+Syndrome:+Results+From+MOR-005,+a+Phase+III+Extension+Study.">Long-term Immunogenicity of Elosulfase Alfa in the Treatment of Morquio A Syndrome: Results From MOR-005, a Phase III Extension Study.</a> Clinical Therapeutics. 39, (2017).</li><li>Schweighardt, B., 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=Immunogenicity+of+Elosulfase+Alfa,+an+Enzyme+Replacement+Therapy+in+Patients+with+Morquio+A+Syndrome:+Results+from+MOR-004,+a+Phase+III+Trial.">Immunogenicity of Elosulfase Alfa, an Enzyme Replacement Therapy in Patients with Morquio A Syndrome: Results from MOR-004, a Phase III Trial.</a> Clinical Therapeutics. 37, (2015).</li><li>Mellman, I. R. A., Plutner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internalization+and+Degradation+of+Macrophage+Fc+Receptors+Bound+to+Polyvalent+Immune+Complexes.">Internalization and Degradation of Macrophage Fc Receptors Bound to Polyvalent Immune Complexes.</a> The Journal of Cell Biology. 98, 1170-1177 (1984).</li><li>Wang, 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=Neutralizing+antibodies+to+the+therapeutic+enzymes:+considerations+for+testing,+prevention+and+treatment.">Neutralizing antibodies to the therapeutic enzymes: considerations for testing, prevention and treatment.</a> Nature Biotechnology. 26, 901-908 (2008).</li></ol>

The authors are employees and shareholders of BioMarin Pharmaceutical Inc.

Neutralizing antibodies or other factors that prevent the ERT uptake through CI-M6PR have the potential to impact ERT safety or efficacy1. Therefore, it is important to evaluate NAbs in subject samples using a robust model relevant to the drug&#39;s mechanism of action. We and others have found that the performance of some bioassay formats (e.g., receptor dimerization, luciferase expression, etc.) does not align with health authority recommendations for assay precision, reproducibility, and sensitivity29. In the bioassay method described here, Jurkat cells that express endogenous CI-M6PR are employed to monitor NAbs specific to the lysosomal ERT uptake. Combined with a flow cytometry readout, this assay uses a physiological cell model with suitable assay precision, sensitivity, reproducibility, and high sample throughput. NAb detection with a flow cytometry readout has also been applied to other indications. For example, methods have been developed to detect pre-existing antibodies against hepatitis E and adeno-associated virus (AAV)30,31.
Critical steps in the protocol include selecting a suitable fluorophore, incorporating an LQC that monitors assay sensitivity, ensuring a sufficient level of cell viability, and maintaining strict adherence to the incubation times. In the example presented here, fluorophores (e.g., Alexa Fluor 488, Alexa Fluor 647, and CypHer 5e), conjugated to a lysosomal ERT, varied widely in their performance. Alexa Fluor 647, which was also the brightest fluorophore tested32, was selected for further assay development. We recommend that several fluorophores be evaluated early on in the development of the assay to provide the best sensitivity and dynamic range. Assay sensitivity during sample testing should be monitored by including an LQC that is close enough to the sensitivity limit that it will fail in 1% of the testing runs1. Together with the HQC, the LQC also acts as a system suitability QC to monitor assay drift over time3. Optimal cell viability and performance is achieved by preparing a cell bank of single-use aliquots and qualifying in new cell banks based on comparable QC results3,18. Cell and fluorophore-conjugated drug incubation times are also central to a consistent assay performance since drug uptake increases with the amount of time the drug is incubated with cells. To minimize day-to-day variability in drug uptake, sample data may be normalized to pooled matrix control samples on each plate (e.g., the cut point control samples in the method above). Another important factor in producing consistent data with this method is to monitor plate uniformity and minimize possible edge effects. An initial experiment may include performing the assay using a single QC across the whole plate, while more robust experiments may be performed during assay validation (e.g., precision and accuracy)1. This demonstrates the importance of the plate map layout33. As shown in the plate map example, quality control samples placed on both sides of the plate monitor a uniformity that can be tracked over time with Levey-Jennings charts34.
While this assay monitors NAbs and other factors that may inhibit lysosomal ERT uptake, additional experiments may be conducted to confirm an uptake inhibition due to antibodies. One option is to treat samples with the protein A/G/L, which non-specifically binds immunoglobulin35. If samples continue to test positive after protein A/G/L depletion, a non-antibody inhibitory factor may be responsible for blocking the drug uptake. The method described here was designed to measure antibody-mediated uptake inhibition, and the positive control and other assay parameters should be reconsidered if a high percentage of subject samples with non-antibody inhibitory factors are found.
The assay may also be further characterized to demonstrate fluorophore-labeled drug traffics to the appropriate cellular compartment based on the drug&#39;s mechanism of action. In the example presented here, an ERT is expected to bind CI-M6PR on the cell surface and traffic it to the lysosome. We previously reported the results of several experiments indicating that nearly all of the fluorescent signal observed in the method results from fluorophore-labeled ERT in the lysosome8. We found that the fluorophore-labeled ERT signal was eliminated following a treatment of the cells with cytochalasin B, which disrupts internalization through the inhibition of actin reorganization. Also, experiments that quenched external fluorophore with trypan blue, or slowed internalization by placing cells at 4 &#176;C, indicate that the fluorophore-labeled ERT is rapidly internalized and very little fluorescence is due to an ERT bound at the cell surface. Lysosomal targeting was confirmed by visualizing the co-localization of pH-sensitive lysotracker dye with the fluorophore-conjugated drug using confocal microscopy. A specificity for uptake through CI-M6PR may also be verified using exogenous M6P to compete with labeled drugs for receptor binding. Similar experiments should be performed to verify the uptake kinetics and cellular localization of fluorophore-conjugated drugs in other assays.
This cell-based assay platform has been used to study NAbs for therapeutic drugs that utilize CI-M6PR receptor-mediated endocytosis. We recently reported results using this assay platform that demonstrated no correlation between the development of an NAb and drug efficacy for elosulfase alfa36,37. To validate the assay for clinical sample testing, the parameters, including assay sensitivity, precision, selectivity, specificity, drug tolerance, robustness, and cut points, should be evaluated according to established guidances3,4. It should also be noted, for lysosomal ERTs, that NAbs may develop with the potential to interfere with drug activity through binding near the enzyme catalytic site. In general, we consider monitoring this type of NAb to be of a lower priority since the harsh acidic and proteolytic environment of the lysosome is not favorable to antibody-ERT interactions2,39,40. However, it is possible that proteolytic-resistant NAbs exist and may inhibit the catalytic portion of a drug40. This assay monitors fluorophore-labeled ERT uptake to the lysosome, and a limitation of the assay is the inability to monitor proteolytic-resistant NAbs. If this type of NAb is suspected based on safety or efficacy data, an assay that monitors ERT activity should be developed and used to test samples.
The assessment of immunogenicity is important in understanding the effects of NAbs on drug safety and efficacy. The identification of NAbs capable of inhibiting in vitro drug uptake via CI-M6PR provides an opportunity for understanding NAb activity in vivo. The method presented here utilizes a human cell line that expresses CI-M6PR to measure the interference of fluorophore-conjugated lysosomal ERT cellular uptake. This method has already been used to monitor NAbs for several ERTs intended to treat lysosomal storage diseases. This assay platform may apply to other methods for studying the effects of NAbs on biologic therapeutics that require a cellular internalization for their proper function.

Immunogenicity assessment is an important part of the safety and efficacy monitoring program for any biologic therapeutic product, including ERTs. Patients may develop an immune response that can directly impact drug safety, efficacy, and pharmacokinetic/pharmacodynamic profiles. A subset of these ADA, called NAbs, may inhibit ERT efficacy in two ways: through the inhibition of the ERT uptake into the targeted cell or by inhibiting the ERT catalytic activity. The method presented here is designed to measure NAbs that interfere with the ERT uptake into cells. To fully monitor the safety and efficacy of the therapeutic ERT, continuous monitoring of NAbs is crucial in elucidating any potential correlations with clinical outcomes or pharmacodynamic effects1.
Platforms for evaluating NAbs against protein therapeutics include cell-based, enzymatic activity and ligand-binding assays1. The optimal assay platform is selected based on a variety of criteria: the mechanism of action of the therapeutic product, the assay platform sensitivity, selectivity, precision, and importantly, its ability to mimic the inhibitory action of NAbs in vivo. Ligand-binding assays may be appropriate in certain cases (e.g., when a relevant cell line cannot be identified or if the appropriate sensitivity cannot be achieved in a cell-based assay). However, in the 2016 draft FDA Guidance for Industry document and other industry-accepted white papers, cell-based NAb assays are recommended because they may better reflect the biological mechanism of the drug in vivo1,2,3.
The critical components for developing a flow cytometry cell-based NAb assay include a suitable cell line that responds to drug stimulation, a surrogate positive-control NAb that neutralizes the ERT, a fluorophore-conjugated ERT, and the test species biological matrix4,5,6. The cell line selection is dependent on the ERT mechanism of action and multiple cell lines should be evaluated during the assay development3. In the method described here, human Jurkat T cells were selected for their endogenous CI-M6PR expression on the cell surface and the lack of antibody fragment receptors (FcRs) that indiscriminately bind the Fc region of most antibodies7,8. During the assay development, it is important to establish a negative control for the validation studies and the patient sample testing, such as serum pooled from individuals that have not been treated with the test article1. Cell lines should also tolerate relevant matrices from different species for continuity across the nonclinical, clinical, and post-marketing stages of drug development1. Another component is the selection of the assay&#39;s positive control. The positive control for the ERT cell uptake assay was chosen based on its ability to bind the therapeutic and neutralize the uptake through CI-M6PR9,1. It is often difficult to obtain useful or sustainable amounts of neutralizing antisera from human subjects for use as an assay control, especially in rare disease patient populations2. Alternatives include antisera from hyper-immunized animals, or affinity-purified polyclonal or monoclonal antibodies spiked into the assay relevant matrix1. While using a species-specific matrix, it is possible that inhibitory factors other than antibodies present in the matrix may inhibit the ERT uptake. Another critical component of the assay is the fluorophore-conjugated ERT. The selection of the fluorophore for the ERT conjugation should be evaluated for each ERT, based on the assay&#39;s need for brightness, pH stability, and potential spectral overlap into other channels on the flow cytometer.
The assay described here is an example for measuring a NAb to a therapeutic protein, such as an ERT, that enters the cell via CI-M6PR. Several ERTs, intended to treat lysosomal storage disorders (LSDs), utilize this pathway for cell uptake and lysosomal targeting, including elosulfase alfa for Morquio A syndrome, cerliponase alfa for CLN2 Batten disease, agalsidase alfa for Fabry disease, and alglucosidase alfa for Pompe disease10,11. The purpose of this method is to measure the relative levels of NAbs that interfere with drug binding and internalization via CI-M6PR. This is performed in tiered screening, confirmatory, and titer steps3. Samples are first screened for NAb positivity and then confirmed positive in the confirmatory step. Finally, samples that screen and confirm positive may be serially diluted to generate an antibody titer1. This cell-based flow cytometry drug uptake assay provides a sensitive and mechanistically relevant in vitro method of measuring drug-specific NAbs that may affect a drug&#39;s pharmacological profile. We previously validated the method and tested clinical samples using this platform for the drug elosulfase alfa8. Here we describe the detailed step-by-step protocol that may be applied to other therapeutic proteins or ERTs.

<table><tbody><tr><td>0.22 &amp;micro;m filter unit, 500 mL vacuum filter flasks</td><td>Corning</td><td>CLS430769</td><td></td></tr><tr><td>Round Bottom 96-well culture plates</td><td>Thermo-Nunclon</td><td>163320</td><td></td></tr><tr><td>Sterile reagent reservoirs</td><td>VistaLab</td><td>3054-1004</td><td></td></tr><tr><td>96 well white round bottom polystyrene microplate plate</td><td>Corning</td><td>3605</td><td></td></tr><tr><td>96-well Polypropylene Tubes, 8-Tube Strips</td><td>Corning</td><td>4408</td><td></td></tr><tr><td>Magnetic bead separator tube rack</td><td>V&amp;amp;P Scientific, Inc</td><td>VP 772F2M-1, VP 772F2R-2, VP 772F2R-3</td><td></td></tr><tr><td>DynaMag -2 Magnet</td><td>Thermo Fisher Scientific</td><td>12321D</td><td></td></tr><tr><td>DynaMag -96 Side Skirted Magnet</td><td>Thermo Fisher Scientific</td><td>12027</td><td></td></tr><tr><td>BD FACSCanto II Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD High Throughput Sampler (HTS)</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BioRad TC20 Automated Cell Counter</td><td>BioRad</td><td>1450103</td><td></td></tr><tr><td>Microplate Shaker</td><td>VWR</td><td>12620-928</td><td></td></tr><tr><td>Galaxy MiniStar Microcentrifuge</td><td>VWR</td><td>C1413</td><td></td></tr><tr><td>tissue culture CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Nuaire</td><td>NU-4750</td><td></td></tr><tr><td>Biosafety cabinet</td><td>Labcono</td><td>Purifier Cell Logic +</td><td></td></tr><tr><td>Jurkat Cell Line</td><td>ATCC</td><td>TIB152&amp;trade;</td><td></td></tr><tr><td>Alexa Fluor 647 Protein Labeling Kit</td><td>Thermo Fisher Scientific</td><td>A20173</td><td></td></tr><tr><td>Dynabeads M270 Streptavidin</td><td>Thermo Fisher Scientific</td><td>65306</td><td></td></tr><tr><td>EZ-Link NHS-LC-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>21343</td><td></td></tr><tr><td>RPMI-1640 1x Medium</td><td>Life Technologies</td><td>A10491-01-500mL</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Pen-Strep (100x) liquid formulation</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>1x DPBS without calcium and magnesium</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>4% Para-formaldehyde</td><td>Electron Microscopy Science</td><td>15735-85</td><td></td></tr><tr><td>LIVE/DEAD Fixable Violet Dead Cell Stain Kit, for 405 nm excitation</td><td>Life Technologies</td><td>L34955</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379-100mL</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>3059</td><td></td></tr><tr><td>Pooled human matrix (&lt;em&gt;e.g.&lt;/em&gt; human serum or cerebrospinal fluid)</td><td>Bioreclamation IVT</td><td>request a quote from website</td><td></td></tr><tr><td>VWR Polyester Plate Film</td><td>VWR</td><td>60941</td><td></td></tr><tr><td>Seal &amp;amp; Sample Aluminum Foil</td><td>Beckman Coulter</td><td>538619</td><td></td></tr></tbody></table>

detection of antibodies that neutralize the cellular uptake of enzyme replacement therapies with a cell-based assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

On the first day of the method, a frozen aliquot of Jurkat cells was thawed and plated, and the test samples were prepared. Figure 1 shows an example plate map. On the second day, the samples were mixed with the Jurkat cells and incubated at 37 &#176;C for approximately 3 h and 15 min. The cells were then washed, fixed with PFA, and analyzed on a flow cytometer. Figure 2 shows example flow cytometer settings.
The flow cytometry gating strategy was designed to measure the amount of fluorophore-conjugated drug in live single Jurkat cells24 (Figure 3). The cells were separated from the debris by drawing a gate that excludes cellular debris [usually, low forward scatter (FSC)] and dead cells [usually, high side scatter (SSC)], leaving only live cells for analysis25. Single cells were then separated from cell clusters by drawing a gate that excludes the high FSC area and the low FSC height26. The cells were treated with a viability stain that labeled any dead or unhealthy cells without an intact membrane, and an additional gate was created to exclude the dead cells22. The MFI of the live single cells was used for the analysis of the fluorophore-conjugated ERT uptake. As an example of potential screening assay results, the matrix spiked with a high amount of surrogate anti-drug antibody positive control [e.g., high-quality control (HQC)] inhibited fluorophore-conjugated ERT uptake, resulting in a low MFI of approximately 300 (Figure 3). In contrast, cells incubated with the matrix in the absence of the positive control antibody should have a higher MFI (MFI = 37,830 in Figure 3), demonstrating the uptake of the fluorophore-conjugated ERT.
The neutralizing positive control antibody for the example here was selected based on the mechanism of action of the drug (i.e., the ERT uptake through CI-M6PR). A panel of fluorophores was also tested and compared for optimal assay sensitivity and dynamic range1. CypHer 5e was tested due to its increased fluorescence at an acidic pH, which might be relevant to some ERTs as an additional measure of lysosomal targeting of the drug27. A green fluorescent dye (e.g., Alexa Fluor 488), and a far-red fluorescent dye (e.g., Alexa Fluor 647) were also examined. An example of how different fluorophores might perform is presented in Figure 4a and 4b. In the example, the Alexa Fluor 647 ERT had the best performance due to its superior sensitivity (e.g., the highest %SI at the lowest PC concentration) and wide dynamic range (~3 orders of magnitude).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57777/57777fig4v2.jpg" /> 
Figure 4: Example results from different fluorophore-ERT conjugates. (A) During the assay development, a positive control (PC) dilution curve should be evaluated using different fluorophore-conjugated ERTs. In the example shown here, two fluorophore-conjugated ERTs (Alexa Fluor 488 and CypHer 5e) at 6.25 &#181;g/mL and a brighter fluorophore-conjugated ERT (Alexa Fluor 647) at 1.56 &#181;g/mL were tested in the presence of increasing concentrations of positive-control NAbs. (B) This panel shows the curves from panel A graphed, using MFI to show the significant increase in the dynamic range using an Alexa Fluor 647-conjugated ERT. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57777/57777fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The method described is a tiered approach for detecting, confirming, and interpolating a quasi-quantitative level of NAb titer3. To establish the assay is ready for validation, the parameters including assay sensitivity, precision, selectivity, specificity, drug tolerance, robustness, and cut points should be evaluated according to established guidances1,3.
Samples were considered potentially positive in this assay when %SI values greater than the screening cut point (SCP) were obtained. The SCP was determined statistically by testing drug-na&#239;ve samples from a representative population and measuring a relative decrease in signal intensity (SI)3. Guidance&#39;s (for instance of the FDA) recommend that the SCP is established at the 95th percentile of the normally distributed data set, and methods for calculating the SCP have been described in detail elsewhere1. Any sample that decreased the assay signal (measured as an increase in the %SI) at or above the SCP was determined to be potentially positive, and samples with results higher than the SCP (with no change or decrease in the %SI) are considered negative.
Samples that screened positive (%SI above the SCP) were tested in the confirmatory assay to determine the specificity of NAbs to the ERT. The confirmatory assay was performed by pre-incubating samples with ERT-conjugated magnetic beads to remove drug-specific antibodies or inhibitory factors. The RR (the ratio of confirmatory MFI to screening MFI) was evaluated to determine the number of NAbs removed from the sample. An RR higher than the calculated threshold of positivity [i.e., the confirmatory cut point (CCP)] indicated the presence of anti-drug NAbs. As in the screening assay, the CCP should first be established by evaluating treatment-na&#239;ve samples from a representative population in the confirmatory assay. CCP was based on a statistically determined 1% false-positive rate and was the threshold designating a positively confirmed sample (Figure 5)3.
Samples that screened and confirmed positive were serially diluted and tested in the titer assay to determine the relative level or titer of NAbs in each sample. The highest dilution at which a sample tests positive when it crossed a designated threshold [e.g., the titer cut point (TCP)] is the sample titer (Figure 5)1,3.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57777/57777fig5.jpg" /> 
Figure 5: Example sample testing results. These panels show examples of samples that tested positive or negative by screening either above or below an SCP of 17.51 %SI. Positive samples were then tested in the confirmatory assay using drug-conjugated magnetic beads to deplete the drug-specific antibody from the samples prior to testing in the assay. Samples with a recovery ratio (RR) greater than the confirmatory cut point (i.e., RR = 1.315) were considered to be confirmed positive. Samples that confirmed positive were diluted until the signal crossed the titer cut point (TCP) to establish the titer (dilution factor) at which the sample result equals the titer cut point. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57777/57777fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Assay repeatability and variability were tested over several days and with more than one analyst. The QCs were initially prepared in one batch and sub-aliquoted for 1x use. Over the course of 3 d, two analysts performed the assay with several sets of QCs to demonstrate the precision of the assay. In the example data shown, the %CV of the inter- and intra-assay precision for the QCs are less than the FDA guidance&#39;s recommendation of %CV &#60; 20% (Figure 6)1.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57777/57777fig6.jpg" /> 
Figure 6: Example of QC precision data generated over three days with two analysts. The precision data were calculated by an analysis of variance (ANOVA) using the formula presented in DeSilva et al.28. The intra-batch (within runs) and inter-batch (between runs) statistics are reported as %CV. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57777/57777fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay at JoVE.com

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

detection-antibodies-that-neutralize-cellular-uptake-enzyme

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

8.7K Views.  BioMarin Pharmaceutical Inc. Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

Video: Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Medicine

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an anti-TNF&#945; mAb. In addition, this protocol can be used to evaluate other anti-TNF&#945; molecules, such as fusion proteins. The cellular model employed here is sensitive to TNF&#945;-mediated apoptosis when an additional stress factor is induced in cell culture conditions (e.g., serum deprivation). This procedure exemplifies how to execute this analytical assay, highlighting the key operations relating to the sample preparation, cell dilution, apoptosis induction, and spectrophotometric measurements that are critical to ensure successful results. This protocol reveals the best-performance conditions relating to apoptosis induction and efficient signal recording, leading to low uncertainty values.

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method

A protocol for the determination of the relative anti-apoptotic activity of an anti-TNF&#945; mAb using a neutralization mechanism with WEHI 164 cells is presented here. This protocol is useful for comparing the neutralization strength of different molecules with the same biological functionality.

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

Thyroid Cancer Cells as Effectors in an ADCC Bioassay for Cancer Immunotherapy

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.

Author Spotlight: Optimizing Antibody-Based Cancer Treatments via Antibody-Dependent, Cell-Mediated Cytotoxicity Assay

Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Biology

Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay

Source: Gu, Y. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57227">Electrochemiluminescence Assays for Human Islet Autoantibodies.</a>&#160;J. Vis. Exp. (2018)
This video showcases the electrochemiluminescence detection of islet autoantibodies in human serum. The antibodies in the serum bridge the ruthenium-conjugated antigen to the biotinylated capture antigen, forming complexes that are detected by electrochemiluminescence measurements.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Buffer Preparation

	Labeling ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Assessing Functional SARS-CoV-2 Neutralizing Antibodies

This video showcases a neutralization assay measuring serum efficacy in neutralizing SARS-CoV-2 viruses. It involves pseudotyped viruses, serum dilution, and luminescence-based quantification, revealing the neutralization titer.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Pseudotype-based neutralization ...

Immunodiagnostics

Specialized Assays

A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

On day 1 of the MN assay, thaw the sera in a 37 degrees Celsius water bath, and remove immediately after thawing. Heat-inactivate the human sera for 30 minutes in a 56 degrees Celsius water bath, as described in the text protocol. Then, place the sera on ice, and add the virus diluent to the sera to achieve a 1 to 10 pre-dilution.
To test with one virus, add 50 microliters of virus diluent to rows B through H, except columns 11 and 12. Then, add 100 microliters of 1 of 10 diluted sera to columns A1 to A10. Perform a two-fold serial dilution from rows A through H, and discard the last 50 microliters at row H.
For the virus control, add 50 microliters of virus diluent to wells A12, B12, C12, and D12. For the cell control, add 100 microliters of virus diluent to wells E12, F12, G12, and H12. For the control sera, add 100 microliters of diluted control sera to well A, column 11, and add 50 microliters of virus diluent to wells B11 through H11. Proceed to serially dilute down. Cover the plates and incubate at 37 degrees Celsius, 5% carbon dioxide, until ready for the virus addition.
For virus addition, dilute the virus to 100 TCID50 in 50 microliters with virus diluent. Add 50 microliters of diluted virus to all wells, except for column 11 on the back titration plates, and the cell control wells E12, F12, G12, and H12 on all plates. Add 50 microliters of virus diluent to all wells in column 11. Then, add 50 microliters of the virus at 100 TCID50 in 50 microliters to the first well.
Next, mix and transfer 50 microliters to successive wells to perform two-fold serial dilutions. Change the pipette tips between wells to avoid virus carryover. Discard 50 microliters from well H11, then add 50 microliters of virus diluent to column 11 to bring the final volume to 100 microliters. Tap the plates to mix. Incubate the plates at 37 degrees Celsius, 5% carbon dioxide for one hour before performing MDCK cell addition on day one and ELISA on day two as before.
After adding the primary and secondary antibodies, as described in the text protocol, perform substrate addition and plate reading. Wash the plates five times with 300 microliters of wash buffer and tap on a lint-free wipe. Add 100 microliters of freshly prepared OPD substrate to each well. Then, incubate the plates at room temperature until the virus control wells reach an optical density at 490 nanometers between 0.8 to 3, with the cell control at a low background OD less than 0.2. Next, add 100 microliters of the stop solution to all wells.
Read the OD of the wells at 490 nanometers, using a microplate spectrophotometer. Finally, determine the neutralizing antibody titer of each serum sample, as described in the text protocol.

EoE Sub Articles

Assessing Functional Neutralizing Antibody Responses Against a Panel of SARS-CoV-2 Variants of Concern Using Standardized, Quantitative Pseudotype Neutralization Assays

To begin, add 50 microliters of DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin to rows B to H, columns 1 to 12 of a white 96-well plate.
Add 5 microliters of convalescent sera into wells of row A, columns 2 to 10, in a total volume of 100 microliters DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin. Add 5 microliters of positive and negative antisera into wells A11 and A12 as controls.
Remove 50 microliters of the solution from the wells of row A and perform two-fold serial dilutions of all the wells beneath it. For each dilution, use a multichannel pipette to mix the solutions 5-10 times by pipetting up and down without creating air bubbles.
After completing the serial dilution, discard the final 50 microliters from the last well of each column.
Centrifuge the plate for 1 minute at 500 rotations per minute to ensure no liquid is left on the walls of the well. Calculate the amount of DMEM required to dilute the SARS-CoV-2 PV as described in the text manuscript.
Mix the diluted PV solution in a pipette basin and aliquot 50 microliters into each well, except for wells A6 to A12 which comprise the cell-only control. The A1 to A6 wells serve as PV-only control.
Centrifuge plate for 1 minute at 500 rotations per minute to ensure no virus is left on the walls of the well. Then, incubate the plates at 37 degrees Celsius with 5 percent carbon dioxide for 1 hour to allow the serum to bind with the SARS-CoV-2 glycoprotein.
Next, prepare a plate of HEK 293T/17 target cells transfected 24 hours earlier with ACE2 and TMPRSS2 plasmids. Remove the culture media from the plate, and wash it twice with 2 milliliters of PBS, by adding it to one side of the dish to avoid cell detachment.
Discard the PBS and add 2 milliliters of trypsin to the plate. Place the plate in an incubator until the cells are detached.
After cells have detached, add 6 milliliters of DMEM with 10 percent FBS and 1 percent Penicillin-Streptomycin to the plate to quench trypsin activity, and resuspend cells gently.
Count the cells using an automated cell counter or by counting the chamber slide. Add 1 x 104 cells aliquoted in a total volume of 50 microliters to each well and incubate the plate at 37 degrees Celsius with 5 percent carbon dioxide for 48 to 72 hours.
After incubation, remove the culture media from all wells, and add 25 microliters of a 1:1 mix of PBS and a commercial luciferase assay reagent. Read the plate using a commercial luciferase assay system with a microplate luminometer.
Using the raw data generated by the luminometer, calculate the IC50 neutralizing antibody titers as described in the text manuscript.