Funding was provided by the Joint Science and Technology Office &#8211; Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).

Plate 20,000 differentiated mouse ES cells (MES) / well in a 96 well Poly-D lysine coated plates and maintain in motor neuron terminal differentiation media for 5-7 days.
1. Compound Administration and Intoxication with BoNT/A
Perform all of the following work in a BSL2 enclosure to maintain compliance with CDC/NIH guidelines.
<ol>
	<li>Prepare 10 mM stock solutions of each library compound in 100% DMSO in polypropylene 96 well plates. Use the 10 mM stock to prepare an intermediate dilution plate that is 10-fold greater than the desired screening concentration. Prepare 100 uM intermediate plate by dispensing 10 mM stock into 96-well plate and dilute it to 100 uM using culture media. Dispense 10 &#181;l of the compounds on to 90 &#181;l of media on the cells11. Test the compounds at 10 uM final concentration and ensure the final percentage of DMSO does not exceed 0.5%.</li>
	<li>Perform all studies that utilize live cells and active toxin under biosafety level 2 conditions. Replace the old media in the 96-well plates with 80 &#181;l of fresh terminal differentiation media using a 12 channel manual pipette. Carry out aspiration and dispense steps by rows to avoid exposure of the cells without media to the ambient air.</li>
	<li>Pretreat cells with compounds prior to application of toxin by addition of 10 &#181;l of 10x compounds from the source plate using a 12 channel pipette, followed by mixing by aspiration (twice). For each 96-well plate, treat two columns of 6 wells with 10x DMSO diluted in the media to serve as high signal and low signal controls. The column without BoNT treatment exhibits the highest level of the full length SNAP-25 (high signal control). Treat the other column with BoNT alone (without any compounds) as the low signal control. Use low control to observe the efficiency of BoNT cleavage of SNAP-25 and its detection with the BACS antibody (Figure 2).</li>
	<li>Incubate cells with compounds for 30 min at 37 &#176;C and 6% CO2 in the cell culture incubator.</li>
	<li>Prepare Botulinum neurotoxin A from a 1 mg/ml commercial source in Phosphate buffered saline (PBS) to a final 10x working stock by diluting with terminal differentiation media.</li>
	<li>Initiate intoxication by adding 10 &#181;l of 10x BoNT/A stock into wells designated for treatment and low signal controls using a multichannel pipette (Figure 2). Treat the wells designated as high control with media alone.</li>
	<li>Decontaminate all plastic ware and other disposables that comes in contact with BoNT/A during the study by immersion in 0.825% hypochlorite solution in water for at least 20 min. Perform all procedures in biosafety cabinets and decontaminate working areas both before and after experiments with 5% detergent disinfectant cleaner such as MicroChem solution.</li>
	<li>Label the plates clearly to denote toxin use and incubate plates treated with 1 nM/L BoNT/A for 4 hr at 37 &#176;C and 6% CO2 in a cell culture incubator. Display appropriate signage to inform colleagues that toxin is in use in the laboratory.</li>
	<li>Stop BONT/A proteolytic cleavage by methanol fixation. Remove media containing toxin and compounds from each well with a multichannel pipette and discard in 10% bleach solution. Add ice cold methanol (100 &#181;l) directly to each well.</li>
	<li>Incubate plates for 15 min at room temperature (RT) to allow fixation to occur.</li>
	<li>After fixation, safely handle the discarded reagents (considered neutralized) with biosafety level 1 protocols and discard accordingly.</li>
	<li>Discard methanol and use 100 &#181;l of PBS to wash the cells and rehydrate them prior to immunostaining. Perform two additional PBS washes.</li>
	<li>After the final wash, leave PBS in the wells, seal plates with microplate adhesive film and store at 4 &#176;C until analysis. Store plates at 4 &#176;C for several days.</li>
</ol>
2. Immunostaining
The immunostaining procedure is a labor-intensive, multistep operation that includes repetitive reagent dispense/aspirate cycles and extensive plate washing which can lead to the potential introduction of significant intraplate and interplate variability. A semi-automated approach is applied to save the time of laboratory personnel, increase assay throughput, and minimize immunostaining variability.
<ol>
	<li>Use an automated workstation equipped with a 96-well head capable of transferring volumes up to 250 &#181;l and integrated with a robotic gripper arm to move labware into different locations on the modular deck (see Material and Equipment) for this protocol.
	<ol>
		<li>The modular deck is designed to host different types of labware and can support up to 11 items at any one time. The automated microplate handler is equipped with a dual magazine microplate stacker in order to hold and manipulate up to 50 plates per cycle.</li>
		<li>The automated workstation is located inside of a Class II biosafety cabinet designed for laboratory automation equipment to provide sterility and safety. For automated immunostaining, the deck is arranged as shown in Figure 3 to enable reagent access as needed and without human intervention.</li>
	</ol>
	</li>
	<li>Pre-load plates containing fixed cells into the plate magazine and transfer to the deck as needed for liquid handling operations. Use the 96 pipette head fitted with 250 &#181;l tips to aspirate PBS from the wells down to 10 &#181;l. Permeabilize cellular membranes to facilitate antibody binding to intracellular targets with permeabilization buffer (0.1% Triton-X 100). Dispense permeabilization buffer (100 &#181;l) into each well before returning the plates to the second storage magazine of the microplate stacker for a 15 min incubation at room temperature (RT).</li>
	<li>Remove permeabilization buffer and perform plate washing with PBS (twice) as previously described in step 1.13.</li>
	<li>After the last wash step, remove the PBS buffer and dispense 100 &#181;l of blocking buffer containing 1% horse serum and 0.1% Tween 20 in PBS into all microplates before returning them to the plate magazine for 1hr incubation at RT.</li>
	<li>Use two primary antibodies for immunostaining: a rabbit polyclonal anti-mouse BACS antibody, for detection of full length SNAP-25 and a mouse monoclonal anti-III tubulin antibody for detection of neurons (see Material and Equipment). After 60 min of incubation, remove blocking buffer and dispense 50 &#181;l of 4ug/ml of BACS antibody and 0.5 &#181;g/ml of III-tubulin in blocking buffer into all plates.</li>
	<li>Incubate for 1hr at RT remove the primary antibodies and wash 3 times with 100 &#181;l of PBS containing 0.05% Tween (PBST).</li>
	<li>Use an anti-mouse IgG labeled with Alexa Fluor 647 to visualize the -III tubulin and anti-rabbit IgG tagged with Alexa Fluor 568 to visualize the BACS antibody. Prepare both antibodies as a 2 &#181;g/ml dilution in blocking buffer and dispense 50 &#181;l of the mixture into each well. 
	NOTE: The secondary antibodies selected for immunostaining are labeled with different fluorophores to allow detection of their emitted fluorescent light at distinct wavelengths using different channels within the detector of the High Content Imager.</li>
	<li>Incubate for 1 hr at RT. Aspirate the secondary antibodies and wash 3 times with 100 &#181;l of PBST.</li>
	<li>After the final wash, add 100 &#181;l of PBS containing Hoechst dye (32 &#181;M) to stain the DNA for nuclei detection.</li>
	<li>Seal the plates with a light impermeable film to protect the fluorophores from photobleaching. Plates can be stored at 4&#160;&#176;C for weeks without significant loss of signal.</li>
</ol>
3. Imaging
NOTE: Perform image acquisition using High Content Imaging System (See Materials and Equipment).
<ol>
	<li>Specification of High Content Imager Definitions:
	<ol>
		<li>Select Plate type, layout, number of fields, objective: Greiner u clear, 96 well format, 16, 20X water respectively.</li>
		<li>Select channels: nucleus excitation EX: 405&#160;nm as channel 4; cytoplasm EX: 644 nm as channel 2; antigen specific antibody channel EX: 561&#160;nm as channel 3 and Set up excitation power of each laser, Setup experiment as 2 exposures, Exposure 1 with one excitation at 644 nm, Exposure 2 with two excitations at 405&#160;nm and 561&#160;nm. Select binning as Bin2.</li>
		<li>Use high control wells for exposure optimization with maximum intensity as SNAP-25 channel and low control wells for minimal intensity.</li>
		<li>Adjust the exposure, so that the intensity of each channel is about half of the saturation level (2,000-2,500 relative units).</li>
		<li>Identify the optimal Z plane on cell mask channel using delta correction script. See Figure 5 for results after immunostaining and imaging. Export data to the server where the image analysis software is residing.</li>
	</ol>
	</li>
</ol>
4. Image Analysis (Figure 6)

NOTE: The following steps describe application of the Columbus software algorithms.
<ol>
	<li>Select an appropriate algorithm to segment the primary objects (nuclei). If required, adjust background threshold and contrast parameters. The Columbus software provides 4 nuclei detection methods. Select the method that segment nuclei&#160;accurately by visually inspecting segmented nuclei (in Figure 6a method M is selected).</li>
	<li>Select the neurite algorithm (CSIRO Neurite Analysis 2) to segment the neurites If required, adjust background threshold and contrast parameters to remove background. See Figure 6b for parameters used to detect neurites.</li>
	<li>Identify the neurite regions (neurite segments) based on secondary objects (neurites) using a -3 pixel expansion of the &#946;-III tubulin channel (Alexa Flour 640) as mask (Figure 6c).</li>
	<li>Calculate intensity of the signal channels (SNAP-25, (Alexa Flour 568) for the neurites region and &#946;-III tubulin channel (Figure 6d &#38; 6e).</li>
	<li>Select desired parameters for export, such as neurite length, mean intensities of SNAP-25, &#946;-III tubulin, nuclei number, neurite segment number, etc. (Figure 6f).</li>
	<li>Transfer plates from plate stackers using the robot and load into the High Content Imager for image acquisition.</li>
</ol>
5. Data Analysis:
To assess the robustness of the designed assay, calculate the following parameters from the plate-based experiment.
<ol>
	<li>Signal (S) to background (B):S/B = mean intensity of high signal control) / mean intensity of low signal control</li>
	<li>Coefficient of Variation (CV) of signal and background (to measure the uniformity of the specific signal): CV = (Standard Deviation (SD) / mean of sample)*100(%), to determine the variability in liquid handling, reagents, etc.</li>
	<li>Signal to Noise: S = (mean intensity of signal &#8211; mean intensity of low signal control) / SD of low control
	<ol>
		<li>Calculate the Z&#8217; factor with intoxication of one half of a 96-well plate and a mock intoxication of the other half. Z&#8217; = 1 - 3 * (SD of high signal control + SD of low signal control) / (mean of high signal control &#8211; mean of low signal control). For the further analysis of the screening data, normalize some of the primary raw parameters on a plate basis to allow comparison between plates and between different days of experiments.</li>
	</ol>
	</li>
	<li>Percent of cleavage, reflecting the reduction of the signal associated with full length SNAP-25 detected by specific antibody (BACS) : % Cleavage = 100% * (mean intensity of high signal control - intensity of sample) / mean intensity of high signal control.</li>
	<li>Percent of Inhibition of cleavage: % Inhibition = 100% * (intensity of sample &#8211; mean intensity of low signal control) / (mean intensity of high signal control - mean intensity of low signal control).</li>
</ol>

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E., 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=Pharmacophore+Refinement+Guides+the+Rational+Design+of+Nanomolar-Range+Inhibitors+of+the+Botulinum+Neurotoxin+Serotype+A+Metalloprotease.">Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease.</a> ACS medicinal chemistry letters. 1, 301-305 (2010).</li><li>Kiris, E., 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=Embryonic+stem+cell-derived+motoneurons+provide+a+highly+sensitive+cell+culture+model+for+botulinum+neurotoxin+studies,+with+implications+for+high-throughput+drug+discovery.">Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.</a> Stem cell research. 6, 195-205 (2011).</li><li>Pellett, S., 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=Neuronal+targeting,+internalization,+and+biological+activity+of+a+recombinant+atoxic+derivative+of+botulinum+neurotoxin+A.">Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A.</a> Biochemical and biophysical research communications. 405, 673-677 (2011).</li><li>McNutt, P., Celver, J., Hamilton, T., Mesngon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell-derived+neurons+are+a+novel,+highly+sensitive+tissue+culture+platform+for+botulinum+research.">Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research.</a> Biochemical and biophysical research communications. 405, 85-90 (2011).</li><li>Fischer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimodal+modulation+of+the+botulinum+neurotoxin+protein-conducting+channel.">Bimodal modulation of the botulinum neurotoxin protein-conducting channel.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 1330-1335 (2009).</li></ol>

Krishna P Kota is an employee of Perkin Elmer Inc. Waltham, MA, that produces instruments and software used in this manuscript. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, the U.S. Department of Defense, the U.S. Department of the Army, or the institutions and companies affiliated with the authors.

The high potency of Botulinum neurotoxins and the relative ease of their weaponization has resulted in their classification as Category A (highest priority) biothreat agents by the U.S. Centers for Disease Control and Prevention. Unfortunately, there are no FDA approved therapeutics to counter BoNT intoxication after the toxin has been internalized by the motor neurons. Any druggable mechanism that promotes neuronal recovery from BoNT intoxication could lead toward the development of a potential therapy to protect both the armed services and public against this biological threat. In this article, we present a detailed HCI assay protocol for screening inhibitors of lethal toxins such as BoNT/A. An unusual aspect of this assay is scrupulous handing of toxins and implementation of strict biosafety protocols to ensure laboratory safety. Extreme care must be exercised while active toxin is in use. BoNT/A inactivation via methanol fixation and microplate decontamination with 10%bleach and 5% surface wipes are key steps that allow microplates to be removed from biosafety cabinets for downstream evaluation in biosafety level 2 laboratory environments.
Due to the ever expanding size of compound libraries coupled with slow motor neuron expansion (cell number), HCI assays for neurotoxin antagonists tend to run over several weeks. This requires that the variability between the plates and across time must be eliminated or minimized. In this protocol we provide automation methods for immunostaining, image acquisition and analysis to reduce the assay variability. Further reliability and consistency can be achieved by the automation of routine tasks associated with this protocol including cell plating, washing, and fixation. Our group is currently evaluating the impact of these improvements with the intent of incorporating them into our protocol in the near future.
In this report, we describe a SNAP-25 cleavage assay using high content imaging to measure relative fluorescence of intact SNAP-25 in motor neurons. This assay uses the BACS and &#946;-III tubulin antibodies to detect full-length SNAP-25 and &#946;-III tubulin respectively11. &#946;-III tubulin is used as a marker to specifically identify neurons (Figure 4). While BoNT/A cleaves SNAP-25 and reduces the SNAP-25 signal, small molecules that inhibit any part of this process improve SNAP-25 signal in the imaging assay. As this assay depends on the fluorescence signal generated from the SNAP-25 distributed across many tiny neurites, high quality images are absolutely necessary. Hence, automated microscopes that produce high resolution confocal images are recommended (Figure 4). We routinely capture 6-16 fields/well in a 96-well format while using the 20X water-immersion objective. The number of field&#8217;s imaged are dependent upon the overall number of neurites required to generate statistically significant results.
Modular image analysis algorithms were utilized to extract multiparameter data (Figure 6). The Columbus modular image analysis algorithms are relatively easy to generate for simpler analysis scenarios. For more complicated analysis or design of specific parameters for complex readouts, Acapella based scripts can be used within the Opera environment as well as inside the context of Columbus. In addition to the fluorescence intensities obtained from the SNAP-25 and &#946;-III tubulin channels, we routinely collect other parameters such as total cell number (indirect measure for cytotoxicity), neurite length, neurite branching, and other endpoints to quantify neuronal health during the assay. The raw endpoint data are exported to statistical analysis software for further data analysis and hit selection (Figure 7). The data presented here demonstrates the ability of the HCI assay to be efficiently utilized for phenotypic screening in search for BoNT/A inhibitors using a physiologically relevant motor neuron model.

The bacterium Clostridium botulinum produces Botulinum neurotoxin, one of the most potent biological toxins known to man1. There are 7 distinct BoNT serotypes (BoNT/A-G). BoNT/A-Gall induce paralysis at the neuromuscular junction due to SNARE complex proteolysis 2,3. SNARE proteolysis prevents neurotransmitter vesicle-membrane fusion and therefore blocks neurotransmitter exocytosis4. The specific SNARE target depends upon the particular BoNT serotype involved in the intoxication process. BoNT/A and BoNT/E cleave SNAP-25 whereas BoNT/C cleaves both SNAP-25 and syntaxin5. The remaining serotypes cleave synaptobrevin (also called Vesicle-associated membrane protein (VAMP). BoNT/A was chosen for assay development as it is responsible for a high proportion of naturally occurring botulism and has the longest duration of action6. Development of small molecule therapeutics against BoNT/A is a major goal for our drug discovery program and has utilized traditional target-based methods to identify active site proteolytic inhibitors7,8-10. However, the creation of active site inhibitors with broad spectrum activity against multiple serotypes and post-exposure efficacy will likely be challenging.
We have therefore implemented an innovative, phenotypic drug discovery approach that uses BoNT SNAP-25 cleavage as a functional endpoint to identify small molecules that can block BoNT-mediated motor neuron intoxication. SNAP-25 is required for neurotransmitter release, as degradation of SNAP-25 is predictive of paralysis and lethality in vivo. For example, cell-based screening could lead to discovery of new modulators of cellular factors responsible for toxin inactivation or inhibition of toxin pathways inside targeted cells. An important factor in phenotypic assay development is the selection of physiologically relevant biological models. We and others have described mouse embryonic stem (ES) cell-derived motor neurons that recapitulate the immunologic character of primary motor neurons, including the expression of SNAP-2511-13. Importantly, these cellular systems are highly sensitive to BoNT/A intoxication and demonstrate dose-dependent cleavage of SNAP-25 in response to increasing concentrations of toxin11,12. The differentiated motor neurons are also produced in quantities that are sufficient for high throughput plate based analysis and allowed the design of an array of cellular assays.
The phenotypic assay is an immunofluorescence method utilizing two distinct antibodies to quantify cleavage of endogenously expressed full length SNAP-25 during BoNT/A intoxication of mouse motor neuron culture. A carboxyl terminal BoNT/A cleavage-sensitive (BACS) antibody that recognizes only full length SNAP-25 allows the assessment of BoNT/A mediated proteolysis of SNAP-25 expression in mouse motor neurons10. A schematic diagram of the HCI assay is depicted in Figure 1.

<table><tbody><tr><td>Botulinum neurotoxin A&amp;nbsp;</td><td>Metabiologics</td><td>NA</td><td>No catalog number</td></tr><tr><td>Microtitre plates</td><td>Greiner&amp;nbsp;</td><td>655946</td><td>Poly-D-Lysine 96-well plates</td></tr><tr><td>BACs antibody</td><td>Lampire Biological</td><td>NA</td><td></td></tr><tr><td>Microchem</td><td>National&amp;nbsp;</td><td>0255</td><td></td></tr><tr><td>Methanol</td><td>Thermo Scientific</td><td>A412-20</td><td></td></tr><tr><td>Formaldehyde</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Horse serum</td><td>Invitrogen</td><td>16050</td><td></td></tr><tr><td>PE JANUS MDT Mini Automated Workstation</td><td>Perkin Elmer</td><td>AJMDT01</td><td></td></tr><tr><td>Opera</td><td>Perkin Elmer</td><td>OP-QEHS-01</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>9002-93-1</td><td></td></tr><tr><td>BIII tubulin antibody</td><td>R&amp;amp;D Systems</td><td>BAM1195</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379-1L</td><td></td></tr><tr><td>Hoechst 33342dye&amp;nbsp;</td><td>Invitrogen</td><td>3570</td><td></td></tr><tr><td>Antimouse IgG</td><td>Invitrogen</td><td>A21236</td><td></td></tr><tr><td>Anti rabbit IgG</td><td>Invitrogen</td><td>A10042</td><td></td></tr><tr><td>Columbus Image analysis software</td><td>Perkin Elmer</td><td>Ver 2.4</td><td></td></tr><tr><td>Spotfire</td><td>Perkin Elmer</td><td>Ver 5.5</td><td></td></tr><tr><td>Clorox bleach</td><td>Fisher Scientific</td><td>18-861-284</td><td></td></tr><tr><td>PlateStack</td><td></td><td></td><td></td></tr></tbody></table>

a high content imaging assay for identification of botulinum neurotoxin inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Data from high and low controls created two distinct populations with the difference of two medians exceeding 3 standard deviations (Figure 7A). The goal of the screening process is to find the compounds within the sample population with values closer to positive control population, assuming a normal distribution within the sample population (Figure 7B, (i)). Data points that are 3 standard deviations beyond the mean are considered statistically different from the noise and classified as an active &#8220;hit&#8221; (Figure 7B(ii), red boxes). The compounds that are classified as &#8220;hits&#8221; will be subjected to further confirmation testing in future studies. Figure 7B (ii) shows the detection of positive hits in the population of the samples from a representative subset of the screening plates. All 4 points that had values lower than the (median samples - 3*SD of the samples) belonged to the same inhibitor that was spotted in different locations within 4 different screening plates. This inhibitor demonstrated robust SNAP-25 protection against BoNT/A, as shown in Figure 5.
<img alt="Figure 1" src="/files/ftp_upload/51915/51915fig1highres.jpg" /> 
Figure 1: Workflow for compound screening in the BoNT/A HCI assay.
<img alt="Figure 2" src="/files/ftp_upload/51915/51915fig2highres.jpg" /> 
Figure 2: The 96-well plate map for the HCI assay. High control wells (pink) were treated with 0.5% DMSO as control only and low control wells (blue) were treated with 1 nM BoNT/A and 0.5% DMSO. Sample wells (green) were treated with 1nM toxin and 10 &#181;M of compounds in 0.5% DMSO. The outer columns and rows (grey) were not used due to incompatibility with the optimized plate format and the inability of the 20X water-Immersion objective to image edge wells.
<img alt="Figure 3" src="/files/ftp_upload/51915/51915fig3highres.jpg" /> 
Figure 3: The design of the automated work station deck for the immunostaining assay. Blocking buffer, primary antibody, secondary antibody and cellular stains were dispensed in 96-well polypropylene plates to reduce dead volume reagent loss. PBS was dispensed in a tray capable of holding 300 ml. The manifold used for liquid waste aspiration is shown on the inset image.
<img alt="Figure 4" src="/files/ftp_upload/51915/51915fig4highres.jpg" /> 
Figure 4: Screen shot of the High Content Imager experimental set up.
<img alt="Figure 5" src="/files/ftp_upload/51915/51915fig5highres.jpg" /> 
Figure 5: High content imaging of SNAP-25 cleavage in mouse ES-derived motor neurons. Cells were treated with 1 nM BoNT/A with and without addition of 1 &#181;M of inhibitor (Toosendanin)14 or left untreated without toxin. SNAP-25 was detected with the BACS antibody. In another channel, neurons were detected using a &#946;-III-tubulin antibody to create mask of neurites for SNAP-25 image analysis. This is a single representative image from one of the 16 images taken from a given well. Blue indicates nuclei stained with Hoechst 33342, red is the BACS signal and green is the &#946;-III-tubulin signal. Images were obtained with the Opera as described. Size bar: 10 &#181;m
<img alt="Figure 6" src="/files/ftp_upload/51915/51915fig6highres.jpg" /> 
Figure 6: Screen shots showing various steps in image analysis pipeline.
<img alt="Figure 7" src="/files/ftp_upload/51915/51915fig7highres.jpg" /> 
Figure 7: Statistical visualization of the data arising from the HCI screen. (A). (i) Box plot analysis of the % Cleavage calculated from control wells; shown as median values with their respective SD (n = 16). Pink represents the high signal control (HSC); blue represents the low signal control (LSC). Z&#8217; = 0.97 (ii) Histogram distribution of the same values as in (i) to demonstrate the distance between two control populations. Median and 3 SD were calculated from the statistical values associated with the corresponding box-plot. (B). Distribution of the 192 compound treated samples from the same experiment. (i) Box-plot demonstrating the statistical values for the % cleavage for samples in comparison to controls as well as statistically significant outliers. (ii) and (iii), Histogram distribution of the same values in population of all data from the screen (green color). Hits (highlighted in red square), were selected from the outliers that have % cleavage values less than 3SD from the median of the sample population (&#60;25.89 % cleavage). The four hits highlighted during selection (iii) on the histogram of all data points have similar values to the high signal control and represented the same compound, spiked into the plate for the tests, a known BoNT/A inhibitor (Toosendanin) that blocked SNAP-25 cleavage.

Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

a-high-content-imaging-assay-for-identification-botulinum-neurotoxin

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.6K Views.  Perkin Elmer Inc.. Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Video: A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

Chemistry

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

Medicine

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Immunology and Infection

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biology

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Cancer Research

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Using TLC-Direct Bioautography Assay to Identify Natural Products for Biocontrol

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Author Spotlight: Discovering New Biopesticides from Bioactive Soil Microbe-Derived Natural Products

We describe the use of thin-layer chromatography direct bioautography assay and liquid chromatography-mass spectrometry to identify microbial natural products that display antagonism against fungal pathogens using the pathogen Sclerotinia sclerotiorum and biopesticidal Bacillus isolates as a model system.

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are ...

Biochemistry

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

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