The authors would like to thank Keith Tarpley of the US EPA Office of Research and Development-Research Triangle Park (ORD-RTP) Graphic and Media team for development, taping, and editing of the instructional video. We would also like to thank Deborah Pritchett from ProteinSimple for helpful conversations regarding optimization of our data. JM Guynn was supported by the Oak Ridge Institute for Science and Education Research/Participation Program at the US Environmental Protection Agency.

NOTE: Ensure that all reagents and samples are prepared according to the manufacturer&#39;s protocol, outlined below. Please wear proper personal protection equipment during this procedure, which includes nitrile gloves, lab coat, closed-toed shoes, and safety goggles. A table of specific materials, reagents and equipment required is provided separately. Total protein concentration of samples should be determined beforehand using established methodologies that are compatible with the lysate buffer used, such as Bradford assay11.
1. Preparation of samples and reagents from the standard pack as supplied by the manufacturer
<ol>
	<li>To prepare the 400 mM working solution of dithiothreitol (DTT), add 40 &#181;L deionized water to the clear tube containing the supplied stock from the manufacturer. It is important to avoid introducing bubbles to the solution by mixing the solution with slow and gentle pipetting.</li>
	<li>Add 20 &#181;L 10X sample buffer and 20 &#181;L of the prepared 400 mM DTT solution to the pink tube containing fluorescent 5X master mix (see the Table of Materials). 
	NOTE: The Master Mix (MM) is sealed by the manufacturer with a foil cover and must be pierced by a pipette tip. Mix gently by slow pipetting to avoid splashing DTT in the pipette.</li>
	<li>Next, add 16 &#181;L deionized water, 2 &#181;L of supplied 10X sample buffer, and 2 &#181;L of the prepared 400 mM DTT solution to the white biotinylated ladder tube supplied by the manufacturer. Mix gently and transfer into a 0.6 mL tube for denaturing.</li>
	<li>Prepare 0.1x sample buffer by diluting the supplied 10X solution 1:100 with water. Prepare enough 0.1x sample buffer to dilute all samples.</li>
	<li>Calculate the amount of 0.1x sample buffer that is added to a sample, which will depend on the final desired concentration of the total protein. Mix 1 part 5x fluorescent MM with 4 parts diluted sample to achieve the desired final protein concentration.
	<ol>
		<li>Calculate volumes as follows: (i) Volume 5x fluorescent MM = (Desired final protein concentration)/5; (ii) Volume of protein stock = (Desired final protein concentration x Total volume needed)/ Protein stock concentration; (iii) Volume 0.1x sample buffer = Total volume - 5x&#160;MM volume - Protein stock volume.</li>
	</ol>
	</li>
</ol>
2. Denaturation of samples and ladder
<ol>
	<li>Place prepared samples and biotinylated ladder in a 95 &#176;C heat block for 5 min. Vortex tubes immediately after incubation, execute a spin for ~5 s in a tabletop centrifuge, and place on ice. 
	NOTE: Some proteins may require gentler denaturing conditions (e.g., 70 oC for 10 min) to prevent protein aggregation and improve migration in the capillary matrix. Consider this option if there is heavy smearing at the higher molecular weights (see video for example).</li>
</ol>
3. Preparation of antibodies
<ol>
	<li>As determined by optimization (see Representative Results and video), prepare the desired dilution(s) of primary antibody (e.g., 1:50, 1:100) in the provided Antibody Diluent 2. 
	NOTE: Antibodies are generally used at higher concentrations for capillary-based immunoassay than for traditional western blotting. The supplied secondary antibody is ready to use without dilution.</li>
</ol>
4. Preparation of Luminol-S and Peroxide
<ol>
	<li>Prepare a 1:1 mixture of luminol-S and peroxide.</li>
	<li>Vortex to mix and store on ice. 
	NOTE: It is important that this mixture is prepared fresh for each experimental assay. 250ul total mix is needed to run a full plate.</li>
</ol>
5. Preparation of the assay plate
<ol>
	<li>As shown in Figure 1, load the samples and reagents prepared above into the assay plate. See detailed instructions below for each row. To minimize evaporation from the wells, ensure that the plate lid is replaced between reagent additions.</li>
	<li>In row A, pipet 5 &#181;L of Biotinylated Ladder into well A1. For the remaining row, pipet prepared samples (5 &#181;L each) into wells A2-A25. 
	NOTE: It is imperative that the A1 well always contains ladder, as the first capillary in the cartridge is optimized for running this standard.</li>
	<li>In row B, pipet 10 &#181;L of Antibody Diluent 2 into each well (B1-B25).</li>
	<li>In row C, pipet 10 &#181;L of Antibody Diluent 2 into well C1. In the remaining row C wells, pipet 10 &#181;L of primary antibody (wells C2-C25).</li>
	<li>In row D, pipet 10 &#181;L of Streptavidin-HRP into well D1. In the remaining row D wells, pipet 10 &#181;L of secondary antibody (wells D2-D25).</li>
	<li>In row E, pipet 10 &#181;L of the freshly prepared luminol-peroxide mix into each well (E1-E25).</li>
	<li>Finally, add 500 &#181;L wash buffer per compartment to each of the top 3 rows of buffer wells. 
	NOTE: It is important to minimize bubble formation by pipetting gently and not expelling the final volume from the tip as bubbles may interfere with the capillary loading and run.</li>
	<li>Once all wells are loaded, centrifuge the plate at ~1000 x g for 5 min at room temperature to remove bubbles and ensure the liquid is in the bottom of the wells. Pop any visible bubbles with a small pipette tip or clean needle (e.g., 25 gauge sterile &#34;rep top&#34; needle).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55911/55911fig1.jpg" /> 
Figure 1. Pipetting template for assay plate. Color coding represents proper reagents and samples (up to 24 total) added to the assay plate. Add biotinylated ladder to well A1 (orange), prepared samples from wells A2 up to A25 (light blue), Antibody Diluent 2 to wells B1-B25 and C1 (light green), primary antibody to wells C2 up to C25 (blue), streptavidin-HRP to well D1 (dark pink), secondary antibody to wells D2 up to D25 (dark green), and luminol-peroxide mix to wells E1 up to E25 (purple). Wash buffer is added to the first three rows of the larger mid-plate wells (dark blue). <a href="//cloudfront-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55911/55911fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Starting the capillary immunoassay instrument
<ol>
	<li>Ensure that the instrument and the accompanying computer are turned on. No warm up time is needed.</li>
	<li>Open the instrumentation software on the computer. First, select the &#34;Assay&#34; tab on the right of the window and &#34;New Assay&#34; under the &#34;File Menu&#34; on the left. Select the size, size range (e.g., 12-230 kDa), and cartridge type (e.g., 25 capillary) that was used for the particular assay. 
	NOTE: One may also input assay parameters, including protein concentration, antibody type, and dilution, if desired, but these are not required to start the assay.</li>
	<li>Open the door on the instrument by pushing the button on top of the orange panel.</li>
	<li>Carefully remove the capillary cartridge from its packaging. Without touching the glass capillaries themselves, place the cartridge into the cartridge holder. Verify cartridge seating by observing the interior light change from orange to blue.</li>
	<li>Hold the plate firmly on the bench and carefully peel off the evaporation seal from the lower portion of the plate. Pop any bubbles seen in these separation matrix wells with a small pipet tip or clean needle (e.g., 25 gauge sterile &#34;rep top&#34; needle).</li>
	<li>Place the assay plate on the plate holder, ensuring it is fully seated, and close the instrument door.</li>
	<li>Click the &#34;Start&#34; button in the software. 
	NOTE: If no Start button appears, the connection with the instrument has been lost. Choose Instrument from the top left menu, then Connect. A pop-up should appear with the instrument serial number; select this, then click Connect. The Start button should now appear.</li>
	<li>When the run is complete, discard the plate. Remove the cartridge and place in a sharps container for disposal, along with any needles that may have been used to pop bubbles. Leave the power on to avoid connection problems if the instrument is being used regularly (e.g., at least weekly).</li>
</ol>
7. Experiment analysis
<ol>
	<li>Before analysis, ensure the following quality checks are performed.
	<ol>
		<li>Verify fluorescent standards by selecting the &#34;Show Standards&#34; icon. Check that the standards are correctly identified in the &#34;Graph View&#34; tab. If they are incorrect, go to the &#34;Single View&#34; icon, right click on the correct peak, and select &#34;Force Standard&#34;, or right click on the incorrect peak and select &#34;Not a Standard&#34;. Perform this check for each new capillary.</li>
		<li>Verify biotinylated ladder by clicking on the &#34;Samples&#34; and &#34;Single View&#34; icons. Select the ladder in the experiment tab. If a peak is incorrectly identified, right click on it in &#34;Graph View&#34; and select &#34;Remove Peak&#34;. 
		NOTE: For example, the biotinylated ladder used for the 12 - 230 kDa kit should show 12, 40, 66, 116, 180, and 230 kDa&#160;sizing peaks. If this step is not performed, the sizing of the sample peaks will be incorrectly calculated, producing spurious results.</li>
		<li>View and analyze sample peaks. Derive data from the peaks table, including molecular weight, peak area, peak height, and signal to noise (S/N), as needed for experimental calculations.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Bradley, H. L., Sabnis, H., Pritchett, D., Bunting, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hematopoietic stem cell protocols. 1185, (2014).</li><li>Guo, L., Eldridge, S., Furniss, M., Mussio, J., Davis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes+(hiPSC-CMs)+to+Monitor+Compound+Effects+on+Cardiac+Myocyte+Signaling+Pathways.">Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways.</a> Curr Protoc Chem Biol. 7 (3), 141-185 (2015).</li><li>Chen, J. Q., 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=Absolute+quantitation+of+endogenous+proteins+with+precision+and+accuracy+using+a+capillary+Western+system.">Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system.</a> Anal Biochem. 442 (1), 97-103 (2013).</li><li>Chen, J. Q., Wakefield, L. M., Goldstein, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+nano-immunoassays:+advancing+quantitative+proteomics+analysis,+biomarker+assessment,+and+molecular+diagnostics.">Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics.</a> J Transl Med. 13, 182 (2015).</li><li>Liu, 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=The+Application+of+a+Novel+Nanovolume+Capillary+Electrophoresis-+Based+Protein+Analysis+System+in+Personalized+&amp;+Translational+Medicine+Research.">The Application of a Novel Nanovolume Capillary Electrophoresis- Based Protein Analysis System in Personalized &amp; Translational Medicine Research.</a> J Bioanal Biomed. S3 (004), (2013).</li><li>ProteinSimple Western. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Size Assay Development Guide, Revision 1. , (2014).</li><li>Eaton, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+modern+quantitative+fluorescent+western+blotting+with+troubleshooting+strategies.">A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.</a> J Vis Exp. (93), (2014).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western++blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Silva, J. M., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fastest+Western+in+town:+a+contemporary+twist+on+the+classic+Western+blot+analysis.">The fastest Western in town: a contemporary twist on the classic Western blot analysis.</a> J Vis Exp. (84), e51149 (2014).</li><li>Thorn, C. F., 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=Doxorubicin+pathways:+pharmacodynamics+and+adverse+effects.">Doxorubicin pathways: pharmacodynamics and adverse effects.</a> Pharmacogenet Genomics. 21 (7), 440-446 (2011).</li><li>Ernst, O., Zor, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearization+of+the+bradford+protein+assay.">Linearization of the bradford protein assay.</a> J Vis Exp. (38), (2010).</li><li>Taira, N., Yoshida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+of+p53+tumor+suppressor:+determinants+of+its+functional+targets.">Post-translational modifications of p53 tumor suppressor: determinants of its functional targets.</a> Histol Histopathol. 27 (4), 437-443 (2012).</li><li>Thompson, T., 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=Phosphorylation+of+p53+on+key+serines+is+dispensable+for+transcriptional+activation+and+apoptosis.">Phosphorylation of p53 on key serines is dispensable for transcriptional activation and apoptosis.</a> J Biol Chem. 279 (51), 53015-53022 (2004).</li><li>Currier, J. M., Cheng, W. Y., Menendez, D., Conolly, R., Chorley, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+Gene+Biomarker+at+the+Tipping+Point+of+Adaptive+and+Adverse+Responses+in+Human+Bronchial+Epithelial+Cells.">Developing a Gene Biomarker at the Tipping Point of Adaptive and Adverse Responses in Human Bronchial Epithelial Cells.</a> PLoS One. 11 (5), e0155875 (2016).</li><li>Moser, A. C., Hage, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis-based+immunoassays:+principles+and+quantitative+applications.">Capillary electrophoresis-based immunoassays: principles and quantitative applications.</a> Electrophoresis. 29 (16), 3279-3295 (2008).</li><li>Shintani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Capillary Electrophoresis Applications. , (1997).</li><li>. Wes reagents and consumables :: ProteinSimple Available from: <a target="_blank" href="https://www.proteinsimple.com/consumables_sw_wes.html">https://www.proteinsimple.com/consumables_sw_wes.html</a> (2017)</li></ol>

The authors declare that they have no competing financial interests. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory and approved for publication. The content does not necessarily reflect the views of the US EPA nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

For decades, there has been sustained interest in the development of capillary electrophoretic-based immunoassay methods because of low sample and reagent expenditure, decreased processing time when compared to traditional methods, and high compatibility to automate the procedure4,15. There are a number of different protocols for the separation of proteins that have utilized capillaries, including electrophoretic, electrokinetic, polymer sieving, and isoelectric methods, which isolate proteins by different properties (respectively, electrostatic charge, partition equilibrium, sieving properties of the separating matrix, and pH)16. Here, we describe an antibody-based (or immunoassay) capillary electrophoresis method, using a polymer sieving separation, that has been automated and commercially adopted3. Advantages of this system include ease of use and operation, standardized and commercially available reagents, and reliable, sensitive measurements that require less reagent and samples compared to traditional protein assays such as western blot, enzyme-linked immunoassay, and other formats3,4,5. It has been noted in previous assessments of this technology that the size range of protein that can be assessed has been limited by the available separation matrix4, however recent offerings have expanded the measurable range from 2 to 440 kDa17. In addition, some lysate buffers are known to be incompatible with the assay18, therefore selection of the experimental reagents used must be considered beforehand.
A major advantage of an automated procedure with commercially available components is consistency of results through standardized methods and reagents. This minimizes chances of assay failure by automating critical steps within the procedure. However, it is important to note that certain practices must be adhered to during the protocol to minimize issues with the capillary electrophoretic-based immunoassay. First, it is critical that the luminol-S/peroxide mixture is prepared fresh and immediately before plate loading. Consistent timing will result in consistent luminescence after the oxidizing agent is added, which will result in consistent measurements for a particular antibody assay after assay. Furthermore, it is important that non-expired reagents are used, which primarily affects the potency of the oxidizing agent. Additionally, it is important that the loading order of samples, antibodies, and other reagents be strictly followed (see Figure 1). Any reagent pipetted out of place will result in assay failure and a wasted run.
Besides these critical steps, primary issues experienced with the technique can generally be overcome through optimization. Indeed, these conditions are specific to each model system/antibody combination and therefore should be determined empirically for each new assay. In this article, we focus on three common optimization procedures: exposure time, lysate titration, and primary antibody dilution. The ability to generate measurable results depends on analysis of an exposure time when the luminol substrate is not being rapidly depleted, as substrate depletion results in loss of signal. Lysate titration determines the linear dynamic range of each assay, which can differ with different model systems, as well as different antibodies, even for the same protein target. Antibody dilutions chosen at or near saturating concentrations ensure changes in signal will not be affected by a shortage of free antibody, but only by differing amount of available protein target epitope. Other considerations during the optimization process may include antibody incubation time and stacking/sample loading time. In most cases the default settings for the instrument offer the best balance of resolution and sensitivity. However, in some cases, poor resolution or sensitivity can be improved by adjusting these parameters.
Capillary electrophoretic-based immunoassay methods provide fast, efficient, and reproducible protein measurements. While these methods have primarily been utilized in research and development settings, the consistency of these technologies has potential utility in regulatory and clinical applications. For example, the identification of susceptible subpopulations to environmental toxicants or patients with progression of disease can be based on protein biomarkers measured in accessible matrices, such as blood, urine, and saliva. As reagent and operation costs drop and the number of samples and targets that can be simultaneously assessed increases, we will likely see capillary electrophoretic-based immunoassay methods used for these types of applications.

Capillary electrophoretic immunoassays measure protein expression in cell lysates using size or charge separation systems and provide several advantages over the traditional immunoassays. For example, when compared to western blot, the automated capillary-based procedure eliminates the need for gels, transfer devices, and manual washes. In addition, the absolute amount of protein required is approximately 10 times less, making capillary-based systems ideal for use with rare cell types or limited sample1,2. Results are obtained in as little as 3 h using automated systems and have previously been demonstrated to be more quantitative and reproducible than conventional western blot procedures3,4,5. The process for size-based assays consists of loading samples containing sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and fluorescently labeled molecular weight markers into capillary columns containing stacking and separation matrices. Voltage applied to the capillaries separates the proteins in the samples according to size, and UV light immobilizes the separated proteins to the capillary wall. The capillary is then immuno-probed with target-specific primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody. Luminol and peroxide catalyze chemiluminescent light generation which is measured by a charge coupled device (CCD) camera and analyzed to quantitate protein.
Despite the relative ease and speed of an automated capillary-based electrophoretic immunoassay platform, optimization of assay conditions such as protein concentration, antibody dilution, and exposure time is important for obtaining accurate, reproducible results. In general, the following procedures should be performed to optimize an assay for these systems:
1) A screen should be performed to evaluate and choose antibodies for signal and specificity to the protein target. If available, purified protein or target epitope can be used to assess specificity; however, it is still important to assess potential non-specific signal in total protein sourced from the model system.
2) Next, the dynamic range of the assay needs to be determined. In an ideal situation, signal doubling (measured using peak area) is observed as sample concentration is doubled; however, in practice, a proportional change in signal to input in a predictable manner (e.g., linear fit) is sufficient for protein quantification. Additionally, this optimization will define protein concentration with high signal but still within the linear range for the experimental model.
3) Determine the optimal antibody concentration using the fixed protein concentration chosen in optimization step 2. As the antibody concentration increases, the signal increases until it plateaus at saturation. An antibody concentration near this saturation level is required for accurate measurement of protein concentration.
The process used to optimize protein concentrations, antibody dilutions, and exposure times for an automated capillary-based size assay6 is demonstrated using whole cell extracts isolated from BEAS-2B, an SV-40 transformed human bronchial epithelial cell line. Protein isolation from cell or tissue extracts can be performed using a number of published protocols7,8,9 and will not be covered here. Results of a trial experiment using the optimized conditions are also reported for total and phosphorylated (serine 15, serine 20) p53 in cultures exposed to doxorubicin (a common chemotherapeutic agent that induces cell apoptosis10) at 1.2, 1.8, and 2.4 &#181;g/mL media for 4 h prior to harvest. The p53 peak areas are normalized to &#593;-tubulin, which is used as a loading control.

<table><tbody><tr><td>Wes instrument</td><td>ProteinSimple (Santa Clara, CA)</td><td>004-600</td><td></td></tr><tr><td>P53 DO-1 primary antibody</td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-126</td><td></td></tr><tr><td>phosphorylated p53 (ser 15) primary antibody</td><td>Cell Signaling Technology</td><td>9286</td><td></td></tr><tr><td>phosphorylated p53 (ser 20) primary antibody</td><td>Cell Signaling Technology</td><td>9287</td><td></td></tr><tr><td>alpha-tubulin primary antibody</td><td>Cell Signaling Technology</td><td>3873</td><td>used as a loading control</td></tr><tr><td>Compass Software</td><td>ProteinSimple (Santa Clara, CA)</td><td></td><td>provided with the Wes</td></tr><tr><td>12-230 kDa Master kit</td><td>ProteinSimple (Santa Clara, CA)&amp;nbsp;</td><td>PS MK02 (since replaced by a new kit #)</td><td></td></tr><tr><td></td><td>www.proteinsimple.com/consumables_sw_wes.html</td><td>INCLUDES PART NO:&lt;br/&gt; Wash Buffer (60 mL) 042-202&lt;br/&gt; 10X Sample Buffer (440 &amp;mu;L) 042-195&lt;br/&gt; Pre-Filled Microplates (8) PS-PP01&lt;br/&gt; Capillary Cartridges (8) PS-CC01&lt;br/&gt; Antibody Diluent II (20 mL) 042-203&lt;br/&gt; Luminol-S (1.5 mL) 042-233&lt;br/&gt; Peroxide (1.5 mL) 042-234&lt;br/&gt; Streptavidin-HRP (132 &amp;mu;L) 042-414&lt;br/&gt; Standard Pack (8): Biotinylated ladder, fluorescent 5X master mix, DTT, and empty 0.6 mL tube PS-ST01&lt;br/&gt; Anti-Rabbit Secondary Antibody 042-206&lt;br/&gt; or&lt;br/&gt; Anti-Mouse Secondary Antibody 042-205</td><td></td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cell culture, treatment, and harvest&lt;/strong&gt; (using vendor recommended protocols; protocols not included in manuscript)</td><td></td><td></td><td></td></tr><tr><td>BEAS-2B cells</td><td>American Type Culture Collection (ATCC, Manassas, VA)</td><td>CRL-9609</td><td></td></tr><tr><td>keratinocyte growth medium, KGM Gold</td><td>Lonza Ltd (Basel, Switzerland)</td><td>192152</td><td>for cell culture</td></tr><tr><td>keratinocyte basal medium, KBM Gold</td><td>Lonza Ltd (Basel, Switzerland)</td><td>192151</td><td>serum free medium for chemical dosing</td></tr><tr><td>doxorubicin</td><td>Sigma-Aldrich (St. Louis, MO)</td><td>D1515</td><td></td></tr><tr><td>Coomassie Blue Bradford Assays</td><td>ThermoFisher Scientific (Waltham, MA)</td><td>23200</td><td>for protein quantification</td></tr><tr><td>Nuclear Extract kit</td><td>Active Motif (Carlsbad, CA)</td><td>D1515</td><td>used to prepare whole cell lysates</td></tr><tr><td></td><td></td><td>INCLUDES:</td><td></td></tr><tr><td></td><td></td><td>Lysis Buffer AM1</td><td></td></tr><tr><td></td><td></td><td>1M dithiothreitol (DTT)</td><td></td></tr><tr><td></td><td></td><td>Protease Inhibitor Cocktail</td><td></td></tr><tr><td></td><td></td><td>10X PBS&amp;nbsp;</td><td></td></tr><tr><td></td><td></td><td>Phosphatase Inhibitors</td><td></td></tr></tbody></table>

procedure and key optimization strategies for an automated capillary electrophoretic-based immunoassay method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

Exposure time - Determining signal burnout
Signal burnout can occur when the luminol and peroxide substrate is depleted too quickly. This can be determined by examining the data at different chemiluminescence exposure times. In the analysis software, go to &#34;Edit -&#62; Analysis -&#62; Images&#34;. Exposures range from 5 to 480 s. The y-axis in an electropherogram reports signal/time, so the data from each exposure should have a similar signal/time coefficient. This coefficient decreases with sequentially longer exposures if luminol becomes depleted, as seen with the p53 DO-1 antibody (Figure 2). Because of substrate depletion, this assay can be considered measurable up to the 0.2 &#181;g/&#181;L concentration only at the 5 - 30 s exposures. Therefore, in this example, 15 s was determined to be the optimal data analysis exposure time for p53.
Lysate titration - Determining linear dynamic range
It is important that measurements be taken within the linear dynamic range of each assay, where changes in signal as measured by peak area are proportional to changes in the amount of protein in the sample. Using the optimal exposure time of 15 s chosen in the previous section, assay linearity is demonstrated for both p53 and &#593;-tubulin over greater than a 15-fold range of concentration (Figure 3). In our experience, a R2 value of &#62;0.9 of a linear regression fit is considered acceptable for a dilution range of purified protein of known quantity (if assay is an absolute quantitative measurement) or sample lysate of unknown target protein (if assay is a relative quantitative measurement).
Optimization of antibody dilution
Using antibodies at saturating concentrations helps ensure that any signal changes measured are due only to changes in protein amount. As a demonstration, two BEAS-2B cell line whole cell extracts (0.2 &#181;g/&#181;L total protein loaded into the assay) were probed with serially diluted &#593;-tubulin antibody concentrations ranging from 1:25 - 1:800 (Figure 4). Chemiluminescent signal (here, measured as peak area) was plotted against antibody dilution. Saturation was observed near the 1:50 dilution where the curve begins a noticeable plateau.
Experimental trial - Doxorubicin treatment in BEAS-2B cells
Using optimized assay conditions, BEAS-2B cell culture was treated with three different concentrations of doxorubicin (1.2, 1.8, and 2.4 &#181;g/mL) for 4 h (Figure 5, Table 1). Activation of p53 through post-translational modifications mediates several cellular responses, including cell cycle arrest, senescence, and apoptosis12. Specifically, the phosphorylation of serine 15 has been attributed to transcriptional activation of p53, resulting in apoptosis after doxorubicin treatment13. In this demonstration, &#593;-tubulin normalized peak areas are presented as fold of control. Interestingly, 3.5 to 4-fold increases in p53 phosphorylation at serine 15 and 2-fold increases in the level of p53 phosphorylated at serine 20 were observed after 4 h exposure to doxorubicin. These results indicate activation of p53; however, no dose-response is seen for the concentrations chosen (conversely, the lowest dose tested elicited the highest response). Total p53 did not demonstrate a clear treatment response in this model system. We have previously observed activation of p53 phosphorylation in the absence of increased levels of total p53 under similar conditions in zinc-treated BEAS-2B cells14.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55911/55911fig2v3.jpg" /> 
Figure 2. Exposure image comparison to detect signal burnout. Lane views show decreasing protein concentrations for BEAS-2B lysates probed with p53 DO-1 antibody at a 1:500 dilution. Chemiluminescence signal coefficients, reported as peak heights in the instrument software, are superimposed. Unlike the peak heights, the visual band intensities are automatically generated and adjusted by the instrument to aid viewing of the bands and are not comparable from one panel to another. Note the decrease in chemiluminescence signal as exposure time increases, with the signal beginning to disappear (split peak) at the two longest exposures, indicating substrate depletion. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55911/55911fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55911/55911fig3v3.jpg" /> 
Figure 3. Lysate titration showing the lane views. Lysate titration showing the lane views (A) of BEAS-2B lysate when probed with 1:500 p53 DO-1 or 1:50 &#593;-tubulin. Unlike the peak area values, the visual band intensities are automatically generated and adjusted by the instrument to aid viewing of the bands and are not comparable from one panel to another.&#160;Linear regression analysis (B) confirms the assays are linear over the entire range tested, from 0.01 to 0.20 &#181;g/&#181;L and 0.025 to 0.40 &#181;g/&#181;L, with R2 values of 0.999 and 0.985, respectively. Total protein concentrations in the middle of the linear range were chosen to accommodate potential target protein variation in either direction (e.g., 0.2 &#181;g/&#181;L for &#945;-tubulin).&#160;<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55911/55911fig3v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55911/55911fig4.jpg" /> 
Figure 4. &#945;-tubulin antibody dilution curves for two separate BEAS-2B protein lysates with and without baseline normalization. A definite departure from linearity is seen at the 1:50 (0.02) dilution, indicating saturation. 1:50 was therefore chosen as the optimal dilution for this antibody. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55911/55911fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55911/55911fig5.jpg" /> 
Figure 5.Effect of 4 h doxorubicin (DXN) treatment on total and serine phosphorylated p53 protein expression of BEAS-2B cells. Peak areas are normalized to &#945;-tubulin and plotted as fold of control (CTL). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55911/55911fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/55911/55911tbl1.jpg" /> 
Table 1. Effect of 4 h doxorubicin (DXN) treatment on total and serine phosphorylated p53 protein expression of BEAS-2B cells. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55911/55911tbl1large.jpg" target="_blank">Please click here to view a larger version of this table.</a>

Watch this Scientific Journal Video about Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method at JoVE.com

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

procedure-key-optimization-strategies-for-an-automated-capillary

Universidad San Sebastian

Research

JoVE Journal

Biology

10.9K Views.  U.S. Environmental Protection Agency. A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

Video: Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

Biochemistry

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Environment

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Bioengineering

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Immunology and Infection

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...