Name: detection of human leukocyte antigen biomarkers in breast cancer utilizing label-free biosensor technology
Uploaded: 2015-03-24T13:00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology at JoVE.com

These studies were supported in part by the Development Corporation of Abilene which provides laboratory space and salary support for Experimmune, a Center for Immunotherapeutic Development at Texas Tech University Health Sciences Center and Resonant Sensors Incorporated.

Ethics statement: De-identified human tissue and plasma samples were obtained from Hendrick Medical Center under an Institutional Review Board approved protocol.
1. Addition of Biotinylated Antibody
NOTE: Monitoring of this step is optional. See alternative protocol if not monitoring.
<ol>
	<li>Obtain commercially available avidin derivative coated plates (see Materials and Equipment Table).</li>
	<li>Add 50 &#181;l phosphate buffered saline (PBS) pH 7.2 to wells. 
	NOTE: Preincubate PBS at 28 &#176;C to minimize temperature related resonance shifts.</li>
	<li>Open bioassay scanner software and follow the Setup Wizard to define the experimental procedure which includes selection of blanks, standards and samples in the plate layout, temperature setting (28 &#176;C), number of scans per min (1), and length of experiment (150 min to accommodate for preparative steps).</li>
	<li>Insert the prepared assay plate into the label free bioassay scanner and start the first scan. When the first scan is complete, select &#8220;self reference to start&#8221;. The baseline is the initial set of scans collected at the start of the experiment. If this is the first read performed on this specific plate, allow the scanner to run to equilibrate temperature and eliminate drift in baseline. See Figure 2 for a screenshot of a baseline followed by sample addition.</li>
	<li>After the baseline has stabilized, or at least 5 scans, pause the read and eject the plate.</li>
	<li>Remove PBS by dumping in the sink and add 50 &#181;l RL21A biotinylated antibody at [10 &#181;g/ml in PBS pH 7.2] to all but control wells on the plate. Add 50 &#181;l PBS to the control wells.</li>
	<li>Insert the plate back into the bioassay scanner and resume reading until saturation is reached, approximately 1.5 hr.</li>
	<li>Pause the read and eject the plate.</li>
	<li>Wash the plate 3 times with 200 &#181;l/well phosphate buffered saline + 0.05% Tween 20 (PBST) followed by 3 rinses with 200 &#181;l/well PBS pH 7.2. Repeat with PBS. Tap excess PBS from plate on paper towels prior to moving to the next step. 
	NOTE: This step can be performed on an automatic plate washer or wash the plate 3 times with 200 &#181;l PBST by dumping liquid into a sink and replacing PBST with a pipette.</li>
	<li>Add 50 &#181;l PBS pH 7.2 to all active wells.</li>
	<li>Insert the plate back into the bioassay scanner and resume reading to monitor post-wash resonance.</li>
	<li>Stop the read and eject the plate.</li>
</ol>
Alternative protocol for Step 1:
<ol start="13">
	<li>Add 50 &#181;l RL21A biotinylated antibody at [10 &#181;g/ml PBS] to all wells.</li>
	<li>Incubate at room temperature for 1.5 hr or overnight at 4 &#176;C.</li>
	<li>Wash the plate 3 times with 200 &#181;l/well PBST followed by 3 rinses with 200 &#181;l/well PBS as in step 1.9.</li>
</ol>
2. Addition of Analyte
<ol>
	<li>Remove the PBS and add 50 &#181;l per well of appropriate assay buffer. For this assay, commercially available normal human serum was used diluted 1:20 in PBS.</li>
	<li>Open bioassay scanner software and follow the Setup Wizard to define experimental procedure.</li>
	<li>Insert the plate into the bioassay scanner and begin a baseline read. If this is the first read performed on this specific plate, let the scanner run to equilibrate temperature and eliminate drift in baseline.</li>
	<li>After the baseline has stabilized, or at least 5 scans, pause the read and eject the plate.</li>
	<li>Remove the assay buffer from the wells.</li>
	<li>Spike controls with HLA A*02:01 monomer bearing relevant (FLSL) or irrelevant (SLLV, YLEV, or KVL) peptides at concentrations ranging from [0.625-10 &#181;g/ml] in assay buffer. Add 50 &#181;l of standards to the control wells of the plate</li>
	<li>Add 50 &#181;l of analyte in assay buffer to the sample wells of the plate. For this assay, spiked commercially available human serum was used.</li>
	<li>Insert the plate back into the bioassay scanner and resume reading until saturation is reached, approximately 30-60 min.</li>
	<li>Pause the read and eject the plate.</li>
	<li>Wash the plate 3 times with 200 &#181;l/well PBST followed by 3 rinses with 200 &#181;l/well PBS as in step 1.9.</li>
	<li>Add 50 &#181;l assay buffer to all active wells.</li>
	<li>Insert the plate back into the bioassay scanner and resume reading to monitor post-wash resonance.</li>
	<li>Stop the read and eject the plate. 
	NOTE: The assay buffer could be PBS, Tissue culture media, or human serum diluted 1:20 in PBS depending on the goal of the assay.</li>
</ol>
3. Data Analysis
<ol>
	<li>Analyze using the bioassay scanner software or export raw data files to preferred statistical analysis software. The bioassay scanner software automatically generates the binding curve based on the plate layout provided during experimental setup. 
	NOTE: If not using the bioassay scanner software for analysis, follow steps 3.2-3.4.</li>
	<li>Subtract the baseline and negative control from each subsequent data point.</li>
	<li>Calculate the mean and standard deviation for replicate wells at each time point.</li>
	<li>Generate a graph of the binding curve or select end point data for bar graphs.</li>
</ol>

<ol>
	<li>Shastri, N., Schwab, S., Serwold, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Producing+nature's+gene-chips:+the+generation+of+peptides+for+display+by+MHC+class+I+molecules.">Producing nature's gene-chips: the generation of peptides for display by MHC class I molecules.</a> Annu Rev Immunol. 20, 463-493 (2002).</li><li>Peaper, D. R., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+MHC+class+I+assembly+and+peptide+binding.">Regulation of MHC class I assembly and peptide binding.</a> Annu Rev Cell Dev Biol. 24, 343-368 (2008).</li><li>Hawkins, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+HLA-presented+fragment+of+macrophage+migration+inhibitory+factor+is+a+therapeutic+target+for+invasive+breast+cancer.">An HLA-presented fragment of macrophage migration inhibitory factor is a therapeutic target for invasive breast cancer.</a> J Immunol. 186 (11), 6607-6616 (2011).</li><li>Verma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR+mimic+monoclonal+antibody+targets+a+specific+peptide/HLA+class+I+complex+and+significantly+impedes+tumor+growth+in+vivo+using+breast+cancer+models.">TCR mimic monoclonal antibody targets a specific peptide/HLA class I complex and significantly impedes tumor growth in vivo using breast cancer models.</a> J Immunol. 184 (4), 2156-2165 (2010).</li><li>Verma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR+mimic+monoclonal+antibodies+induce+apoptosis+of+tumor+cells+via+immune+effector-independent+mechanisms.">TCR mimic monoclonal antibodies induce apoptosis of tumor cells via immune effector-independent mechanisms.</a> J Immunol. 186 (5), 3265-3276 (2011).</li><li>Bassani-Sternberg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+plasma+HLA+peptidome+as+a+potential+source+for+cancer+biomarkers.">Soluble plasma HLA peptidome as a potential source for cancer biomarkers.</a> Proc Natl Acad Sci U S A. 107 (44), 18769-18776 (2010).</li><li>Hickman, H. D., Yewdell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mining+the+plasma+immunopeptidome+for+cancer+peptides+as+biomarkers+and+beyond.">Mining the plasma immunopeptidome for cancer peptides as biomarkers and beyond.</a> Proc Natl Acad Sci U S A. 107 (44), 18747-18748 (2010).</li><li>Neethling, F. 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=Assessing+vaccine+potency+using+TCRmimic+antibodies.">Assessing vaccine potency using TCRmimic antibodies.</a> Vaccine. 26 (25), 3092-3102 (2008).</li><li>Mittendorf, E. 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=Clinical+trial+results+of+the+HER-2/neu+(E75)+vaccine+to+prevent+breast+cancer+recurrence+in+high-risk+patients:+from.">Clinical trial results of the HER-2/neu (E75) vaccine to prevent breast cancer recurrence in high-risk patients: from.</a> US Military Cancer Institute Clinical Trials Group Study I-01 and I-02. Cancer. 118 (10), 2594-2602 (2012).</li><li>Winter, H., Fox, B. A., Ruttinger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+of+cancer+vaccines.">Future of cancer vaccines.</a> Methods Mol Biol. 1139, 555-564 (2014).</li><li>Magnusson, R., Wawro, D., Zimmerman, S., Ding, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resonant+photonic+biosensors+with+polarization-based+multiparametric+discrimination+in+each+channel.">Resonant photonic biosensors with polarization-based multiparametric discrimination in each channel.</a> Sensors. 11 (2), 1476-1488 (2011).</li><li>Kaja, 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=Detection+of+novel+biomarkers+for+ovarian+cancer+with+an+optical+nanotechnology+detection+system+enabling+label-free+diagnostics.">Detection of novel biomarkers for ovarian cancer with an optical nanotechnology detection system enabling label-free diagnostics.</a> J Biomed Opt. 17 (8), 081412-081411 (2012).</li><li>Magnusson, R., Wang, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+principle+for+optical+filters.">New principle for optical filters.</a> Appl. Phys. Lett. 61, 1022-1024 (1992).</li><li>Wawro, D., Tibuleac, S., Magnusson, R., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+fiber+endface+biosensor+based+on+resonances+in+dielectric+waveguide+gratings.">Optical fiber endface biosensor based on resonances in dielectric waveguide gratings.</a> Proc. SPIE. 3911, 86-94 (2000).</li><li>Altman, J. D., 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=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274 (5284), 94-96 (1996).</li></ol>

The authors, Debra Wawro Weidanz and Robert Magnusson, are respectively Chief Executive Officer and Chief Technology Officer at Resonant Sensors Incorporated, which produces the bioassay plate reader and assay plates used in this article.&#160;

The method described herein introduces a biomarker screening system for cancer diagnostics that would enable rapid detection of soluble peptide/HLA complexes in patient serum, plasma, urine or saliva samples in a high throughput 96- or 384-well format. This assay system utilizing T cell receptor mimicking monoclonal antibodies and a label-free detection system cuts the analysis time to less than 1 hr compared to 3.5 hr by conventional ELISA. This high-throughput approach enables rapid stratification of patients to clinical trials for therapeutic cancer vaccines or biopharmaceuticals. Current methods for the detection of these disease targets require affinity purification and HPLC-MS/MS of individual patient samples, a cumbersome and time consuming process. Although this method is amenable to conventional ELISA techniques, there is concern that secondary detection reagents such as the anti-&#946;2 microglobulin antibody could alter the structure of the nascent HLA molecule and inhibit binding to the TCRm limiting detection. Label-free detection alleviates these concerns. This procedure could be modified for use on other label-free systems such as a surface plasmon resonance system. However, this would greatly reduce the higher throughput capabilities compared to the bioassay system used in this study.
This protocol can be effectively modified for the detection of non-HLA biomarkers in patient serum and plasma with adherence to a few critical steps. Monitoring of the initial antibody coating process is recommended for optimization of the surface, as a &#60;200 pm shift will likely result in low signal for the subsequent detection step. The baseline and post-wash reads are useful for the endpoint analysis as the abundance of serum proteins may mask binding of low concentration analyte. Finally, temperature variation of samples can result in well to well variation. It is recommended that all samples be pre-incubated and the label free biosassay unit temperature controller be set at 2-3 &#176;C above room temperature. Refrigerated samples should be allowed enough time to reach the operating temperature.
Future modifications of this protocol will include multiplexing of TCRm on the plate surface. The bioassay system is currently amenable to spotting of wells at a density of at least 8-plex. By introducing 4-8 TCRm to each well, per test sample volumes are reduced and enable the collection of increasingly complex data per patient. This capability could further inform patients with regard to therapeutic options.

The class I Human Leukocyte antigen (HLA) samples peptides of 8-11 amino acids in length derived from the proteasomal degradation of the intracellular protein repertoire and presents these peptides on the surface of every nucleated cell1,2. The HLA complex is &#8220;nature&#8217;s biomarker&#8221;, indicating the health of each cell (Figure 1) to circulating cytotoxic T lymphocytes (CTL). Recognition of disease associated peptides results in elimination of the offending cell by CTL. Thus, the adaptive immune response is highly effective at eliminating viral infection and tumor. However, the immune system exerts pressure which often promotes selection for viruses or tumor capable of evading an effective CTL response. T-cell receptor mimicking (TCRm) monoclonal antibodies (mAbs) which recognize specific peptide/HLA-A*02:01 complexes such as those derived from breast cancer associated proteins, macrophage migration inhibitory factor (MIF19-27)3 and NY-ESO-1157-165, enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response4,5. This representative work is focused on the HLA-A*0201 molecule, which is expressed by up to 30% of the population. However, the identification of other relevant HLA/peptide complexes, followed by the production of TCRm enables the development of targeted immunotherapies and companion disease diagnostics, expanding the utility of this work to a much broader population.
A portion of the peptide/HLA complexes bound for the cell surface is released into the plasma. Due to the highly complex nature of peptide/HLA pools, current methods for mining the immunopeptidome for HLA associated biomarkers are time consuming. This process requires the affinity purification of soluble HLA from plasma, separation of HLA associated peptides by reverse phase high pressure liquid chromatography (RP-HPLC) and peptide sequencing by tandem mass spectrometry (MS/MS)6,7. Although this process is a viable option for the discovery of novel therapeutic targets, it is a cumbersome process as a companion diagnostic tool for HLA associated targets already in the therapeutic vaccine or biopharmaceutical pipeline8-10. TCRm directed against these targets provide the tools for rapid companion diagnostic development aimed at stratifying patients into relevant clinical trials and provide more informed therapeutic options.
In this work, a breakthrough biomarker screening system for cancer diagnostics is presented which utilizes TCRm and a label-free bioassay system that monitors biological interactions with minimal processing steps. The label-free bioassay system is based on a method, which utilizes the guided-mode resonance (GMR) effect that occurs in dielectric waveguide gratings11-14. When broadband light contacts the diffractive grating in the plates required for this system, two specific wavelengths are reflected. The binding interaction between an immobilized receptor and its ligand is monitored in real time by tracking the resonance wavelength shift with a spectrum analyzer12. Separate resonance peaks occur for incident TE (electric vector normal to the plane of incidence) and TM (magnetic vector normal to the plane of incidence) polarization states providing multiple data points that can potentially increase detection accuracy11,14. Using antibody pre-sensitized plates in this label-free assay system, the assay run time with data analysis is approximately 45 min. In addition, multiple patient samples can be interrogated in a 96- or 384-well format, a clear advantage over an HPLC-MS/MS based format.

<table border="0" cellpadding="0" cellspacing="0" width="1191">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:521px;"><a name="RANGE!A1:D29">Name of the Material/Equipment</a></td>
			<td style="width:195px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description (optional)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ResoSens Label-free Bioassay Detection System</td>
			<td>Resonant Sensors Incorporated</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Avidin Sensitized Bionetic 96-well Plates</td>
			<td>Resonant Sensors Incorporated</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ResoVu software</td>
			<td>Resonant Sensors Incorporated</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RL21A Biotinylated TCRm mAb&#160;</td>
			<td>PureMHC, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RL9A Biotinylated TCRm mAb&#160;</td>
			<td>PureMHC, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FLSL HLA-A*02:01 Monomer&#160;</td>
			<td>Experimmune</td>
			<td></td>
			<td>full peptide sequence: FLSELTQQL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SLLV HLA-A*02:01 Monomer&#160;</td>
			<td>Experimmune</td>
			<td></td>
			<td>full peptide sequence: SLLMWITQV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">YLEV HLA-A*02:01 Monomer&#160;</td>
			<td>Experimmune</td>
			<td></td>
			<td>full peptide sequence: YLEPGPVTV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KVL HLA-A*02:01 Monomer&#160;</td>
			<td>Experimmune</td>
			<td></td>
			<td>full peptide sequence: KVLEYVIKV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pooled Normal Human Serum&#160;</td>
			<td>ThermoFisher</td>
			<td>BP2657100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MDA-MB-231 Cell Supernatant</td>
			<td>ATCC</td>
			<td>HTB-26</td>
			<td style="width:355px;">Cells grown in IMDM , 10% FBS and 1% penn/strep. Supernatants harvested from 3 day confluent cultures.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#39;s Modification of Dulbecco&#39;s Medium (IMDM)</td>
			<td>Life Technologies</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin&#160;</td>
			<td>Life Technologies</td>
			<td>15070-63</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hydroxide</td>
			<td>ThermoFisher</td>
			<td>5318-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher</td>
			<td>BP665-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline + 0.05% Tween 20 (PBST)</td>
			<td>ThermoFisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,2&#8217;-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS)</td>
			<td>ThermoFisher</td>
			<td>37615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-human &#946;2 microglobulin&#160;</td>
			<td>Dako</td>
			<td>A0072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peroxidase conjugated goat anti-rabbit IgG&#160;</td>
			<td>Jackson Immunoresearch</td>
			<td>111-035-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc&#160; microplates</td>
			<td>ThermoFisher</td>
			<td align="right">439454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy 2 microplate reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immpress Reagent Kit</td>
			<td>Vector</td>
			<td>MP-7402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immpact DAB</td>
			<td>Vector</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin QS</td>
			<td>Vector</td>
			<td>H3403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2a</td>
			<td>MP BioMedicals</td>
			<td>50328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG2b</td>
			<td>MP BioMedicals</td>
			<td>50330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-HLA-A2 (BB7.2)</td>
			<td>Experimmune</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism 5</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of human leukocyte antigen biomarkers in breast cancer utilizing label-free biosensor technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

A representative set of experiments was performed using the well-characterized TCRm, RL21A, a murine IgG2a monoclonal antibody which specifically recognizes a peptide (MIF19-27 or FLSL) within the context of the HLA-A*02:01 molecule3. The cognate peptide/HLA monomer, as well as irrelevant HLA monomer15, was used to demonstrate the described procedure. Figure 3 illustrates the assay format.
RL21A is specific for the FLSL/HLA monomer with little cross reactivity with other peptide/HLA complexes3. This specificity is recapitulated using the label-free bioassay system (Figure 4). The sensitivity of the label free bioassay system for this experiment is in the low nanomolar range as determined by titration of the RL21A antibody on immobilized FLSL/HLA monomer (Figure 5). Although the background signal is significantly increased in serum samples, specific detection of FLSL/HLA monomer in spiked human serum is achieved in Figure 6 and a concentration gradient is readily discernable in these samples. Finally, supernatants from the MDA-MB-231 cell line, previously shown to present the FLSL/HLA-A*02:01 molecule, are shown to contain soluble FLSL monomer using this label free assay platform (Figure 7). Subsequent addition of FLSL/HLA monomer increases the signal on RL21A (Figure 7). Due to the sensitivity of the system, well to well variation including negative resonance shifts can occur as a result of temperature fluctuations as seen as a function of the standard deviations in Figures 4 and 5. These variations can be reduced dramatically by pre-incubation of all samples and the bioassay plate reader at a temperature in slight excess of room temperature (i.e., 28 &#176;C).
In Figure 8, briefly, 96-well polystyrene plates were coated with [10 &#181;g/ml] of RL21A for 2 hr at room temperature, washed with PBST, blocked with 0.5% low fat milk, washed with PBST, and incubated for 2 hr at room temperature with serial dilutions of FLSL HLA monomer in normal human serum diluted 1:10 in PBS to generate a standard curve or patient plasma diluted 1:10 in PBS. The plate was washed with PBST, incubated 1 hr at room temperature with rabbit anti-human &#946;2 microglobulin [1:5,000] to detect intact HLA, and washed in PBST. Plates were then incubated for 30 min with goat anti-rabbit IgG [1:10,000] and washed with PBST. Colorimetric detection was performed using ABTS (2,2&#8217;-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) substrate with a 15 min incubation time and observed at 405 nm on a microplate reader. FLSL/HLA was detected in three patient samples.
In Figure 8B, patient plasma RL.064 was diluted 1:20 in PBS and then added to the plate and monitored on the label-free detector for 60 min. Specific detection of FLSL/HLA complex in patient serum was accomplished. Student&#8217;s t test was performed using graphing and statistics software (p &#60;0.05).
In Figure 8C, tissue sections were stained at 1 &#181;g/ml with mouse anti-HLA-A2 (BB7.2) as a positive control, RL21A, IgG2b and IgG2a respectively as negative controls. Staining was detected using an anti-mouse detection kit, DAB (diaminobenzadine), and hematoxylin QS for nuclear staining as directed by the manufacturer. Staining of tumor tissue by RL21A confirms presentation of FLSL/HLA complexes.
<img alt="Figure 1" src="/files/ftp_upload/52159/52159fig1highres.jpg" /> 
Figure 1: Tumor antigen presentation by the class I human leukocyte antigen. Cancerous transformation is an intracellular disorder. HLA sample intracellular proteins and reveal cancer-related changes at the cell surface. CTL and TCRm are able to recognize cancerous cells through HLA-peptide complexes distinct to those cells.
<img alt="Figure 2" src="/files/ftp_upload/52159/52159fig2highres.jpg" /> 
Figure 2: Screenshot of data acquisition in the bioassay scanner software. Example of data acquisition showing the initial baseline scan followed by RL21A antibody addition to a plate coated with 10 &#181;g/ml of FLSL/HLA complex. Concentrations of antibody are as indicated. Each line represents an individual well monitored over time.
<img alt="Figure 3" src="/files/ftp_upload/52159/52159fig3highres.jpg" /> 
Figure 3: Assay format. Illustration of antibodies immobilized on the diffractive grating surface of the assay plate capturing relevant peptide/HLA complexes in solution.
<img alt="Figure 4" src="/files/ftp_upload/52159/52159fig4highres.jpg" /> 
Figure 4: Specificity of RL21A for FLSL/HLA complex. Demonstration of the specificity of biotinylated RL21A TCRm for its relevant (FLSL) HLA monomer compared to irrelevant HLA monomer (YLEV, SLLV, and KVL). Biotinylated RL21A TCRm [10 &#181;g/ml] was immobilized on the avidin coated assay plate surface and detection was performed using unlabeled relevant or irrelevant HLA monomer [10 &#181;g/ml] in PBS. RL21A was specific for FLSL/HLA Complex. Two-way ANOVA was performed using graphing and statistics software (p &#60;0.001).
<img alt="Figure 5" src="/files/ftp_upload/52159/52159fig5highres.jpg" /> 
Figure 5: Binding sensitivity of RL21A to its cognate peptide/HLA complex. Illustration of the detection limit and binding sensitivity of RL21A to the FLSL/HLA complex. Biotinylated HLA Monomer FLSL [10 &#181;g/ml] was immobilized on the assay plate surface and unlabeled RL21A was added to the plate in serial dilutions in PBS as indicated. Detection for this system was in the low nanomolar range.
<img alt="Figure 6" src="/files/ftp_upload/52159/52159fig6highres.jpg" /> 
Figure 6: Detection of HLA Complexes in Spiked Human Serum. Demonstration of the detection of HLA in human serum. Biotinylated RL21A TCRm [10 &#181;g/ml] was immobilized on the avidin coated assay plate surface. Pooled normal human serum spiked with relevant (FLSL) or irrelevant (SLLV) HLA monomer was diluted 1:20 in PBS and then added to the plate and monitored on the label-free detector for 60 min. Specific detection of FLSL/HLA complex in human serum was accomplished. In general, background signal (irrelevant SLLV monomer) is higher for diluted serum samples compared to purified analytes in PBS (see Figure 4). Two-way ANOVA was performed using graphing and statistics software (p &#60;0.0001).
<img alt="Figure 7" src="/files/ftp_upload/52159/52159fig7highres.jpg" /> 
Figure 7: Soluble HLA complexes were detected in breast cancer cell culture supernatants. The ability to detect HLA in breast cancer cell supernatants is demonstrated. Biotinylated RL21A TCRm, RL9A TCRm (negative control), and an isotype control were immobilized on the assay plate surface. Spiked [5 &#956;g/ml] and unspiked MDA-231 cell culture supernatants were added and binding was monitored for 60 min on the label-free detector. FLSL and SLLV HLA monomer spiked samples represent positive controls for RL9A and RL21A binding. One-way ANOVA was performed using graphing and statistics software (p &#60;0.0001).
<img alt="Figure 8" src="/files/ftp_upload/52159/52159fig8highres.jpg" /> 
Figure 8: Specific detection of FLSL/HLA complexes in patient samples. (A) A traditional Enzyme Linked Immunosorbent Assay (ELISA) was utilized to detect FLSL/HLA specific complexes in patient plasma. (B) Biotinylated RL21A TCRm or IgG2a [10 &#181;g/ml] was immobilized on the avidin assay plate. (C) Breast tumor tissue from patient RL.064 was stained as previously described3 to verify tumor presentation of the FLSL/HLA complexes. <a href="https://www.jove.com/files/ftp_upload/52159/52159fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology at JoVE.com

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

detection-human-leukocyte-antigen-biomarkers-breast-cancer-utilizing

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

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 ...