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><tbody><tr><td>ResoSens Label-free Bioassay Detection System</td><td>Resonant Sensors Incorporated</td><td></td><td></td></tr><tr><td>Avidin Sensitized Bionetic 96-well Plates</td><td>Resonant Sensors Incorporated</td><td></td><td></td></tr><tr><td>ResoVu software</td><td>Resonant Sensors Incorporated</td><td></td><td></td></tr><tr><td>RL21A Biotinylated TCRm mAb&amp;nbsp;</td><td>PureMHC, LLC</td><td></td><td></td></tr><tr><td>RL9A Biotinylated TCRm mAb&amp;nbsp;</td><td>PureMHC, LLC</td><td></td><td></td></tr><tr><td>FLSL HLA-A*02:01 Monomer&amp;nbsp;</td><td>Experimmune</td><td></td><td>full peptide sequence: FLSELTQQL</td></tr><tr><td>SLLV HLA-A*02:01 Monomer&amp;nbsp;</td><td>Experimmune</td><td></td><td>full peptide sequence: SLLMWITQV</td></tr><tr><td>YLEV HLA-A*02:01 Monomer&amp;nbsp;</td><td>Experimmune</td><td></td><td>full peptide sequence: YLEPGPVTV</td></tr><tr><td>KVL HLA-A*02:01 Monomer&amp;nbsp;</td><td>Experimmune</td><td></td><td>full peptide sequence: KVLEYVIKV</td></tr><tr><td>Pooled Normal Human Serum&amp;nbsp;</td><td>ThermoFisher</td><td>BP2657100</td><td></td></tr><tr><td>MDA-MB-231 Cell Supernatant</td><td>ATCC</td><td>HTB-26</td><td>Cells grown in IMDM , 10% FBS and 1% penn/strep. Supernatants harvested from 3 day confluent cultures.</td></tr><tr><td>Iscove&#039;s Modification of Dulbecco&#039;s Medium (IMDM)</td><td>Life Technologies</td><td>12440-053</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>10082-147</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>Life Technologies</td><td>15070-63</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>ThermoFisher</td><td>5318-1</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>ThermoFisher</td><td>BP665-1</td><td></td></tr><tr><td>Phosphate Buffered Saline + 0.05% Tween 20 (PBST)</td><td>ThermoFisher</td><td>BP337-500</td><td></td></tr><tr><td>2,2&amp;rsquo;-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS)</td><td>ThermoFisher</td><td>37615</td><td></td></tr><tr><td>rabbit anti-human &amp;beta;2 microglobulin&amp;nbsp;</td><td>Dako</td><td>A0072</td><td></td></tr><tr><td>Peroxidase conjugated goat anti-rabbit IgG&amp;nbsp;</td><td>Jackson Immunoresearch</td><td>111-035-144</td><td></td></tr><tr><td>Nunc&amp;nbsp; microplates</td><td>ThermoFisher</td><td>439454</td><td></td></tr><tr><td>Synergy 2 microplate reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Immpress Reagent Kit</td><td>Vector</td><td>MP-7402</td><td></td></tr><tr><td>Immpact DAB</td><td>Vector</td><td>SK-4105</td><td></td></tr><tr><td>Hematoxylin QS</td><td>Vector</td><td>H3403</td><td></td></tr><tr><td>IgG2a</td><td>MP BioMedicals</td><td>50328</td><td></td></tr><tr><td>IgG2b</td><td>MP BioMedicals</td><td>50330</td><td></td></tr><tr><td>anti-HLA-A2 (BB7.2)</td><td>Experimmune</td><td></td><td></td></tr><tr><td>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>

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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 ...

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Research

JoVE Journal

Immunology and Infection

14.3K Views.  Texas Tech University Health Sciences Center.  Intact klasse I HLA / peptide complexen worden vergoten door kankercellen, wat neerkomt op een potentieel relevante kanker biomarker. Met behulp labelvrije sensortechnologie en T-cel receptor nabootsen monoklonale antilichamen, detectie van stal MIF / HLA-A * 02: 01 complexen in MDA-MB-231 cel supernatanten, spiked humaan serum en plasma van een patiënt wordt aangetoond, waardoor de ontwikkeling van een nieuwe kankertherapie diagnostisch platform. 

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

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier (MSC) for Lung Cancer Screening

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the outcome of this deadly disease. MicroRNAs (miRNAs) are tissue specific, small, non-coding RNAs regulating gene expression, which may act as extracellular messengers of biological signals derived from the cross-talk between the tumor and its surrounding microenvironment. They could thus represent ideal candidates for early detection of lung cancer. In this work, a methodological workflow for the prospective validation of a circulating miRNA test using custom made microfluidic cards and quantitative Real-Time PCR in plasma samples of volunteers enrolled in a lung cancer screening trial is proposed. In addition, since the release of hemolysis-related miRNAs and more general technical issues may affect the analysis, the quality control steps included in the standard operating procedures are also presented. The protocol is reproducible and gives reliable quantitative results; however, when using large clinical series, both pre-analytical and analytical features should be cautiously evaluated.

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier MSC for Lung Cancer Screening

Here, we present the detailed protocol adopted in the BioMILD screening trial to perform the circulating microRNA signature classifier test for early lung cancer detection.

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the ...

Cancer Research

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

Biochemistry

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Bioengineering

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Biology

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen&#8482; QuantiGene&#8482; Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP&#174; magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&#38;E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

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 ...

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

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