Supported by NIH grants RC1-HL-101102, P01-CA039542, T32-HL007622, the Hartwell Foundation, and the Doris Duke Charitable Foundation. Dr. Paczesny is an investigator of the Eric Hartwell fund and the Amy Strelzer Manasevit Research Program.

1. Experiment Day 0: Sample Preparation and ELISA Test Plate Coating with Capture Antibody for IL-2R&alpha;, REG3&alpha; and HGF
<ol>
 <li>Plasma aliquot samples to be analyzed will be pulled, thawed and spun at 12,000 rpm for 10 min to separate the clots at the bottom and lipids on top from the plasma. 150 &mu;l of undiluted plasma will be plated from each sample onto a 96-well V-bottom plate (source plate) by manual pipetting according to predefined maps. The aliquots will be wrapped in parafilm and kept in a humid chamber at 4 &deg;C during the entire process; no longer than 72 hr.</li>
 <li>IL-2R&alpha; and HGF capture antibodies will be reconstituted and diluted per manufacturer specification and 50 &mu;l will plated into each well of respective 96-well high-binding half-well plates which are then sealed and incubated overnight at 4 &deg;C. Alternatively, many plates can be dried at 37 &deg;C and stored at 4 &deg;C for later use, depending on the stability of the protein.</li>
 <li>REG3&alpha; capture antibody will be diluted according to manufacturer protocol using manufacturer coating buffer and 25 &mu;l will be plated in each well of a 384-well Nunc Maxi-Sorp plate which is then sealed and incubated overnight at 4 &deg;C.</li>
</ol>
2. Experiment Day 1: IL-2R&alpha; ELISA (Figure 1)
<ol>
 <li>The IL-2R&alpha; test plate is washed, blocked with BLOTTO in TBS, and the standard is reconstituted and an 8-point standard curve is prepared per manufacturer protocol.</li>
 <li>After washing the plate following the blocking step, 50 &mu;l of undiluted plasma is plated in duplicate from the source plate to the ELISA test plate, and 50 &mu;l of each standard is plated in duplicate. The plate is sealed and incubated for 2 hr at room temperature on a plate rotator set at 300 rpm.</li>
 <li>The plasma is reclaimed from the IL-2R&alpha; ELISA test plate and placed back in the undiluted plasma source plate. The ELISA is completed per manufacturer protocol (with volumes adjusted for half-well plates) and the optical density of each well will be read using a plate reader set to 450-570 nm, and the data saved and analyzed.</li>
</ol>
3. Experiment Day 1: REG3&alpha; ELISA (Figure 1)
<ol>
 <li>10 &mu;l of undiluted plasma will be transferred to a separate v-bottom source plate. 90 &mu;l of manufacturer-provided dilution buffer is added to each well to create a 1:10 dilution source plate.</li>
 <li>The REG3&alpha; ELISA is performed per manufacturer protocol (with volumes adjusted for 384-well plates) and the optical density of each well will be read using a plate reader set to 450-620 nm, and the data will be saved and analyzed.</li>
</ol>
4. Experiment Day 1: Elafin and TNFR1 Test Plate Coating with Capture Antibody
<ol>
 <li>Elafin and TNFR1 capture antibodies will be reconstituted and diluted per manufacturer specification and 50 &mu;l will plated into each well of respective 96-well high-binding half-well plates which are then sealed and incubated overnight at room temperature for Elafin, and at 4 &deg;C for TNFR1.</li>
</ol>
5. Experiment Day 1-2: HGF ELISA (Figure 1)
<ol>
 <li>After completing the IL-2R&alpha; ELISA and assuring the test does not need to be repeated, 60 &mu;l of undiluted plasma will transferred to a new source plate and then 60 &mu;l of 1% BSA in 1 x PBS is added to each well to make a 1:2 diluted plasma source plate.</li>
 <li>The HGF test plate is washed, blocked with BLOTTO in TBS, and the standard is reconstituted and an 8-point standard curve is prepared per manufacturer protocol.</li>
 <li>After washing the plate following the blocking step, 50 &mu;l of 1:2 diluted plasma is plated in duplicate on the ELISA test plate, and 50 &mu;l of each standard is plated in duplicate. The plate is sealed and incubated overnight at room temperature on a plate rotator set at 300 RPM.</li>
 <li>The 1:2 diluted plasma is reclaimed from the HGF ELISA test plate and placed back in the 1:2 diluted plasma source plate. The ELISA is completed per manufacturer protocol (with volumes adjusted for half-well plates) and the optical density of each well will be read using a plate reader set to 450-570 nm, and the data saved and analyzed.</li>
</ol>
6. Experiment Day 2: Elafin ELISA
<ol>
 <li>10 &mu;l of undiluted plasma will be transferred to a new source plate and then 190 &mu;l of 1% BSA in 1 x PBS will be added to each well to make 200 &mu;l of plasma dluted 1:20.</li>
 <li>The Elafin ELISA as performed according to manufacturer protocol (with volumes adjusted for half-well plates), and the optical density of each well will be read using a plate reader set to 450-570 nm, and the data saved and analyzed.</li>
</ol>
7. Experiment Day 2: TNFR1 ELISA
<ol>
 <li>25 &mu;l of 1% BSA in PBS will be added to the 1:20 source plate (now containing 100 &mu;l of 1:20 plasma) to obtain 125 &mu;l of 1:25 diluted plasma.</li>
 <li>The TNFR1 ELISA is completed per manufacturer protocol (with volumes adjusted for half-well plates) and the optical density of each well will be read using a plate reader set to 450-570 nm, and the data saved and analyzed.</li>
</ol>
8. Experiment Day 2: IL-8 ELISA
<ol>
 <li>60 &mu;l of 1:2 diluted plasma will transferred to a new source plate and then 180 &mu;l of IL-8 diluent is added to each well to make a 1:6 diluted plasma source plate.</li>
 <li>The IL-8 ELISA is completed per manufacturer protocol (with volumes adjusted for half-well plates) and the optical density of each well will be read using a plate reader set to 450-570 nm, and the data saved and analyzed.</li>
</ol>
Once all ELISAs have been completed, unused stock plasma will be replaced into the thawed aliquots and frozen for future use.

<ol>
	<li>Paczesny, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomarker+panel+for+acute+graft-versus-host+disease.">A biomarker panel for acute graft-versus-host disease.</a> Blood. 113, 273-278 (2009).</li><li>Paczesny, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elafin+is+a+biomarker+of+graft-versus-host+disease+of+the+skin.">Elafin is a biomarker of graft-versus-host disease of the skin.</a> Science Translational Medicine. 2, 13ra12 (2010).</li><li>Ferrara, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+islet-derived+3+alpha+is+a+biomarker+of+gastrointestinal+graft-versus-host+disease.">Regenerating islet-derived 3 alpha is a biomarker of gastrointestinal graft-versus-host disease.</a> Blood. , (2011).</li><li>Ferrara, J. L., Levine, J. E., Reddy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft-versus-host+disease.">Graft-versus-host disease.</a> Lancet. 373, 1550-1561 (2009).</li><li>Miyamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+concentration+of+the+soluble+interleukin-2+receptor+for+monitoring+acute+graft-versus-host+disease.">Serum concentration of the soluble interleukin-2 receptor for monitoring acute graft-versus-host disease.</a> Bone Marrow Transpl. 17, 185-190 (1996).</li><li>Holler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+tumor+necrosis+factor+alpha+in+acute+graft-versus-host+disease+and+complications+following+allogeneic+bone+marrow+transplantation.">Role of tumor necrosis factor alpha in acute graft-versus-host disease and complications following allogeneic bone marrow transplantation.</a> Transplant. Proc. 25, 1234-1236 (1993).</li><li>Okamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+hepatocyte+growth+factor+in+serum+in+acute+graft-versus-host+disease.">Increased hepatocyte growth factor in serum in acute graft-versus-host disease.</a> Bone Marrow Transpl. 28, 197-200 (2001).</li><li>Uguccioni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+interleukin-8+serum+concentrations+in+beta-thalassemia+and+graft-versus-host+disease.">Elevated interleukin-8 serum concentrations in beta-thalassemia and graft-versus-host disease.</a> Blood. 81, 2252-2256 (1993).</li><li>Osuchowski, M. F., Siddiqui, J., Copeland, S., Remick, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+ELISA+to+profile+multiple+cytokines+from+small+volumes.">Sequential ELISA to profile multiple cytokines from small volumes.</a> J. Immunol. Methods. 302, 172-181 (2005).</li><li>Osuchowski, M. F., Remick, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+repetitive+use+of+samples+to+measure+multiple+cytokines:+the+sequential+ELISA.">The repetitive use of samples to measure multiple cytokines: the sequential ELISA.</a> Methods. 38, 304-311 (2006).</li><li>Schweitzer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+profiling+on+microarrays+by+rolling-circle+amplification.">Multiplexed protein profiling on microarrays by rolling-circle amplification.</a> Nat. Biotechnol. 20, 359-365 (2002).</li></ol>

No conflicts of interest declared.

The sequential ELISA method presented here allows for measurement of multiple plasma proteins on small volumes of plasma which may be difficult to obtain and/or irreplaceable such as samples from human subjects with rare diseases or plasma samples obtained from mice9,10. The sequential ELISAs are typically performed in the order of increasing plasma dilution factor, with ELISAs requiring plasma diluted &ge; 1:10 typically not needing to be reclaimed, although this can be done if desired. The ability to perform sequential ELISA is limited by ELISA kits/protocols in which the plasma is mixed with other reagents or for which different dilution buffers are required for the plasma; this precludes the ablility to re-use a sample due to concerns that an incompatible buffer/reagent will interfere with the performance of a particular test. With careful planning, 10 or more ELISAs may be performed on the same plasma sample.
Individual laboratories may need to adjust plasma dilutions in order to have interpretable results based upon expected plasma concentrations of the protein of interest in the samples from the test subjects. Differences in laboratory equipment may result in the need to optimize incubation and colorimetric development times, number of washes and/or wash soak times in order to optimize any given ELISA.
To increase high-throughput capacity and accuracy and to perform analyses in a cost-effective manner, the use of a robotic liquid handling platform capable of analysis on 384 well plates and an automated plate washer with stacking unit are recommended. This equipment can increase the accuracy and precision of analysis performed by multiple users, and help provide consistency of analysis to reduce inter- and intra-assay variation.
We used sequential ELISA over available multiplex platforms for two reasons: 1) Most of the antibody pairs for novel proteins cannot easily be conjugated on beads or other material as well as time consuming and expensive; 2) individual ELISA assays are more precise than multiplex microarray or beads, secondary to an absence of cross-reactivity11 . If a reliable method is established to perform multiplexed, bead-based microarrays, it may be able to replace the sequential ELISA process, but may be limited by the ability to conjugate the antibodies to beads and/or by the number of proteins desired to be analyzed.

Acute graft-versus-host disease (GVHD), a leading cause of non-relapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (HSCT), is measured by dysfunction in three organ systems: the skin, liver and gastrointestinal (GI) tract4. Acute GVHD typically occurs between two and eight weeks after transplant but may occur later, and is often clinically indistinguishable from other post-HSCT complications such as conditioning regimen toxicity, infection or medication side effects. Through the use of proteomic strategies and high-throughput validation using sequential ELISA, we have identified 6 proteins whose concentrations are elevated at the onset of clinical manifestations of GVHD. IL-2R&alpha;, TNFR1, HGF and IL-8, when combined into a 4-biomarker panel can diagnose GVHD at the onset of clinical symptoms and can predict post-HSCT survival independently of GVHD severity1. Elafin, a biomarker for GVHD of the skin, can discriminate between GVHD rash and rash from other causes such as drug eruptions and can predict transplant survival2. We have recently identified REG3&alpha; as a biomarker of GVHD of the lower gastrointestinal tract, the target organ most associated with NRM. Plasma REG3&alpha; concentration can reliably identify GVHD as the cause for post-HSCT diarrhea and correlate to histologic severity of GVHD on diagnostic intestinal biopsies. REG3&alpha; concentrations at GI GVHD onset can also predict responsiveness to GVHD therapy and NRM3. The incorporation of these validated GVHD biomarkers into clinical care is currently being investigated in clinical trials.
These experiments were performed on small plasma aliquots collected from patients receiving HSCT between 2000 and 2010 at the time of GVHD onset that are irreplaceable and of limited quantity. Due to the precious nature of these samples, we have developed a method of measuring multiple plasma protein concentrations in an efficient, reproducible manner to eliminate excess freeze-thaw cycles, thaw time and plasma usage. This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents. This manuscript focuses on the technological aspects of measuring GVHD biomarkers.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Human IL-2 R alpha Duoset</td>
 <td>R&amp;D Systems</td>
 <td>DY223</td>
 <td></td>
 </tr>
 <tr>
 <td>Human HGF Duoset</td>
 <td>R&amp;D Systems</td>
 <td>DY294</td>
 <td></td>
 </tr>
 <tr>
 <td>Human IL-8 OptEIA KIT II</td>
 <td>Becton Dickinson</td>
 <td>550999</td>
 <td></td>
 </tr>
 <tr>
 <td>Ab-Match ASSEMBLY Human PAP1 (REG3&alpha;) Kit</td>
 <td>MBL International</td>
 <td>5323</td>
 <td></td>
 </tr>
 <tr>
 <td>Ab-Match UNIVERSAL Kit</td>
 <td>MBL International</td>
 <td>5310</td>
 <td></td>
 </tr>
 <tr>
 <td>Human sTNFRI/TNFRSF1A Duoset</td>
 <td>R&amp;D Systems</td>
 <td>DY225</td>
 <td></td>
 </tr>
 <tr>
 <td>Human Trappin-2/Elafin Duoset</td>
 <td>R&amp;D Systems</td>
 <td>DY1747</td>
 <td></td>
 </tr>
 <tr>
 <td>96-well polystyrene conical bottom plates</td>
 <td>Thermo Scientific</td>
 <td>249570</td>
 <td>Used for plasma source plates</td>
 </tr>
 <tr>
 <td>Costar half-well high-binding 96-well plates</td>
 <td>Corning</td>
 <td>3690</td>
 <td>For IL-2R&alpha;, HGF, TNFR1 and elafin ELISAs</td>
 </tr>
 <tr>
 <td>Nunc 384-well MaxiSorp plates</td>
 <td>Nunc</td>
 <td>464718</td>
 <td>For REG3&alpha; Elisa</td>
 </tr>
 <tr>
 <td>HyClone Phosphate Buffered Saline, 1x</td>
 <td>Thermo Scientific</td>
 <td>SH30256.02</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin, Fraction V, Heat Shock Treated</td>
 <td>Fisher Scientific</td>
 <td>BP1600-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Blocker BLOTTO in TBS</td>
 <td>Thermo Scientific</td>
 <td>37530</td>
 <td>Blocking agent for IL-2R&alpha; HGF and TNFR1 ELISAs</td>
 </tr>
 <tr>
 <td>Dulbecco's Phosphate-Buffered Saline</td>
 <td>Gibco</td>
 <td>21600-069</td>
 <td>Wash buffer for IL-2R&alpha;, HGF, Elafin and TNFR1 ELISAs</td>
 </tr>
 <tr>
 <td>TMB Peroxide Susbtrate</td>
 <td>Kirkegaard and Perry Laboratories</td>
 <td>50-76-00</td>
 <td></td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Acros Organics</td>
 <td>233362500</td>
 <td>Wash buffer for IL-2R&alpha;, HGF, Elafin and TNFR1 ELISAs</td>
 </tr>
 <tr>
 <td>Sulfuric Acid</td>
 <td>Sigma-Aldrich</td>
 <td>84720</td>
 <td>(Diluted to 2N) for stop solution</td>
 </tr>
 </tbody>
</table>

high throughput sequential elisa for validation of biomarkers of acute graft-versus-host disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

The biomarker workflow and timeline are detailed in Table 1 and Table 2, respectively. Once completed, concentrations of 6 different proteins have now been quantified on the same plasma sample using a total of 150 &mu;L of plasma. Plating the samples in duplicate per test allows for internal quality assurance, with CV's less than 10% being optimal. If performing the sequential ELISA on multiple plates, consistent optical densities of the high standard are preferred and allow for improved inter-plate reliability of measurements; standard curve ODs can be compared between plates to look assess for inconsistency in ELISA performance (Figure 2). Development times using tetramethylbenzidine colorimetric substrate and high concentration ODs observed for each biomarker in our laboratory are listed in Table 3.
<img src="/files/ftp_upload/4247/4247fig1.jpg" alt="Figure 1"/> 
 Figure 1. Workflow for IL-2R&alpha;, REG3&alpha; and HGF ELISAs. After the plasma samples have been plated on the IL-2R&alpha; ELISA test plates it is reclaimed to make dilution source plates for other ELISAs. For the HGF ELISA, the plasma is reclaimed to prepare the 1:6 dilution plate for IL-8. <a href="https://www.jove.com/files/ftp_upload/4247/4247fig1large.jpg" target="_blank"> Click here for larger figure</a>.
<img src="/files/ftp_upload/4247/4247fig2.jpg" alt="Figure 2"/> 
 Figure 2. Optical Densities for the standard curve of 7 different ELISA plates measuring REG3&alpha; concentrations corresponding to the 1084 patients tested for the initial REG3&alpha; GI GVHD biomarker report3. Consistent ODs between plates assure consistent protein concentration measurements between plates. Concentrations of proteins in plasma samples are calculated by comparing sample optical densities to the standard curve optical densities.
<table border="1">
 <tbody>
 <tr>
 <td>Experiment Day 0</td>
 <td>1. Prepare samples</td>
 </tr>
 <tr>
 <td></td>
 <td>2. IL-2R&alpha;, HGF and REG3&alpha; capture Ab</td>
 </tr>
 <tr>
 <td>Experiment Day 1</td>
 <td>1. IL-2R&alpha;ELISA</td>
 </tr>
 <tr>
 <td></td>
 <td>2. REG3&alpha;ELISA</td>
 </tr>
 <tr>
 <td></td>
 <td>3. HGF ELISA (through sample plating)</td>
 </tr>
 <tr>
 <td></td>
 <td>4. Elafin and TNFR capture Ab</td>
 </tr>
 <tr>
 <td>Experiment Day 2</td>
 <td>1. HGF ELISA completion</td>
 </tr>
 <tr>
 <td></td>
 <td>2. Elafin ELISA</td>
 </tr>
 <tr>
 <td></td>
 <td>3. TNFR1 ELISA</td>
 </tr>
 <tr>
 <td></td>
 <td>4. IL-8 ELISA</td>
 </tr>
 <tr>
 <td></td>
 <td>5. Refreeze unused plasma</td>
 </tr>
 </tbody>
</table>
Table 1. GVHD Biomarker Workflow Overview
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Day 0</td>
 <td colspan="3">Sample preparation and overnight capture body incubation</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td colspan="2">ELISA</td>
 <td>IL-2R&alpha;</td>
 <td>REG3&alpha;</td>
 <td>HGF</td>
 <td>Elafin</td>
 <td>TNFR1</td>
 <td>IL-8</td>
 </tr>
 <tr>
 <td>Time (hr)</td>
 <td>0.0</td>
 <td>Blocking</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>1.0</td>
 <td>Samples Plated</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>3.0</td>
 <td>Reclaim plasma samples; Detection Ab</td>
 <td>Blocking; Prepare Samples (1:10 dilution) </td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>4.0</td>
 <td></td>
 <td>Samples plated (1:10)</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>5.0</td>
 <td>Streptavidin-HRP</td>
 <td>Detection Ab</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>5.5</td>
 <td>TMB</td>
 <td>Streptavidin-HRP</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>6.0</td>
 <td>Plate Reading</td>
 <td>TMB</td>
 <td>Blocking; Prepare samples (1:2 dilution)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>6.5</td>
 <td></td>
 <td>Plate Reading</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>7.0</td>
 <td></td>
 <td></td>
 <td>Samples plated (1:2)</td>
 <td>Capture Ab (overnight incubation)</td>
 <td>Capture Ab (overnight incubation)</td>
 <td></td>
 </tr>
 <tr>
 <td>Day 2</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Time (hr)</td>
 <td>0.0</td>
 <td></td>
 <td></td>
 <td>Reclaim plasma, Detection Ab</td>
 <td>Blocking; Prepare samples (1:20)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>1.0</td>
 <td></td>
 <td></td>
 <td></td>
 <td>Sample plating (1:2)</td>
 <td>Blocking; Further dilution of 1:20 samples to 1:25 dilution</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>2.0</td>
 <td></td>
 <td></td>
 <td>HRP</td>
 <td></td>
 <td>Sample plating (1:25)</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>2.5</td>
 <td></td>
 <td></td>
 <td>TMB</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>3.0</td>
 <td></td>
 <td></td>
 <td>Plate Reading</td>
 <td>Detection Ab</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>3.5</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td>Prepare samples (1:6 dilution)</td>
 </tr>
 <tr>
 <td></td>
 <td>4.0</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td>Detection Ab</td>
 <td>Sample plating (1:6)</td>
 </tr>
 <tr>
 <td></td>
 <td>5.0</td>
 <td></td>
 <td></td>
 <td></td>
 <td>HRP</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>5.5</td>
 <td></td>
 <td></td>
 <td></td>
 <td>TMB</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>6.0</td>
 <td></td>
 <td></td>
 <td></td>
 <td>Plate Reading</td>
 <td>HRP</td>
 <td>Detection Ab</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td>TMB</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>7.0</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td>Plate Reading</td>
 <td>TMB</td>
 </tr>
 <tr>
 <td></td>
 <td>7.5</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td>Plate reading</td>
 </tr>
 <tr>
 <td colspan="2">After completion</td>
 <td colspan="6">Replace source plasma into aliquots and freeze for later use</td>
 </tr>
 </tbody>
</table>
Table 2. Timeline for performing ELISAs.
<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td>Plasma Dilution factor</td>
 <td>High Standard Concentration</td>
 <td>Substrate Development Time (min)</td>
 <td>High OD</td>
 <td>Curve</td>
 </tr>
 <tr>
 <td>IL-2R&alpha;</td>
 <td>1:1</td>
 <td>2,000 pg/ml</td>
 <td>5</td>
 <td>1</td>
 <td>Linear</td>
 </tr>
 <tr>
 <td>HGF</td>
 <td>1:2</td>
 <td>4,000 pg/ml</td>
 <td>22</td>
 <td>2.1</td>
 <td>4-parameter</td>
 </tr>
 <tr>
 <td>IL-8</td>
 <td>1:6</td>
 <td>200 pg/ml</td>
 <td>12</td>
 <td>2.7</td>
 <td>4-parameter</td>
 </tr>
 <tr>
 <td>REG3&alpha;</td>
 <td>1:10</td>
 <td>100 ng/ml</td>
 <td>12</td>
 <td>1.7</td>
 <td>4-parameter</td>
 </tr>
 <tr>
 <td>Elafin</td>
 <td>1:20</td>
 <td>2,000 pg/ml</td>
 <td>20</td>
 <td>1.9</td>
 <td>4-parameter</td>
 </tr>
 <tr>
 <td>TNFR1</td>
 <td>1:25</td>
 <td>800 pg/ml</td>
 <td>8</td>
 <td>2.7</td>
 <td>Linear</td>
 </tr>
 </tbody>
</table>
Table 3. ELISA Details for 6 GVHD Biomarkers.

Watch this Scientific Journal Video about High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease at JoVE.com

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

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15.7K Views.  University of Michigan. High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Video: High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...