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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Design of Custom Array Layout and Production
<ol>
	<li>Generate a spreadsheet of peptide sequences with corresponding allele callings. The array has 20 rows and 30 columns and can hold up to 20x30=600 spots for peptides. Depending on the total number of peptides recorded in step 1.5 from one particular donor, and based on our past experience with two examples that we had tried, the 600 spot array can hold all the sequences generated from ~2 separate transplant cases.
	<ol>
		<li>Rationally plan the most efficient way to fit the sets of peptides within the 600-spot format of the array.</li>
		<li>If more than one subject can fit into one whole array, insert empty rows after the first donor&#39;s sequences to match the total number of rows that can be divided by 30 (the number of spots in a row on the array).</li>
		<li>Immediately following the last empty row for the first set of sequences, paste in the entire column of sequence contents from the second transplant case.</li>
		<li>Repeat these steps until the array is filled and no more cases can be added without exceeding the 600 spots. If entire empty rows are left to fill at the bottom, &#34;move&#34; the empty row to be positioned between the two donor sets. This wide space left between cases makes cutting of the membrane after completion of the synthesis easier - following step 2.2. below.</li>
	</ol>
	</li>
	<li>Program the peptide sequences in the context of the array layout. The program of the SPOT synthesizer takes a text format of the sequences (one row, one peptide sequence), which can be directly obtained by saving the resulting spreadsheet from step 1.1.4 as a simple text document (without the extra column(s) for allele names, aa positions&#160;etc.).</li>
	<li>Run automated peptide array synthesis via the SPOT synthesizer. The entire operation of the synthesis is detailed in Kudithipudi et al. Note that Fmoc synthesis of two arrays takes ~4-5 days.</li>
</ol>
2. Probe and Reprobe Antisera from a Time Series of an Individual Transplant Recipient.
NOTE: The 600-spot membrane array has the dimensions ~7 cm x 13 cm. After synthesis, the arrays can be stored at room temperature as dry membranes for at least two years when shielded from direct light. Avoid excessive folding of the membrane to preserve its longevity for repeated use.
<ol>
	<li>Rehydrate the membrane array with ethanol and visualize peptide spots stained with Ponceau S.
	<ol>
		<li>Rehydrate the membrane array carrying the peptides following a stepwise procedure optimized in Li et al.
		<ol>
			<li>Immerse the array membrane in 20 mL of 100% ethanol.</li>
			<li>Add 20 mL distilled water to dilute the solution to 50% ethanol and incubate at room temperature for 15 min.</li>
			<li>Change the immersion solution to 40 mL of 100% water three times and incubate 15 min each time.</li>
			<li>Wash in a proper working buffer, e.g., 20 mL of TBST (Tris-buffered saline with 0.1%), three times for 5 min each.</li>
		</ol>
		</li>
		<li>Ponceau S staining of the array to visualize synthetic peptides
		<ol>
			<li>Add 20 mL of pre-formulated Ponceau S solution directly to the hydrated array and incubate for ~30 s with shaking.</li>
			<li>Run distilled water continuously over the membrane to de-stain the background Ponceau S color. During the process, peptide spots of red color may become visible.</li>
			<li>Separate the portions of the array for each donor by carefully cutting between the sections of the array for individual cases. Mark the orientation of each array.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Pre-block array.
	<ol>
		<li>Block the membrane in 20 mL of 5% non-fat milk dissolved in TBST buffer. This milk-based solution will further de-stain the Ponceau S color. Replace the milk buffer several times in order to achieve the clearest peptide images.</li>
		<li>For record-keeping purposes, take a photo of the Ponceau S image of the array using a hand-held camera (example in Figure 1).</li>
		<li>Continue blocking of the membrane in 5% non-fat milk at 4 &#176;C overnight or at room temperature for 2 h with rocking.</li>
	</ol>
	</li>
	<li>Incubate array with transplant antiserum. 
	NOTE: It should be noted that a phenomenon known as prozone or the Hook effect may result from complement-dependent interference in serum antibody analysis. To circumvent this problem, an optional serum pretreatment step can be considered with either ethylenediaminetetraacetic acid (EDTA) or heat inactivation of serum samples.
	<ol>
		<li>Remove the blocking buffer and wash the membrane with 20 mL of TBST three times the next day for 5 min each time.</li>
		<li>Add 20 &#181;L of the crude serum of the recipient to 20 mL of 2.5% milk in TBST buffer and incubate with the membrane for 2-3 h at room temperature. Note that in this initial round of probing, it is recommended that the last post-transplant serum (or likely most sensitized serum) in the time series is first used. This is based on the assumption that earlier specimens in the series, particularly from pre-transplant time points, have less variety of alloantibodies that tend to develop over time. This way, any possibility of signal interference from &#34;carry-over&#34; between probing rounds can be clearly distinguished from truly developed alloantibody reactivity due to immune responses against the graft.</li>
	</ol>
	</li>
	<li>Wash the array using 20 mL of TBST and incubate it with a secondary antibody.
	<ol>
		<li>Wash the membrane three times for 10 min each time.</li>
		<li>Incubate with goat anti-human IgG-HRP (Horseradish peroxidase) secondary antibody at 1:10,000 dilution in TBST buffer supplemented with 1% milk for another 2 h.</li>
	</ol>
	</li>
	<li>Wash and develop the blot.
	<ol>
		<li>Wash the membrane three times with TBST for 10 min each time.</li>
		<li>Perform enhanced chemiluminescence (ECL) using 5 mL of luminol solution freshly mixed with 5 mL of peroxide solution to develop the membrane (for 1 min).</li>
		<li>Visualize ECL signals using a suitable imager (i.e., ChemiDoc Imaging Systems or Azure C600).</li>
	</ol>
	</li>
	<li>Scan and quantify the blot.
	<ol>
		<li>Save the developed images (Figure 2, lower image) and perform quantification of spot intensity.</li>
	</ol>
	</li>
</ol>
3. Compare Antiserum Reactivity across a Clinical Time Series.
<ol>
	<li>Strip the array.
	<ol>
		<li>At this point, keep the membrane wet at all times.</li>
		<li>Strip the membrane by incubating with 20 mL of commercial stripping buffer at 37 &#176;C for 20 min, and then wash the membrane with TBST three times for 10 min each. Then repeat the blocking (step 3.2) and probing (step 3.3) steps using a different serum of the patient taken at a different time point.</li>
	</ol>
	</li>
	<li>Block stripped membrane. 
	Note: Following stripping, the membrane can be reused for another round of probing of a different serum from the same patient in a time series.
	<ol>
		<li>Block the membrane using 5% milk buffer as before (see step 2.2).</li>
	</ol>
	</li>
	<li>Reprobe a different antiserum from the same time series (repeat steps from 2.3-2.6; example in Figure 2, upper image). 
	NOTE: The stripped array can then be used to reprobe another serum specimen. Since the peptides are covalently conjugated to the supporting matrix of the membrane, we showed that the array can be reused for up to 20 rounds of stripping and reprobing cycles without losing its performance.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peptide array</td>
			<td>INTAVIS Bioanalytical Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S solution</td>
			<td>Sigma-Aldrich</td>
			<td>P7170</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat milk</td>
			<td>Bio Rad Laboratories</td>
			<td>1706404</td>
			<td></td>
		</tr>
		<tr>
			<td>TBST</td>
			<td>Santa Cruz Biotechnology</td>
			<td>10711454001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgG&#8211;HRP</td>
			<td>ThermoFisher Scientific</td>
			<td>A18811</td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Substrate</td>
			<td>Bio Rad Laboratories</td>
			<td>1705061</td>
			<td></td>
		</tr>
		<tr>
			<td>Restore Western Blot Stripping Buffer</td>
			<td>Thermo Scientifics</td>
			<td>21059</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc gel imaging system</td>
			<td>Bio Rad Laboratories</td>
			<td>1708265</td>
			<td></td>
		</tr>
	</tbody>
</table>

a peptide array-based detection of anti-donor hla alloantibodies in organ recipients

Source: Liu, P., et al. <a href="https://app.jove.com/v/56278">Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation.</a> J. Vis. Exp. (2017).
This video demonstrates an assay to detect anti-donor HLA alloantibodies in organ recipients. A peptide array is synthesized to represent potential epitopes for antibody-mediated organ rejection. The array is incubated with the recipient&#39;s serum, containing alloantibodies that bind to their target epitopes. Antibody binding is detected using chemiluminescence to identify donor-specific HLA epitopes that incite alloantibody reactivity.

<img alt="Figure 1" src="/files/ftp_upload/22113/22113_Figure1.jpg" /> 
Figure 1. Image of an array section stained with Ponceau S
Note disparity in color intensity is due to differences in amino acid composition among peptides, while peptide concentrations among all spot areas are the same. (The image is an illustrative example, not the one from this actual study.)
<img alt="Fig...

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Design of Custom Array Layout and ...

Take a peptide array derived from an organ donor&#39;s human leukocyte antigen or HLA sequence comprising different peptides on cellulose membrane.
Each peptide carries at least one amino acid mismatch between the donor and recipient HLA sequence, representing a potential epitope for antibody-mediated organ rejection.
Treat with a blocking buffer to prevent non-specific antibody interaction.
Add recipient serum collected post-organ transplantation, containing alloantibodies.
These antibodies, formed against the HLA of the donor, bind to their target epitope.
Add a peroxidase-conjugated secondary antibody that binds to the alloantibody.
Add chemiluminescent substrate and hydrogen peroxide. The peroxidase utilizes hydrogen peroxide to oxidize the substrate, emitting light.
Scan the membrane to obtain an image.
Add a buffer to strip the bound antibodies. Reprobe with pre-transplantation recipient serum and obtain an image.
Compared to the pre-transplantation image, dark spots on the post-transplantation image indicate donor-specific HLA epitopes that incite alloantibody reactivity.

a-peptide-array-based-detection-anti-donor-hla-alloantibodies-organ

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

40 ビュー. 出典: Liu, P., et al. Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation. J. Vis. Exp. (2017年)。 このビデオは、臓器レシピエントの抗ドナーHLA同種抗体を検出するアッセイを示しています。ペプチドアレイは、抗体を介した臓器拒絶反応の潜在的なエピトープを表すために合成されます。アレイは、標的エピトープに結合する同種抗体を含むレシピエントの血清?...

ビデオ: 臓器レシピエントにおける抗ドナーHLA同種抗体のペプチドアレイベースの検出 - 概念

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

ペプチドマイクロアレイによるヒストン抗体特異性の解析

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

JoVE Journal

生化学

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

24時間臓器保存と連続テレメトリを用いた豚モデルにおける異形性腎臓自動移植

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

医学

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

アレルギー診断のための好塩基球活性化試験

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

記録

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients