an assay to detect autoantibodies in human serum using a hippocampal neuronal culture

An Assay to Detect Autoantibodies in Human Serum using a Hippocampal Neuronal Culture

For the enzymatic dissociation of the hippocampus at 1 milliliter of 2.5% trypsin in the 50-milliliter tube containing the hippocampi, after bringing the volume in the tube to 5 milliliters with HBSS, incubate the tube for 15 minutes in the water bath at 37 degrees Celsius. To further dilute the trypsin, add 10 milliliters of pre-heated HBSS, and place the tube back in the water bath set at 37 degrees Celsius for 5 minutes.
Next, use a 1,000-microliter micropipette to transfer the hippocampi appearing as a mucous mass to a 50-milliliter tube, and incubate the tissue with 6 milliliters of pre-heated HBSS for 5 minutes, as demonstrated. To dissociate the tissue mechanically, transfer the hippocampal mass to a 2-milliliter tube with conical bottom. After adding 1 milliliter of pre-heated DMEM media, homogenize the pellet with a 1,000-microliter micropipette by gently aspirating up and down.
Ensuring not to create bubbles, repeat the up-and-down aspiration 10 to 20 times with a pre-pulled glass pipette, with its tip in contact with the conical bottom of the tube, until the mixture is translucent. Count the cells before distributing them evenly on a 3.5-centimeter dish with crossed-shaking movements. Once done, place the dish in the incubator with 5% carbon dioxide.
For fluorescent immunostaining of 14 days in-vitro cells, add the sample containing anti-NMDAR antibodies to the coverslips, and incubate at 37 degrees Celsius and 5% carbon dioxide. After one hour, treat the sample with 4% formaldehyde in PBS, as a fixation solution, at room temperature for 5 minutes. Then wash the sample thrice with PBS for 5 minutes each, followed by addition of secondary antibody goat anti-human Alexa Fluor 488 at 1 to 1,000 dilution for 1 hour at room temperature. Next, mount the coverslips containing the sample with a liquid-mounting medium, and aspirate any remaining liquid.

an-assay-to-detect-autoantibodies-human-serum-using-hippocampal

Nanyang Technological University

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JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
PART 1: Establishment of dissociated, fetal, rodent hippocampal neuronal cultures
1. Preparative steps
<ol>
	<li>Poly-L-Lysine (PLL) coating of the plating surfaces (3 days before dissection)
	<ol>
		<li>Prepare the borate buffer by adding 2.38 g of boric acid and 1.27 g of borax to 500 mL of distilled water and stirring for 15 min until dissolved. Under a hood, sterilize the buffer solution by filtering (0.2 &#181;m pore size), label, and store at room temperature (RT). 
		NOTE: This solution is stable and can be used for 6 months. 
		&#8203;CAUTION: When preparing borate buffer, wear recommended personal protective equipment.</li>
		<li>Prepare the PLL stock solution (100 mg/mL) by adding 1 g of PLL to 10 mL of distilled water and stirring until dissolved. Prepare 500 &#181;L aliquots of PLL stock solution. To reach a final concentration of 1 mg/mL, add 500 &#181;L of PLL aliquot to 50 mL of borate buffer and swirl until dissolved, and sterilize the solution by filtering (0.2 &#181;m pore size). 
		NOTE: This solution is stable and can be used for 6 months.</li>
		<li>Prepare the surfaces for coating. Use 12 mm diameter coverslips for immunostaining procedures and glass-bottom dishes for studies that will include the imaging of live neurons. If using coverslips, autoclave and then place five coverslips in each dish (3.5 cm diameter). 
		NOTE: The thickness of the coverslip affects the quality and intensity of the signal of the images acquired. Most microscope objectives are designed for #1.5 coverslips. Choose the appropriate coverslips according to the imaging setup and techniques.</li>
		<li>Under a hood, add 1.5 mL of PLL solution to each dish (glass-bottom dish or 3.5 cm dish with five coverslips). Make sure all the coverslips are submerged and stored at RT for 24 h.</li>
		<li>2 days before dissection, aspirate the PLL solution and wash with sterile endotoxin-free water, making sure the coverslips are submerged. Keep water in the dishes and store at RT for 24 h.</li>
		<li>1 day before dissection, aspirate the water and replace it with NB + B27 culture media (see below), and place the dishes in an incubator at 37 &#176;C for 24 h.</li>
	</ol>
	</li>
	<li>Preparation of culture media and stock solution (1 day before dissection)
	<ol>
		<li>Dulbecco&#39;s Modified Eagle Medium (DMEM): To 500 mL of DMEM High Glucose (4.5 g/L) without L-Glutamine and phenol red, add 50 mL of horse serum, 50 mL of fetal bovine serum (FBS), 10 mL of L-glutamine (200 mM), 10 mL of sodium pyruvate (100 mM), 10 mL of penicillin-streptomycin (10,000 U/mL). Filter (0.2 &#181;m pore size) and store in 5 mL aliquots at 4 &#7506;C with a tightly closed cap. 
		NOTE: This solution is stable and can be used for 1 month.</li>
		<li>Neurobasal (NB) medium supplemented with B27 (NB + B27): To 50 mL of NB medium without phenol red, add 1 mL of B27 supplement.</li>
		<li>Hibernate-E medium supplemented with B27 (Hibernate + B27): To 50 mL of Hibernate-E medium, add 1 mL of B27 supplement.</li>
		<li>Extracellular Physiological Solution (EPS): To 1 L of sterile water, add the following at the specified final concentrations: NaCl (140 mM), KCl (3.5 mM), N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid) (HEPES, 10 mM), glucose (20 mM), and CaCl2 (2 mM). Stir until dissolved and adjust the pH to 7.4 by adding NaOH (0.5 M) or HCl (0.5 M). Under the hood, sterilize by filtering (0.2 &#181;m pore size) and store at 4&#176;C.</li>
	</ol>
	</li>
	<li>Equipment and other laboratory materials (day of dissection)
	<ol>
		<li>Set a water bath to 37 &#176;C. Heat the following aliquots: 50 mL of Hanks&#39; Balanced Salt Solution (HBSS), and 5 mL of DMEM.</li>
		<li>Sterilize the following tools by immersing in a beaker containing absolute ethanol (Figure 1B): forceps, curved forceps, scissors, fine-curved forceps, fine-straight forceps, surgery scissors, fine-angled forceps, and precision spring scissors. 
		NOTE: To protect the tools, place a soft material (e.g., science precision wipes) at the bottom of the beaker.</li>
		<li>Fill two trays with ice and place the following items.
		<ol>
			<li>Ice tray 1 (Figure 1C): Place surgical tools in the beaker with absolute ethanol, a beaker with HBSS for rinsing the surgical tools, a 10 cm dish with HBSS to place the embryos, a 6 cm dish with HBSS to place the heads of the embryos, and a 6 cm dish with Hibernate + B27 to place the brains of the embryos.</li>
			<li>Ice tray 2: Place a 1 mL aliquot of trypsin 2.5% and a 3.5 cm dish with Hibernate + B27 for the dissected hippocampus. 
			NOTE: The time needed for this procedure depends on the investigator&#39;s experience and the number of embryos to be dissected. Therefore, the ice must remain frozen for the needed amount of time. Polystyrene trays are recommended for this purpose.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Dissection and seeding (Figure 1)
<ol>
	<li>Hippocampal isolation 
	NOTE: Pregnant rats with embryos at embryonic day 18 (E18) are euthanized immediately before the protocol starts in accordance with the local ethics committee of the University of Barcelona, following European (2010/63/UE) regulations about the use and care of experimental animals. In this protocol, carbon dioxide (CO2) inhalation was used as the method of euthanasia.
	<ol>
		<li>Dissect the rat at the level of the abdominal peritoneum using forceps and scissors. Extract the uterus with the E18 embryos and place it in a 10 cm dish that has been chilled on ice. 
		&#8203;NOTE: It is important to avoid the rat hairs attaching to the embryos. It is recommended to use copious amounts of 70% ethanol to sterilize the abdomen. From this step onward, work inside the hood on ice tray 1.</li>
		<li>Open the embryonic sacs and transfer the embryos to the 10 cm dish with HBSS, making sure the embryos are fully immersed.</li>
		<li>Remove the embryo head with scissors and place it in the 6 cm dish with HBSS. Repeat this process with all the embryos. 
		NOTE: To avoid contamination, all tissue except for the embryo heads should be discarded in a container with a screw cap.</li>
		<li>Hold the embryo head with fine-curved forceps and pierce the orbits with a pair of fine-straight forceps, entering at a 45&#176; angle, and then release the fine-curved forceps. 
		NOTE: The forceps must not go through the brain, so it is important to maintain the angle when entering.</li>
		<li>Dissect the skin and skull with the surgery scissors, starting from the occipital bone to the frontal bone. Remove the brain with a pair of fine-curved forceps and place it in the 6 cm dish with Hibernate + B27. Repeat this process until all the brains are recovered.</li>
		<li>Sagittally separate the telencephalons with the fine-straight forceps. 
		NOTE: From this step onward, the dissection of the hippocampus is carried out under a stereomicroscope. It is recommended to use articulated lamps so that the light illuminates the dissection surface from the sides. It is also recommended to use a black background as this provides more contrast, allowing to better distinguish the hippocampus.</li>
		<li>Prepare a 10 cm dish by placing drops of Hibernate + B27 at 1 cm distances (e.g., in a circle). Place one telencephalon per drop of Hibernate + B27 and visualize through the dissecting scope (1.25x objective magnification is recommended).</li>
		<li>Carefully peel the meninges and remove the thalamus to better visualize the hippocampus.</li>
		<li>Dissect the hippocampus with the precision spring scissors and place it in the 3.5 cm dish with Hibernate + B27 in ice tray 2. Repeat for every telencephalon until all the hippocampi are collected.</li>
		<li>Carefully collect all the hippocampi with a Pasteur pipette and transfer them to a 50 mL tube. 
		NOTE: Take the minimum volume of Hibernate + B27 when collecting the hippocampi so as not to dilute the trypsin.</li>
	</ol>
	</li>
	<li>Cell dissociation
	<ol>
		<li>Enzymatic dissociation of the hippocampus
		<ol>
			<li>In the 50 mL tube containing the hippocampi, add 1 mL of 2.5% trypsin and bring it to a 5 mL volume with HBSS. Incubate for 15 min in the water bath at 37 &#176;C.</li>
			<li>To dilute the trypsin, add 10 mL of pre-heated HBSS and incubate for 5 min in the water bath at 37 &#176;C.</li>
			<li>Transfer the hippocampi that now appear as a mucus mass to a 50 mL tube with a 1,000 &#181;L micropipette, add 6 mL of pre-heated HBSS, and incubate for 5 min in the water bath at 37 &#176;C.</li>
		</ol>
		</li>
		<li>Mechanical dissociation of the hippocampus
		<ol>
			<li>Transfer the mass to a 2 mL tube with conical bottom, taking the minimal volume. Add 1 mL of pre-heated DMEM media. Homogenize the pellet with a 1,000 &#181;L micropipette by gently aspirating up and down. 
			NOTE: It is critical to avoid generating bubbles when aspirating as bubbles may lyse the cells.</li>
			<li>Repeat the up and down aspiration with a pre-pulled glass pipette (10x-20x), with its tip in contact with the conical bottom of the tube. Avoid generating bubbles. At the end of this step, the mixture must be translucent.</li>
			<li>Once homogenously mixed such that the mixture is translucent, transfer the mixture to a tube containing 4 mL of DMEM media at 37 &#176;C and homogenize with a glass pipette by pipetting up and down.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell seeding
	<ol>
		<li>Count the cells. The number of neurons in the solution can be counted according to standard laboratory procedures. 
		NOTE: In imaging experiments for antibody detection and calcium activity, a concentration of 50,000 cells per 3.5 cm dish is optimal.</li>
		<li>Keeping the cells suspended, withdraw the calculated volume, and plate in 3.5 cm dishes containing PLL-coated coverslips or in the PLL-coated glass-bottom dishes.</li>
		<li>Evenly distribute the cells on the dishes by softly shaking in crossed movements (back and forth, then side to side; repeat as needed) and place the dishes in the CO2 (5%) incubator. 
		NOTE: It is important to use crossed movements to avoid the cells settling in the periphery of the dish.</li>
		<li>Every week, add approximately 1 mL of NB + B27, so the culture does not dry. 
		NOTE: After 2 weeks (14 days in vitro [div]), the neurons are mature and express all factors to be used for experimentation. The total average number of neurons obtained using this protocol is approximately 2.5 x 106 neurons coming from an average of 12 E18 embryos per pregnant rat.</li>
	</ol>
	</li>
</ol>
PART 2: Using hippocampal neuronal cultures for antibody detection in neuronal cell surface proteins

NOTE: This part of the protocol demonstrates how hippocampal cultures are used to identify anti-NMDAR antibodies in the serum and/or CSF of patients with anti-NMDAR encephalitis. Fluorescent immunostaining is used to visualize the reactivity in the live neurons, but other visualization methods could also be used. An appropriate control for this experiment would be serum or CSF from a healthy individual. When using human samples, keep in mind that approval from the institutional ethical committee may be required.
3. Live fluorescent immunostaining
<ol>
	<li>Use 14 div cells that have been grown on coverslips (50,000 cells per 3.5 cm dish containing five coverslips).</li>
	<li>Inside the hood, rinse the coverslips with NB pre-heated to 37 &#176;C.</li>
	<li>Add the sample that in this case contains anti-NMDAR antibodies (used as a primary antibody) diluted in NB media at 1:200 for serum or 1:2 for CSF and incubate 1 h at 37 &#176;C (inside the CO2 (5%) incubator).</li>
	<li>Rinse carefully with phosphate-buffered saline (PBS) 3x at RT.</li>
	<li>Add fixation solution (4% formaldehyde in PBS) and incubate at RT for 5 min. 
	CAUTION: When handling 4% formaldehyde, work inside the hood and wear recommended personal protective equipment.</li>
	<li>Wash 3x with PBS for 5 min each.</li>
	<li>Add secondary antibody Goat anti-Human AF488 at 1:1000 dilution and incubate at RT for 1 h. 
	NOTE: During the incubation, protect from light exposure by covering with aluminum foil.</li>
	<li>Wash 3x with PBS for 5 min each. Rinse with distilled water</li>
	<li>Mount the coverslips with a liquid mounting medium (e.g., antifading mounting media with DAPI; approximately 7 &#181;L), aspirate any remaining liquid and rinse with distilled water. The neurons are now ready for fluorescence imaging. 
	CAUTION: When handling mounting medium, wear the recommended personal protective equipment.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Cell culture dish</td>
			<td>Nunc</td>
			<td>12-565-020</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm round coverslips</td>
			<td>Fisher</td>
			<td>NC9708845</td>
			<td></td>
		</tr>
		<tr>
			<td>20x NA 0.75 S Fluor air objective</td>
			<td>Nikon</td>
			<td>CFI Super Fluor 20X</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 cm Cell culture dish</td>
			<td>Nunc</td>
			<td>12-565-90</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm Cell culture dish</td>
			<td>Nunc</td>
			<td>12-565-94</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker 100 mL</td>
			<td>Pirex</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Borax</td>
			<td>Sigma-Aldrich</td>
			<td>B9876</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>B0252</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>FST</td>
			<td>11009-13</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM High Glucose (4.5 g/L), without L-Glutamine, without Phenol Red</td>
			<td>Capricorn</td>
			<td>DMEM-HXRXA</td>
			<td></td>
		</tr>
		<tr>
			<td>Female Wistar rat (18-days pregnant)</td>
			<td>Janvier</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biowest</td>
			<td>S181B-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-angled forceps</td>
			<td>FST</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-curved forceps</td>
			<td>FST</td>
			<td>11272-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-straight forceps</td>
			<td>FST</td>
			<td>11251-23</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC filter cube</td>
			<td>Nikon</td>
			<td>Standard Series</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Human AF488</td>
			<td>Invitrogen</td>
			<td>A11013</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Capricorn</td>
			<td>HBSS-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hibernate-E medium</td>
			<td>Gibco</td>
			<td>A12476-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum (HS)</td>
			<td>Thermofisher</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-NMDAR antibody (CSF)</td>
			<td>Patient Sample</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-NMDAR antibody (Serum)</td>
			<td>Patient Sample</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ/Fiji</td>
			<td>NIH</td>
			<td>v1.50i</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Nikon</td>
			<td>Eclipse TE2000-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine (PLL)</td>
			<td>Peptide international</td>
			<td>OKK-35056</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene ice tray</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision spring-scissors</td>
			<td>FST</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold with DAPI (antifading mounting media)</td>
			<td>Molecular Probes</td>
			<td>P36941</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>14068-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Biowest</td>
			<td>L0642-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery scissors</td>
			<td>FST</td>
			<td>14081-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 2.5%</td>
			<td>Gibco</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, sterile endotoxine free</td>
			<td>Sigma-Aldrich</td>
			<td>W3500</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Cunquero, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63829">Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis.</a> J. Vis. Exp. (2022)
This video demonstrates a technique for identifying antibodies against specific antigens in a sample using a rat hippocampal neuronal culture. By exposing cultured neurons to patient samples containing autoantibodies targeting N-Methyl-D-aspartate receptors (NMDARs), the presence of autoantibodies bound to the neuronal surface is revealed through immunostaining.

<img alt="Figure 1" src="/files/ftp_upload/22096/22096_Figure1.jpg" /> 
Figure 1: Visual protocol for primary cultures of hippocampal neurons. (A) Flowchart showing the three parts of the protocol for preparing dissociated-cell cultures of hippocampal neurons from embryonic rats at E18. The protocol is divided into 1. Hippocampal isolation, 2. Cell dissociation, and 3. Cell seeding. (B) Selection of recommended tools for hippocampal isolation grouped into three categories: (1- Forceps, 2- Curved forceps, 3- Scissors) for embryo collection, (4- Fine-curved forceps, 5- Fine-straight forceps, 6- Surgery scissors) for brain extraction, and (7- Fine-angled forceps, 8- Precision spring-scissors) for hippocampal isolation. (C) Schematic representation of ice tray 1 with plates and media needed for the hippocampus isolation. Embryos and heads are placed in HBSS, whereas brains are placed in Hibernate medium + B27.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
PART 1: Establishment of ...

Take rat embryo hippocampi.
Add trypsin. Incubate to mediate tissue matrix breakdown, loosening hippocampal cells.
Dilute with a buffer. Incubate for continued enzymatic digestion with trypsin.
Next, transfer the tissue mass to a media-containing tube. Pipette repeatedly for mechanical dissociation into single cells.
Seed cells on poly-L-lysine-coated coverslips within culture plates.
Incubate. Hippocampal cells adhere to the coated surfaces. The neurons develop a lamella and extend minor neurites.
Further, the neurites grow with axonal and dendritic processes, forming a neuronal network.
Mature neurons express synaptic excitatory receptors, N-Methyl -D-aspartate receptors, or NMDARs.
Post-incubation, wash the coverslips with media.
Add a diluted autoimmune encephalitis patient serum. The autoantibodies bind to neuronal NMDARs.
Wash with buffer and then fix the cells.
Add fluorophore-labeled secondary antibodies that bind to the NMDAR-bound autoantibodies.
Wash with buffer. Place the coverslip over mounting media containing DAPI to stain nuclei.
Microscopically observe fluorescence signals on neurons, indicating the presence of autoantibodies in the serum.

15 visualizações. Um ensaio para detectar autoanticorpos em soro humano usando uma cultura neuronal do hipocampo

Vídeo: Um ensaio para detectar autoanticorpos em soro humano usando uma cultura neuronal do hipocampo - Experimento

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

Personalizado do Peptide matrizes para a deteção de aloanticorpos HLA no transplante de órgãos

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

JoVE Journal

Bioquímica

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Ensaios de eletroquimioluminescência para humano ilhéu auto-anticorpos

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

Imunologia e Infecção

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune encephalitis. Detection of the specific autoantibody against the NMDA receptor in the blood or cerebrospinal fluid (CSF) is essential for the accurate diagnosis of this condition. The NMDA receptor is an ion channel protein complex that contains four subunits, including two mandatory NMDA receptor subunit 1 (NR1) and one or two NMDA receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA receptor subunit 2C (NR2C), or NMDA receptor subunit 2D (NR2D). The epitope of anti-NMDA receptor autoantibody was reported to be present at the extracellular N-terminal domain of the NR1 subunit of the NMDA receptor. The goal of this study is to develop a simple cell-based immunofluorescence assay that can be used as a screening test to detect the presence of autoantibodies against NR1 subunit of the NMDA receptor in the blood to facilitate the clinical and basic research of anti-NMDA receptor autoimmune encephalitis.

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

We ectopically expressed NR1 subunit of NMDA receptor tagged with green fluorescent protein in human embryonic cells (HEK293) as antigen to detect autoantibodies against NMDA receptor in the blood of patients suspected with autoimmune encephalitis. This simple method may be suitable for screening purposes in clinical settings.

Um ensaio simples baseada em célula imunofluorescência para detectar anticorpos contra o Receptor N-metil-D-aspartato (NMDA) no sangue

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

An Assay to Detect Autoantibodies in Human Serum using a Hippocampal Neuronal Culture

Transcrição

An Assay to Detect Autoantibodies in Human Serum using a Hippocampal Neuronal Culture