This work was supported by the Chang Gung Medical Foundation (grant number CMRPG3C1771, CMRPG3C1772, CMRPG3E0631, CMRPG3E0632, and CMRPG3E0633).

The study was approved by the Institutional Review Board of Chang Gung Memorial Hospital at Linkuo, Taoyuan, Taiwan (102-2577A3).
1. Preparation of the NR1-GFP Expression Plasmid
<ol>
	<li>Mix 10 ng of NR1-GFP plasmid with 100 &#181;L of Escherichia coli competent cells strain DH5&#945; in a sterile 1.5 mL centrifuge tube, pipet the mixture gently up and down 4-6 times, and incubate the tube on ice for 20 min.</li>
	<li>Incubate the tube at 42 &#176;C for 1 min in a water bath, remove the tube from the water bath, add 1 mL lysogeny broth (LB) medium into the tube, and incubate the tube at 37 &#176;C in an incubator with shaking for 1 h.</li>
	<li>Spread 50 &#181;L of the mixture onto an LB agar plate containing ampicillin (0.1 mg/mL), and incubate the LB agar plate at 37 &#176;C in an incubator overnight.</li>
	<li>Pick a single colony from the overnight LB agar plate using a sterile tip, and swirl the tip in a in a bacterial culture tube containing 5 mL of LB medium and ampicillin (0.1 mg/mL). Incubate the tube at 37 &#176;C in an incubator with shaking overnight.</li>
	<li>Aliquot 3 mL of the overnight bacterial culture into a 500 mL flask that contains 250 mL sterile LB medium and ampicillin (0.1 mg/mL). Incubate the flask at 37 &#176;C with shaking. Check regularly the optic density (OD) of the bacterial culture at wavelength of 600 nm using a spectrometer until the OD600 reaches 0.6-1.0. 
	NOTE: Mix well 800 &#181;L of the overnight bacterial culture with 200 &#181;L glycerol to prepare the glycerol stock, and store the glycerol stock under -80 &#176;C for future use.</li>
	<li>Prepare NR1-GFP expression plasmid from the 250 mL bacterial culture
	<ol>
		<li>Collect the cells by centrifugation at 6,000 x g for 15 min at 4 &#176;C.</li>
		<li>Resuspend the bacteria pellet in 10 mL resuspension buffer containing RNase A; vortex thoroughly until no clump is seen.</li>
		<li>Add 10 mL lysis buffer to the bacterial suspension, gently invert the mixture 4-6 times, and incubate the lysate at room temperature (RT) for 5 min.</li>
		<li>Add 10 mL pre-chilled precipitation buffer to the lysate, gently invert the mixture 4-6 times.</li>
		<li>Pour the lysate into the barrel of a filter cartridge with the cap on; wait for 10 min at RT.</li>
		<li>Remove the cap from the cartridge outlet nozzle, insert a plunger into the cartridge, and gently press the plunger. Collect the filtered lysate into a 50 mL tube.</li>
		<li>Add 2.5 mL proprietary buffer from the manufacturer to the filtered lysate, mix the mixture by inverting the tube approximately 10 times, and incubate the mixture on ice for 30 min.</li>
		<li>Apply the filtered lysate mixture to a filter column that was pre-equilibrated with 10 mL equilibration buffer. Allow the column to drain by gravity.</li>
		<li>Wash the column with 30 mL wash buffer twice, then elute DNA from the column using 15 mL elution buffer.</li>
		<li>Add 10.5 mL (0.7 volumes) isopropanol to the eluted DNA buffer, mix well, and centrifuge the mixture at 20,000 x g for 30 min at 4 &#176;C.</li>
		<li>Decant the supernatant carefully, wash the DNA pellet with 5 mL endotoxin-free 70% ethanol once, and centrifuge at 20,000 x g for 10 min.</li>
		<li>Decant the supernatant, dry the pellet for 5-10 min in a chemical hood, and dissolve the pellet in 300-500 &#181;L of endotoxin-free buffer.</li>
		<li>Determine the plasmid concentration by measuring the absorption of the solution at UV 260/280 nm using a spectrophotometer. 
		NOTE: Prepare the expression plasmid GFP using the same protocol.</li>
	</ol>
	</li>
</ol>
2. Transfection of HEK293 Cells with NR1-GFP Expression Plasmid
<ol>
	<li>Aliquot 200 &#181;L of 2% gelatin solution to each well of a 48-well culture plate and incubate the plate at 37 &#176;C for at least 30 min.</li>
	<li>Aspirate the gelatin solution from the well, and seed 5 x 104 HEK293 cells in 200 &#181;L of cell culture medium containing 10% fetal bovine serum (FBS) in each well. Incubate the plate at 37 &#176;C in a humidified incubator supplied with 5% CO2 overnight. 
	NOTE: Cells were counted using a hemocytometer.</li>
	<li>On the next day, replace the spent culture medium with 200 &#181;L of fresh culture medium containing 10% FBS per well.</li>
	<li>For each single well, prepare solution A by mixing 100 ng of NR1-tGFP plasmid with 20 &#181;L culture medium, and solution B by mixing 0.8 &#181;L transfection reagent with 20 &#181;L culture medium. Multiply the number of wells needed to prepare the total volume for each experiment.</li>
	<li>Prepare solution C by mixing solution A and solution B, and incubate the mixture at RT for 20-30 min.</li>
	<li>Aliquot 40 &#181;L of solution C to each well containing the HEK293 cells, swirl the plate gently, and incubate the plate at 37 &#176;C in a humidified incubator supplied with 5% CO2 overnight.</li>
	<li>Check the expression of NR1-GFP recombinant protein in the host cells under fluorescent microscope with 40X-200X magnification (excitation/emission: 482/502 nm), and proceed to the cell-based immunofluorescence assay. 
	NOTE: Transfect the expression plasmid GFP into the HEK293 cells using the same protocol.</li>
</ol>
3. Cell-based Immunofluorescence Assay
<ol>
	<li>Aspirate the spent medium from the wells, then wash each well with 200 &#181;L of phosphate buffered saline (PBS) three times.</li>
	<li>Add 200 &#181;L of 4% paraformaldehyde solution to each well; incubate the plate at RT for 15 min.</li>
	<li>Aspirate the paraformaldehyde solution from the wells, and wash each well with 200 &#181;L of PBS three times.</li>
	<li>Add 200 &#181;L of 10% skim milk in PBST (0.1% Tween-20 in PBS) solution to each well, and incubate the plate at RT for 1 h with gentle shaking.</li>
	<li>Aspirate the 10% skim milk in PBST solution from the well, then wash the wells with 200 &#181;L PBST once.</li>
	<li>Incubate the wells with diluted plasma (1:100 in PBST) as the primary antibody with gentle shaking at RT for 1 h. 
	NOTE: Plasma is obtained from the blood of patients suspected to have autoimmune encephalitis.</li>
	<li>Aspirate the diluted plasma from the wells, and wash each well with 200 &#181;L of PBST three times.</li>
	<li>Add 200 &#181;L of goat anti-human IgG conjugated with Alexa Fluor 594 (1:1,000 dilution in PBST) to each well, and incubate the culture plate at RT with gentle shaking for 1 h.</li>
	<li>Aspirate the goat anti-human IgG conjugated with Alexa Fluor 594 solution, and wash each well with 200 &#181;L of PBST three times.</li>
	<li>Add 100 &#181;L glycerol solution (50% glycerol in PBS and 1:100,000 of 4&#39;,6-diamidino-2-phenylindole (DAPI)) to each well.</li>
	<li>Observe and image the cells under a fluorescent microscope using a 10X eyepiece, and 4X, 10X, and 20X objectives. 
	NOTE: Use the correct filter for each dye: for NR1-GFP (excitation/emission = 482/502 nm), for Alexa Fluor 594 (excitation/emission = 561/594 nm), for DAPI (excitation/emission = 358/461 nm). Conduct the negative control using the HEK cells expressing GFP in the same way.</li>
</ol>

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R., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+surface+antibody-mediated+autoimmune+encephalitis.">Neuronal surface antibody-mediated autoimmune encephalitis.</a> Semin Neurol. 34 (4), 458-466 (2014).</li><li>Lancaster, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnosis+and+treatment+of+autoimmune+Encephalitis.">The diagnosis and treatment of autoimmune Encephalitis.</a> J Clin Neurol. 12 (1), 1-13 (2016).</li><li>Pruss, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+analysis+of+NMDA+receptor+antibodies+in+encephalitis+of+unknown+origin.">Retrospective analysis of NMDA receptor antibodies in encephalitis of unknown origin.</a> Neurology. 75 (19), 1735-1739 (2010).</li><li>Leypoldt, F., Armangue, T., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+encephalopathies.">Autoimmune encephalopathies.</a> Ann N Y Acad Sci. 1338, 94-114 (2015).</li><li>Dalmau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA-receptor+encephalitis:+case+series+and+analysis+of+the+effects+of+antibodies.">Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.</a> Lancet Neurol. 7 (12), 1091-1098 (2008).</li><li>Titulaer, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+and+prognostic+factors+for+long-term+outcome+in+patients+with+anti-NMDA+receptor+encephalitis:+an+observational+cohort+study.">Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study.</a> Lancet Neurol. 12 (2), 157-165 (2013).</li><li>Chapman, M. R., Vause, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA+receptor+encephalitis:+diagnosis,+psychiatric+presentation,+and+treatment.">Anti-NMDA receptor encephalitis: diagnosis, psychiatric presentation, and treatment.</a> Am J Psychiatry. 168 (3), 245-251 (2011).</li><li>Gable, M., Glaser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-n-methyl-d-aspartate+receptor+encephalitis+appearing+as+a+new-onset+psychosis:+disease+course+in+children+and+adolescents+within+the+california+encephalitis+project.">Anti-n-methyl-d-aspartate receptor encephalitis appearing as a new-onset psychosis: disease course in children and adolescents within the california encephalitis project.</a> Pediatr Neurol. 72, 25-30 (2017).</li><li>Dalmau, J., Lancaster, E., Martinez-Hernandez, E., Rosenfeld, M. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-methyl-D-aspartate+antibody+encephalitis:+temporal+progression+of+clinical+and+paraclinical+observations+in+a+predominantly+non-paraneoplastic+disorder+of+both+sexes.">N-methyl-D-aspartate antibody encephalitis: temporal progression of clinical and paraclinical observations in a predominantly non-paraneoplastic disorder of both sexes.</a> Brain. 133 (Pt 6), 1655-1667 (2010).</li><li>Suh-Lailam, B. B., Haven, T. R., Copple, S. S., Knapp, D., Jaskowski, T. D., Tebo, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA-receptor+antibody+encephalitis:+performance+evaluation+and+laboratory+experience+with+the+anti-NMDA-receptor+IgG+assay.">Anti-NMDA-receptor antibody encephalitis: performance evaluation and laboratory experience with the anti-NMDA-receptor IgG assay.</a> Clin Chim Acta. 421, 1-6 (2013).</li><li>Mayer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+of+glutamate+receptor+ion+channel+complexes.">Structural biology of glutamate receptor ion channel complexes.</a> Curr Opin Struct Biol. 41, 119-127 (2016).</li><li>Ramberger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+diagnostic+accuracy+of+microscopy+and+flow+cytometry+in+evaluating+n-methyl-d-aspartate+receptor+antibodies+in+serum+using+a+live+cell-based+assay.">Comparison of diagnostic accuracy of microscopy and flow cytometry in evaluating n-methyl-d-aspartate receptor antibodies in serum using a live cell-based assay.</a> PLoS One. 10 (3), e0122037 (2015).</li><li>Wandinger, K. P., Saschenbrecker, S., Stoecker, W., Dalmau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-NMDA-receptor+encephalitis:+a+severe,+multistage,+treatable+disorder+presenting+with+psychosis.">Anti-NMDA-receptor encephalitis: a severe, multistage, treatable disorder presenting with psychosis.</a> J Neuroimmunol. 231 (1-2), 86-91 (2011).</li><li>Dahm, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seroprevalence+of+autoantibodies+against+brain+antigens+in+health+and+disease.">Seroprevalence of autoantibodies against brain antigens in health and disease.</a> Ann Neurol. 76 (1), 82-94 (2014).</li><li>J&#233;z&#233;quel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-+and+single+molecule-based+methods+to+detect+anti-n-methyl-d-aspartate+receptor+autoantibodies+in+patients+with+first-episode+psychosis+from+the+OPTiMiSE+project.">Cell- and single molecule-based methods to detect anti-n-methyl-d-aspartate receptor autoantibodies in patients with first-episode psychosis from the OPTiMiSE project.</a> Biol Psychiatry. , (2017).</li></ol>

The authors have nothing to disclose.

There are several cell-based immunological assays to screen the presence of autoantibodies against NMDA receptor reported in the literature, including live cell-based immunofluorescence assay11, fixed cell-based immunofluorescence assay9, and flow cytometry-based assay14. The live cell-based immunofluorescence assay should be performed shortly after the preparation of cells ectopically expressing the NR1 protein, while the flow cytometry-based assay ...

Anti-NMDA receptor autoimmune encephalitis is a newly recognized disease entity that can occur in patients of all ages, and affects predominantly female patients1,2. It is one of the most frequently diagnosed encephalitis among patients with initial unknown etiology of encephalitis3. Patients affected with anti-NMDA receptor encephalitis usually have prodromal symptoms of a headache or fever, followed by the quick development of consciousness level change and a variety of acute neuropsychiatric symptoms, including agitation, irritability, anxiety, insomnia, hallucinations, delusions, aggression, bizarre behaviors, movement abnormalities, autonomic dysregulation, and seizure attacks4,5. Early recognition of this condition and timely treatment with immunotherapy are important for a better outcome and even full recovery in affected patients6. Hence, it is suggested that anti-NMDA receptor autoimmune encephalitis should be considered as an important differential diagnosis of patients presenting with acute or new-onset psychotic features7,8.
Besides clinical features, detection of autoantibody against the NMDA receptor in the blood or CSF is essential for the accurate diagnosis of anti-NMDA receptor autoimmune encephalitis9. Most of the immunological tests to detect the anti-NMDA receptor autoantibody are available in a few research laboratories10,11, and there is only one commercially available cell-based immunofluorescence assay for screening the anti-NMDA receptor autoantibodies12. The goal of this study is to develop a simple in-house cell-based immunofluorescence assay that can be conveniently used in the laboratory to screen the presence of anti-NMDA receptor autoantibodies to facilitate the clinical research of anti-NMDA receptor autoimmune encephalitis. The NMDA receptor is a heterotetramer ion channel protein complex specifically expressed in the brain. It is made of two compulsory NR1 subunits, and the combination of one or two subunits of NR2A, NR2B, NR2C, or NR2D13. A previous study reported that the main epitope targeted by the antibodies was at the extracellular N-terminal domain of the NR1 subunit5. Hence, in this protocol, we express the human recombinant NR1-subunit protein of NMDA receptor tagged with green fluorescent protein (GFP) in the human embryonic kidney epithelial cell line (HEK293), and develop a cell-based immunofluorescence assay to detect the IgG class of anti-NMDA receptor autoantibodies in the blood.

<table><tbody><tr><td>GRIN1 (GFP-tagged) - Human glutamate receptor, ionotropic, N-methyl D-aspartate 1 (GRIN1), transcript variant NR1-1</td><td>Origene (Rockville, MD, USA)</td><td>RG219368</td><td>NR1-cDNA clone tagged with C-terminal tGFP sequences</td></tr><tr><td>pCMV6-AC-GFP</td><td>Origene (Rockville, MD, USA)</td><td>PS100010</td><td>mammalian vector with C-terminal tGFP tag</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen (Hilden Germany)</td><td>12362</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Invitrogen (Carlsbad, CA, USA)</td><td>11668-019</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher</td><td>31985-070</td><td></td></tr><tr><td>DMEM, High Glucose, Pyruvate</td><td>Thermo Fisher</td><td>11995-065</td><td></td></tr><tr><td>Characterized Fetal Bovine Serum, US Origin</td><td>GE Healthcare Life Sciences</td><td>SH30071.01</td><td></td></tr><tr><td>Goat anti-Human IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td><td>Thermo Fisher</td><td>A-11014</td><td></td></tr><tr><td>Positive control: anti-glutamate receptor (type NMDA)</td><td>Euroimmun AG, L&amp;uuml;beck, Germany</td><td>CA 112d-0101</td><td></td></tr><tr><td>Euroimmun Assay Kit</td><td></td><td></td><td></td></tr></tbody></table>

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.

On average, we could obtain 200-300 &#181;g endotoxin-free expression plasmid NR1-GFP and GFP from 250 mL bacterial culture following the procedures described in section 1 of the protocol. An amount of 100 ng/well of the expression plasmid was used for the transfection of the HEK293 cells cultured in the 48-well plate as described in the section 2 of the protocol. 24-30 h after transfection, the cells expressed the NR1-GFP, and GFP recombinant proteins could be detected under fluorescent ...

Watch this Scientific Journal Video about A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood at JoVE.com

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.

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 ...

a-simple-cell-based-immunofluorescence-assay-to-detect-autoantibody

Research

JoVE Journal

Neuroscience

9.8K Views.  Chang Gung Memorial Hospital-Linkou. Wij hebben ectopically NR1 subeenheid van NMDA receptor gelabeld met groen fluorescent proteïne in menselijke embryonale cellen (HEK293) als antigeen te detecteren autoantilichamen tegen NMDA receptor in het bloed van patiënten verdacht met auto-immune encefalitis. Deze eenvoudige methode kan geschikt voor screeningonderzoek in klinische instellingen zijn.

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

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Immunology and Infection

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Medicine

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Anti-Nuclear Antibody Screening Using HEp-2 Cells

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although IIF is an excellent screening test in expert hands, the technical difficulties of processing and reading IIF slides &#8211; such as the labor intensive slide processing, manual reading, the need for experienced, trained technologists and the use of dark room &#8211; make the IIF method difficult to fit in the workflow of modern, automated laboratories.
The first and crucial step towards high quality ANA screening is careful slide processing. This procedure is labor intensive, and requires full understanding of the process, as well as attention to details and experience.
Slide reading is performed by fluorescent microscopy in dark rooms, and is done by trained technologists who are familiar with the various patterns, in the context of cell cycle and the morphology of interphase and dividing cells. Provided that IIF is the first line screening tool for SARD, understanding the steps to correctly perform this technique is critical.
Recently, digital imaging systems have been developed for the automated reading of IIF slides. These systems, such as the NOVA View Automated Fluorescent Microscope, are designed to streamline the routine IIF workflow. NOVA View acquires and stores high resolution digital images of the wells, thereby separating image acquisition from interpretation; images are viewed an interpreted on high resolution computer monitors. It stores images for future reference and supports the operator&#8217;s interpretation by providing fluorescent light intensity data on the images. It also preliminarily categorizes results as positive or negative, and provides pattern recognition for positive samples. In summary, it eliminates the need for darkroom, and automates and streamlines the IIF reading/interpretation workflow. Most importantly, it increases consistency between readers and readings. Moreover, with the use of barcoded slides, transcription errors are eliminated by providing sample traceability and positive patient identification. This results in increased patient data integrity and safety.
The overall goal of this video is to demonstrate the IIF procedure, including slide processing, identification of common IIF patterns, and the introduction of new advancements to simplify and harmonize this technique.

Indirect immunofluorescent (IIF) assays have traditionally been used for the detection of antinuclear antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of systemic autoimmune rheumatic diseases (SARD). This protocol demonstrates how to effectively perform the IIF technique to accurately detect these autoantibodies.

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

Biology

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis

Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as &#34;autoimmune encephalitis&#34; (AE). There are currently 17 known AE syndromes, and all are associated with antibodies against the neuronal cell surface or synaptic proteins. The clinical syndromes are complex and vary according to the type of associated antibody. The best-known of these diseases is anti-N-methyl D-aspartate receptor (NMDAR) encephalitis, which is a prominent neuropsychiatric disorder associated with severe memory and behavioral impairments. The associated antibodies react with the GluN1 subunit of the NMDAR at the N-terminal domain. The approach most frequently used for the discovery and characterization of AE antibodies includes the culture of dissociated, fetal, rodent hippocampal neurons. During the process of antibody characterization, live neurons in culture are exposed to patients&#39; serum or CSF, and the detection of reactivity indicates that the serum or CSF samples of the patient contain antibodies against neuronal surface antigens. Hippocampal cultures can also be used to determine whether the antibodies in patients are potentially pathogenic by examining if they cause structural or functional alterations of the neurons. The level of success of these studies depends on the quality of the cultures and on the protocols used to obtain and detect the reactivity of patient samples. This article provides an optimized protocol for primary cell culture of fetal rat hippocampal neurons combined with immunostaining to determine the presence of antibodies in the serum or CSF of patients. An example of how to examine the potential pathogenic effects of NMDAR antibodies using cultured neurons and calcium imaging is also presented.

Autoimmune encephalitis is a new category of antibody-mediated diseases of the central nervous system. Hippocampal neurons can be used to discover and characterize these antibodies. This article provides a protocol for primary cell culture and immunostaining to determine autoantibodies in the serum and cerebrospinal fluid of patients.

Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as ...

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

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