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

Chia-Hsiang Chen; Yu-Syuan Chang

doi:10.3791/56676

וזמינותו פשוטה מבוססת-תא Immunofluorescence לאיתור נוגדן עצמי כנגד הקולטן N-מתיל-D-אספרטט (NMDA) בדם

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Neuroscience

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JoVE Journal Neuroscience

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

וזמינותו פשוטה מבוססת-תא Immunofluorescence לאיתור נוגדן עצמי כנגד הקולטן N-מתיל-D-אספרטט (NMDA) בדם

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07:20 min

January 9, 2018

DOI: 10.3791/56676-v

Chia-Hsiang Chen^1,2, Yu-Syuan Chang¹

¹Department of Psychiatry,Chang Gung Memorial Hospital-Linkou, ²Department and Graduate Institute of Biomedical Sciences,Chang Gung University

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Overview

This study investigates the detection of autoantibodies against the NMDA receptor in patients suspected of autoimmune encephalitis using a cell-based assay. Human embryonic kidney cells (HEK293) expressing the NR1 subunit tagged with green fluorescent protein (GFP) serve as the model system. This simple and reliable method provides a potential screening tool for clinical settings.

Key Study Components

Area of Science

Neuroscience
Immunology
Cell biology

Background

Autoimmune encephalitis can present with acute neuro-psychiatric symptoms.
Detection of autoantibodies is crucial for differential diagnosis.
Existing methods may be complicated or lack sensitivity.
This study introduces a straightforward assay for screening autoantibodies.

Purpose of Study

To develop a reliable method for screening NMDA receptor autoantibodies.
To assist in the diagnosis of autoimmune encephalitis.
To evaluate the feasibility of this approach in clinical practice.

Methods Used

This study employs a cell culture platform using HEK293 cells.
HEK293 cells are transfected with the NR1-GFP plasmid to express the NMDA receptor.
The assay involves several incubation and washing steps, along with fluorescence microscopy for detection.
Important steps include preparing gelatin-coated culture plates and using primary and secondary antibodies for detection.
The entire procedure can be completed in approximately four hours.

Main Results

Detection of NR1-GFP expression in HEK293 cells, showing around 30% expression.
Positive detection of autoantibodies indicated by colocalization of NR1-GFP and anti-NMDA antibody signals.
Confirmation of specific binding of antibodies to NR1-GFP highlighted significant results.
The method demonstrates potential utility with clear positive and negative control assays.

Conclusions

This study provides a method to effectively screen for NMDA receptor autoantibodies.
The efficiency of the technique opens avenues for further research in psychiatric conditions.
This approach could facilitate better understanding and diagnosis of acute mental health emergencies.

Frequently Asked Questions

What are the advantages of using HEK293 cells for this assay?

HEK293 cells are easy to culture and transfect, allowing for robust expression of the NR1 subunit and efficient detection of autoantibodies against NMDA receptors.

How is the primary antibody used in the assay?

The primary antibody, diluted in PBST, is incubated with the HEK293 cells to bind any existing autoantibodies in the plasma samples being tested.

What outcomes are measured in this assay?

The assay measures the presence of autoantibodies based on the colocalization of the fluorescence signals from NR1-GFP and the secondary antibody conjugated with Alexa Fluor 594.

Can this method be adapted for other receptors?

Yes, the method can potentially be adapted for other receptor assays by substituting the plasmid to express different receptor subunits once optimized.

What are the limitations of this screening method?

While it is a quick screening tool, it may require further confirmation through methods like western blot analysis to validate the presence of autoantibodies.

אנחנו ectopically לידי יחידת משנה NR1 של קולטן NMDA מתויג עם חלבון פלואורסצנטי ירוק בתאים עובריים אנושיים (HEK293) כמו אנטיגן לזהות עצמיים כנגד קולטן NMDA בדם של חולים חשוד עם דלקת אוטואימונית. שיטה פשוטה זו עשויה להיות מתאימים לסינון למטרות בהגדרות קליניים.

המטרה הכוללת של בדיקה זו היא לזהות נוגדנים עצמיים כנגד קולטן NMDA בדמם של חולים עם חשד לדלקת מוח אוטואימונית. המדד עוזר לענות על שאלות המפתח באבחנה מבדלת של חולים עם תסמינים נוירו-פסיכיאטריים חריפים. היתרון העיקרי של טכניקה זו הוא שמדובר באמצעי סינון פשוט ואמין.

התחל הליך זה על ידי הכנת לוחות תרבות מצופים ג'לטין. יש למנות 200 מיקרוליטר של תמיסת ג'לטין של 2% לכל באר של צלחת תרבית של 48 בארות ולדגור את הצלחת בחום של 37 מעלות צלזיוס למשך 30 דקות לפחות. לאחר 30 דקות יש לשאוב את תמיסת הג'לטין מהבארות.

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