Detection of Anti-MDA5 Autoantibodies Using HeLa Cells and Immunocytochemistry with Light Microscopy

Summary

We provide a step-by-step immunocytochemistry (ICC) methodology for detecting anti-MDA5. This approach involves cell fixation, permeabilization, antibody incubation, and imaging techniques, which allow for the accurate detection of anti-MDA5 autoantibodies, aiding in the diagnosis of rapidly progressing interstitial lung disease in myositis patients.

Abstract

Anti-MDA5 autoantibodies are critical biomarkers for dermatomyositis (DM), amyopathic dermatomyositis (CADM), and polymyositis (PM), particularly in identifying patients at risk of rapidly progressive interstitial lung disease (RP-ILD). Early detection of these autoantibodies is essential to improve patient outcomes, as delayed diagnosis often leads to poor prognoses. Currently, radioimmunoassay is the gold standard for detecting anti-MDA5, but its use is limited by high costs, lengthy procedures, and the need for specialized expertise. Additionally, the blot test, a widely used clinical tool, exhibits a high false-positive rate for MDA5 autoantibodies, potentially compromising diagnostic accuracy. To address these limitations, we propose a non-radioactive, highly sensitive, and standardized confirmatory testing method using Immunocytochemistry (ICC). This protocol involves treating HeLa cells with an MDA5 construct, permeabilizing the cells with Triton X-100 to facilitate binding of primary anti-MDA5 autoantibodies, and detecting bound antibodies using enzyme-conjugated secondary antibodies (e.g., horseradish peroxidase) with DAB chromogen for microscopy imaging. ICC offers a practical, cost-effective, and high-sensitivity approach for visualizing anti-MDA5 autoantibodies within cellular structures. By integrating ICC as a supplementary confirmatory procedure, this study aims to enhance the reliability of anti-MDA5 detection, thereby improving diagnostic and prognostic strategies for RP-ILD in DM, CADM, and PM patients.

Introduction

Idiopathic inflammatory myopathies (IIM), commonly referred to as myositis, are a heterogeneous group of autoimmune disorders characterized by chronic muscle inflammation, variable clinical manifestations, and diverse therapeutic outcomes. Common symptoms include muscle weakness, reduced endurance, and myalgia¹. The primary subtypes of IIM include polymyositis (PM) and dermatomyositis (DM), while clinically amyopathic dermatomyositis (CADM) is distinguished by characteristic skin rashes similar to those seen in DM but with minimal or no muscle involvement. A major complication in PM, DM, and CADM is interstitial lung disease (ILD), which affects approximately 40% of patients and is associated with increased mortality².

The clinical course and prognosis of ILD in IIM vary widely. ILD associated with DM and CADM (DM-/CADM-ILD) tends to be more treatment-resistant and carries a worse prognosis compared to PM-associated ILD. Among these, acute or subacute ILD, which progresses rapidly within three months³, is particularly severe, with a reported five-year survival rate of only 52%, compared to 87% for chronic ILD, which progresses slowly or remains stable. Rapidly progressive ILD (RP-ILD), a subtype of acute/subacute ILD, is characterized by a rapid onset of dyspnea and extensive alveolar damage visible on chest imaging. RP-ILD is a life-threatening condition with a poor prognosis, underscoring the urgent need for early diagnosis and prompt intervention to improve patient outcomes^4,5,6.

Myositis-specific autoantibodies (MSAs) have emerged as critical biomarkers in myositis-associated ILD, with anti-MDA5 autoantibodies playing a particularly significant role. These autoantibodies are frequently detected in patients with PM-/DM-/CADM-ILD and serve as important prognostic indicators for RP-ILD^7,8,9. MDA5 (melanoma differentiation-associated gene 5) is a cytosolic pattern recognition receptor encoded by an interferon-inducible gene that detects viral and mitochondrial double-stranded RNA, initiating interferon-mediated immune responses¹⁰. Although the precise pathogenic mechanisms remain unclear, anti-MDA5 autoantibodies are believed to disrupt MDA5 function, thereby contributing to autoimmune pathogenesis.

Timely detection of anti-MDA5 autoantibodies is essential for the early identification and management of RP-ILD. Traditionally, radiolabeled immunoprecipitation (IP) using ³⁵S-methionine-labeled K562 cell extracts has been considered the gold standard for detecting anti-MDA5 autoantibodies¹¹. However, this method is impractical for routine clinical use due to its high cost, dependence on specialized equipment and trained personnel, strict radioactive waste disposal regulations, and the limited shelf life of radiolabeled reagents. In clinical practice, blot assays are commonly employed as alternatives; however, they are associated with a high false-positive rate for anti-MDA5 autoantibodies^12,13, raising concerns about diagnostic accuracy. Consequently, there is an urgent need for a reliable, non-radioactive confirmatory assay to validate positive results and improve diagnostic confidence.

To address this gap, we propose the use of immunocytochemistry (ICC) as a supplementary confirmatory test for anti-MDA5 autoantibodies. This approach involves transfecting HeLa cells with an MDA5 construct, incubating the cells with patient plasma, and detecting bound anti-MDA5 autoantibodies using enzyme-conjugated secondary antibodies (e.g., horseradish peroxidase) combined with a chromogenic substrate for visualization under light microscopy. ICC provides a non-radioactive, highly sensitive, and standardized platform for visualizing anti-MDA5 autoantibodies within cellular compartments. By integrating ICC with the blot assay, this method aims to reduce false-positive rates, improve diagnostic accuracy, and ultimately enhance clinical management and outcomes for patients.

The objective of this study is to establish a robust, non-radioactive protocol for the detection of anti-MDA5 autoantibodies, addressing the limitations of current detection methods and offering clinicians a practical and reliable tool for the early diagnosis of RP-ILD in patients with PM, DM, and CADM. In the accompanying video narration, this life-threatening course is colloquially described as 'rapid death'; scientifically, it corresponds to rapidly progressive ILD (RP-ILD). This work builds upon existing evidence and has the potential to significantly improve prognostic assessment and therapeutic decision-making in clinical practice.

Protocol

1. Cell culture and transfection

1. Maintain HeLa cells with Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic at 37 °C in a humidified 5% CO₂ atmosphere.
2. Passage the cells every 2-3 days or when the cells reach 90%-100% confluency.
3. Seed 7 × 10⁴ HeLa cells in each well of a 24-well plate (1.9 cm²/well) 24 h before transfection to let the cells reach approximately 90% confluency.
4. Dilute 500 ng of plasmid (pENTER-MDA5) and 1.5 µL of transfection reagent each in 25 µL of reduced serum medium.
5. Add diluted DNA to diluted transfection reagent (1:1 ratio). Mix well and incubate for 5 min.
6. Add the mixture to the cells seeded one day before and agitate to mix the DNA-lipid complex immediately. Confirm that the cells are 80%-90% confluent.
7. Incubate the cells at 37 °C in a humidified 5% CO₂ atmosphere for 24 h and proceed to perform ICC.
   NOTE: We used a commercially available vector (pEnter-MDA5); more information can be found in the Table of Materials. The transfection efficacy is ~50%.
   The success of transfection and expression of the target protein can be determined by transfecting the cells with a fluorescent protein-expressing plasmid and conducting a Western blot to visualize expression of the target protein.

2. Cell fixation

1. After incubation, wash the cells 2x with PBS to remove any remaining media.
   NOTE: Washing can be performed by shaking the plate on an orbital shaker.
2. Fix the cells in 4% paraformaldehyde in PBS. Leave the cells for 15 min at room temperature.
   CAUTION: Paraformaldehyde is toxic. Use appropriate handling guidelines.
3. Aspirate the fixative reagent and use PBS to wash the cells 2x.

3. Cell permeabilization

1. Add Triton X-100 reagent (0.3% in PBS) and let the cells sit for 10 min at room temperature.
2. After 10 min, dispose of the detergent.
3. Add PBS to wash the cells once.

4. Cell blocking

1. Perform blocking with 10% fetal bovine serum in PBS (1x) and incubate the cells for 1 h at room temperature.
2. Remove the blocking reagent, then add PBS to wash the cells once.

5. Hybridization of the patient's antibody

1. Add the diluted patient plasma to the cells. Be sure to dilute the patient sample (1:5,000 dilution in PBS) in PBS before use.
   NOTE: Adjust the dilution to enhance the signal or reduce the background as needed. To ensure unbiased results, the staff conducting the tests had no knowledge of which samples belonged to the patients and which were from healthy individuals.
2. Incubate the sample at 4 °C for 12-16 h (overnight).
3. Following the incubation period, remove the patient sample and wash the cells 3x with PBS.

6. Hybridization of secondary antibody

1. Treat the cells with diluted Horseradish Peroxidase-conjugated Goat Anti-Human IgG (1:250 dilution in PBS), and leave the cells for 1 h at room temperature.
2. An hour later, dispose of the secondary antibodies.
3. Rinse with PBS 3x.

7. Cell staining

1. After washing the cells, stain the cells with 300 µL of DAB working solution/well.
   NOTE: This solution was prepared by mixing DAB chromogen concentrate and DAB diluent according to the manufacturer's instructions.
2. After staining the cells for 5 min, remove the dye, rinse with deionized distilled water, and shake for 5 min.

8. Cell counterstaining

1. Counterstain the cell nucleus with diluted hematoxylin.
   NOTE: We use Hematoxylin Gill II in a 10-fold dilution and wait for 5 min for the staining process.
2. After 5 min, dispose of the hematoxylin, then wash with deionized distilled water for 2 x 5 min.

9. Observation

1. Observe the staining results using an optical microscope.
   NOTE: A true positive shows strong brown staining inside HeLa cells expressing MDA5; this confirms the anti-MDA5 autoantibodies in the patient's sample. A negative result shows HeLa cells with no brown staining, indicating no anti-MDA5 autoantibodies. A false-positive result shows extensive brown staining in all cells regardless of MDA5 expression. This non-specific staining, seen in other autoimmune conditions, must be distinguished from a true positive to avoid misdiagnosis.

Results

The personnel conducting the tests were blinded to the identities of the samples, including the specific patients from whom they were obtained, to minimize potential bias in the evaluation process. However, while the healthy control samples were not subjected to blinding, they consistently produced clear and unequivocal results.

Figure 1A demonstrates the successful expression of MDA5 in HeLa cells ...

Discussion

Our ICC-based technique for identifying anti-MDA5 autoantibodies offers significant advantages over traditional methods such as line blot analysis and radioimmunoassay. Previously, Nombel et al. reported sensitivity and specificity values of 96% and 100%, respectively, by comparing indirect immunofluorescence (IIF) on MDA5-transfected cells with ELISA in a cohort of 23 anti-MDA5-positive dermatomyositis patients and 22 anti-MDA5-negative controls¹⁴. However, this ELISA is not commercially availabl...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates (1.9 cm²/well)	Corning	354541
Antibiotic-antimycotic	Gibco	15240062
beakers	PYREX	1000-600
Conical tubes (15 mL)	Corning	430052
Conical tubes (50 mL)	Corning	430290
Deionized distilled water			Our deionized distilled water is produced in-house using a Milli-Q system (Column 1: Quantum TEX, SN: F4EB53476; Column 2: Prepak, SN: F1CB11442)
Fetal Bovine Serum Characterized	Cytiva	SH30396.03
graduated cylinders (50 mL)	VITLAB	64804
Hematoxylin Gill II	Leica	3801522
Hyclone Dulbecco’s Modified Eagle Medium with high glucose	Cytiva	SH30243.02
Lipofectamin 2000 transfection reagent	Invitrogen	11668019	Transfection reagent
MDA-5 (D74E4) Rabbit mAb	Cell Signaling Technology	5321
Microcentrifuge tubes (1.5 mL Eppendorf tubes)	AXYGEN	142503
Opti-MEM I reduced serum medium	Gibco	31985070
Orbital shaker (optional)	FIRSTEK	S300R
Paraformaldehyde	Sigma-Aldrich		4% in 1x PBS
PBS	PRO TECH	ME222605
pENTER-MDA5	Vigene Biosciences Inc.	CH863586
Peroxidase AffiniPure Goat Anti-Human IgG (H+L)	Jackson ImmunoResearch	109-035-003
pipette controller	Thermo Scientific	130165
Pipettes	GILSON
Pipettes and pipette tips (10 µL)	QSP	104-Q
Pipettes and pipette tips (200 µL)	QSP	T090-Q
Pipettes and pipette tips (1000 µL)	QSP	111-Q
Serological pipettes (5 mL)	SPL	91005
Serological pipettes (10 mL)	SPL	91010
Serological pipettes (25 mL)	SPL	91025
SignalStain DAB substrate kit	Cellsignaling	8059	0.3% in PBS