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

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.

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.

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 transfected with the pENTER-MDA5 construct. To validate transfection efficiency, ICC was performed using a commercial anti-MDA5 antibody. Strong brown cytoplasmic staining was observed in approximately 50% of the transfected cells, indicating robust MDA5 protein expression. In contrast, non-transfected cells processed under identical conditions showed no cytoplasmic staining, confirming the specificity of the antibody and the absence of endogenous MDA5 expression. 

Following the ICC protocol, representative samples from anti-MDA5-positive patients, confirmed by radiolabeled immunoprecipitation, demonstrated clear brown cytoplasmic staining in MDA5-expressing HeLa cells, while non-transfected cells remained unstained (Figure 1B). This staining pattern indicates the presence of anti-MDA5 autoantibodies in patient plasma and aligns with the established gold-standard detection using 35S-methionine-labeled K562 cell extracts.

Patient characteristics are summarized in SupplementalTable S1, which outlines clinical diagnoses, anti-MDA5 antibody status, and classification into three groups: anti-MDA5-positive, false-positive, and healthy control. In addition to antibody status, Supplemental Table S1 now also summarizes patient demographics, including age, sex, and underlying diagnosis for each cohort. Antibody reactivity was graded semi-quantitatively as weak (1+), moderate (2+), or strong (3+) based on band intensity, reflecting increasing antibody levels. This provides clinical context for interpreting ICC results, while maintaining the primary methodological focus of the present study. The study cohort included 10 patients with confirmed anti-MDA5 autoantibodies (positive group), five patients with autoimmune diseases who yielded false-positive results by commercial blot testing (false-positive group), and 10 healthy individuals (healthy control group). Each sample was tested alongside positive and negative controls and verified independently using radiolabeled immunoprecipitation. For samples with clear and conclusive staining results, a single ICC run was deemed sufficient. In cases where staining was ambiguous or borderline, further testing was conducted, including serial dilutions and replicate ICC assays, to ensure accurate interpretation. This approach ensured both methodological rigor and resource efficiency in validating the performance of the ICC-based detection method.

False-positive results are illustrated in Figure 2. In these samples, plasma from patients with autoimmune diseases other than idiopathic inflammatory myopathies (IIM) was used. Unlike Figure 1, brown cytoplasmic staining was observed extensively across all cells, regardless of MDA5 expression status. This staining pattern indicates non-specific binding and highlights the potential for false positives in samples from patients with other autoimmune conditions. Negative results are presented in Figure 3, where plasma from healthy individuals was tested. Virtually no brown cytoplasmic staining was observed in either transfected or non-transfected HeLa cells, indicating the absence of anti-MDA5 autoantibodies in these samples.

Figure 1: Validation of MDA5 expression and detection of anti-MDA5 autoantibodies using ICC. (A) HeLa cells transfected with an MDA5-expressing plasmid were stained using a commercial anti-MDA5 antibody. Strong brown cytoplasmic staining was observed, indicating robust MDA5 protein expression. In contrast, non-transfected cells processed under identical conditions exhibited no staining, confirming both antibody specificity and the absence of endogenous MDA5 expression. (B) HeLa cells transfected with MDA5 were incubated with plasma from patients confirmed to be anti-MDA5 positive by radiolabeled immunoprecipitation. Clear cytoplasmic brown staining was observed, demonstrating the presence of anti-MDA5 autoantibodies in the patient sample. Scale bars = 50 µm. Abbreviation: ICC = immunocytochemistry. Please click here to view a larger version of this figure.

Figure 2: Detection of false-positive signals using plasma from patients with autoimmune diseases other than IIM. Widespread brown cytoplasmic staining was observed in both transfected and non-transfected cells, suggesting non-specific antibody binding. These results illustrate the potential for false-positive detection in line blot assays when using plasma from patients with autoimmune diseases unrelated to anti-MDA5-positive IIM patients. Scale bars = 50 µm. Abbreviation: IIM = idiopathic inflammatory myopathies. Please click here to view a larger version of this figure.

Figure 3: Negative staining results with plasma from healthy control individuals. HeLa cells transfected with MDA5 and incubated with plasma from healthy individuals showed little to no brown cytoplasmic staining. Please click here to view a larger version of this figure.

Supplemental Table S1: Patient characteristics. Please click here to view a larger version of this figure.

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 available and is intended for research use only. Building upon these findings, our study focuses on the development and demonstration of the ICC-based detection protocol. Unlike radioimmunoassay, our ICC approach is non-radioactive, making it safer and more environmentally friendly. In addition to these advantages, the ICC protocol also reduces turnaround time compared to radiolabeled assays, thereby improving efficiency in sample processing and enabling more rapid clinical decision-making. These features position our method as a robust alternative for detecting anti-MDA5 autoantibodies, particularly in the diagnosis of idiopathic inflammatory myopathies (IIM) and rapidly progressive interstitial lung disease (RP-ILD).

The success of our ICC technique relies on several critical steps. First, efficient transfection is paramount, as suboptimal expression can lead to inconsistent results. Second, the fixation process must be carefully controlled to preserve cellular morphology and antigenicity. Third, the incubation with patient plasma requires careful optimization, as variations in incubation time and conditions can significantly impact antibody binding. Finally, the use of a highly specific secondary antibody (rabbit anti-human IgG [HRP]) and the Liquid DAB+ Substrate Chromogen System must be optimized to achieve clear and specific staining. Meticulous attention to each of these steps is essential to ensure reproducibility and accuracy.

During the development of this protocol, we identified several areas where modifications and troubleshooting may be necessary. For instance, the dilution of human plasma used as the primary antibody can be adjusted to optimize signal intensity or reduce background staining. In our protocol, a 1:5,000 dilution was found to balance sensitivity and specificity effectively; however, this may need adjustment depending on the sample. Blocking with 10% fetal bovine serum (FBS) in 1x PBS was effective in minimizing non-specific binding. If background staining is excessive, increasing the patient plasma dilution or extending the washing steps between antibody incubations can help. Conversely, if the signal is faint, reducing the dilution of the patient plasma or optimizing the DAB chromogen application time (typically 5 min) may improve results. Uneven staining can be mitigated by consistent reagent dispersion using an orbital shaker and careful handling during washes and incubations.

Despite its advantages, our ICC-based technique has several limitations. One concern is the potential for false positives due to cross-reactivity with other autoantibodies present in patient samples. Additionally, the method's reliance on cell culture and transfection introduces variability that may affect reproducibility. Skilled personnel are required for both performing the technique and interpreting the results, which limits widespread adoption in clinical settings. Furthermore, while a 1:5,000 dilution was optimal in our study, this may not be universally applicable, requiring adjustment depending on the clinical context.

Importantly, this manuscript is focused on presenting a detailed and reproducible protocol for anti-MDA5 autoantibody detection using ICC, rather than providing a definitive clinical validation. While preliminary comparisons with radiolabeled immunoprecipitation demonstrated high specificity (100%) and sensitivity (96%), comprehensive validation against the gold-standard radioimmunoassay will be addressed in a separate study. ICC-based techniques offer a non-radioactive, cost-effective, and accessible alternative to radiolabeled immunoprecipitation-the current gold standard-which faces major logistical barriers for clinical implementation^14,15,16. Furthermore, the high false-positive rates associated with commercial line blot assays^12,17 underscore the need for reliable confirmatory methods. Within this context, our ICC approach provides a valuable tool for research and diagnostic laboratories, particularly in resource-constrained settings.

The potential applications of our ICC-based technique extend beyond the detection of anti-MDA5 autoantibodies. It could be adapted for use in the identification of autoantibodies associated with other autoimmune diseases, such as systemic lupus erythematosus or rheumatoid arthritis. Furthermore, the method may be useful in cancer research for characterizing tumor-associated antigens or in infectious diseases for detecting pathogen-specific antibodies. The versatility of our ICC-based approach makes it a promising tool for both research and clinical diagnostics, with potential integration into broader diagnostic workflows to improve accuracy and reliability.