Name: Detection of Anti-MDA5 Autoantibodies Using HeLa Cells and Immunocytochemistry with Light Microscopy (Video) | JoVE Journal
Uploaded: 2025-10-31T14:05:00.000+00:00
Duration: 10 min 55 s
Description: Learn immunocytochemistry to detect anti-MDA5 autoantibodies using transfected HeLa cells. This method enables rapid diagnosis of interstitial lung disease in myositis patients.

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5 minutes

This work was supported by grants from Taiwan&#39;s National Science and Technology Council awarded to Jye-Lin Hsu (NSTC 114-2320-B-039-038 and NSTC 113-2320-B-039-032). This study was also supported in part by China Medical University and Hospital grants awarded to Jye-Lin Hsu (CMU113-MF-89 and C1110812016-12).

1. Cell culture and transfection
<ol>
	<li>Maintain HeLa cells with Dulbecco&#39;s Modified Eagle Medium containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic at 37 &#176;C in a humidified 5% CO2 atmosphere.</li>
	<li>Passage the cells every 2-3 days or when the cells reach 90%-100% confluency.</li>
	<li>Seed 7 &#215; 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.</li>
	<li>Dilute 500 ng of plasmid (pENTER-MDA5) and 1.5 &#181;L of transfection reagent each in 25 &#181;L of reduced serum medium.</li>
	<li>Add diluted DNA to diluted transfection reagent (1:1 ratio). Mix well and incubate for 5 min.</li>
	<li>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.</li>
	<li>Incubate the cells at 37 &#176;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.</li>
</ol>
2. Cell fixation
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Aspirate the fixative reagent and use PBS to wash the cells 2x.</li>
</ol>
3. Cell permeabilization
<ol>
	<li>Add Triton X-100 reagent (0.3% in PBS) and let the cells sit for 10 min at room temperature.</li>
	<li>After 10 min, dispose of the detergent.</li>
	<li>Add PBS to wash the cells once.</li>
</ol>
4. Cell blocking
<ol>
	<li>Perform blocking with 10% fetal bovine serum in PBS (1x) and incubate the cells for 1 h at room temperature.</li>
	<li>Remove the blocking reagent, then add PBS to wash the cells once.</li>
</ol>
5. Hybridization of the patient&#39;s antibody
<ol>
	<li>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.</li>
	<li>Incubate the sample at 4 &#176;C for 12-16 h (overnight).</li>
	<li>Following the incubation period, remove the patient sample and wash the cells 3x with PBS.</li>
</ol>
6. Hybridization of secondary antibody
<ol>
	<li>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.</li>
	<li>An hour later, dispose of the secondary antibodies.</li>
	<li>Rinse with PBS 3x.</li>
</ol>
7. Cell staining
<ol>
	<li>After washing the cells, stain the cells with 300 &#181;L of DAB working solution/well. 
	NOTE: This solution was prepared by mixing DAB chromogen concentrate and DAB diluent according to the manufacturer&#39;s instructions.</li>
	<li>After staining the cells for 5 min, remove the dye, rinse with deionized distilled water, and shake for 5 min.</li>
</ol>
8. Cell counterstaining
<ol>
	<li>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.</li>
	<li>After 5 min, dispose of the hematoxylin, then wash with deionized distilled water for 2 x 5 min.</li>
</ol>
9. Observation
<ol>
	<li>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&#39;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.</li>
</ol>

<ol><li>Mehta, P., Machado, P. M., Gupta, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+and+managing+anti-MDA5+dermatomyositis%2C+including+potential+COVID-19+mimicry%5BTitle%5D)+AND+%22Rheumatol+Int%22%5BJournal%5D)">Understanding and managing anti-MDA5 dermatomyositis, including potential COVID-19 mimicry.</a> Rheumatol Int. 41 (6), 1021-1036 (2021).</li>
<li>Tansley, S. L., McHugh, N. J., Wedderburn, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+and+juvenile+dermatomyositis%3A+are+the+distinct+clinical+features+explained+by+our+current+understanding+of+serological+subgroups+and+pathogenic+mechanisms%5BTitle%5D)+AND+%22Arthritis+Res+Ther%22%5BJournal%5D)">Adult and juvenile dermatomyositis: are the distinct clinical features explained by our current understanding of serological subgroups and pathogenic mechanisms.</a> Arthritis Res Ther. 15 (2), 211(2013).</li>
<li>Fujisawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+of+myositis-associated+interstitial+lung+disease%5BTitle%5D)+AND+%22Medicina%22%5BJournal%5D)">Management of myositis-associated interstitial lung disease.</a> Medicina. 57 (4), 347(2021).</li>
<li>Sasaki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+initiation+of+plasma+exchange+therapy+for+a+patient+with+anti-MDA5+autoantibody-positive+dermatomyositis+developing+rapidly+progressive+interstitial+lung+disease%5BTitle%5D)+AND+%22Mod+Rheumatol+Case+Rep%22%5BJournal%5D)">Early initiation of plasma exchange therapy for a patient with anti-MDA5 autoantibody-positive dermatomyositis developing rapidly progressive interstitial lung disease.</a> Mod Rheumatol Case Rep. 5 (1), 87-92 (2021).</li>
<li>Go, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Survival+benefit+associated+with+early+cyclosporine+treatment+for+dermatomyositis-associated+interstitial+lung+disease%5BTitle%5D)+AND+%22Rheumatol+Int%22%5BJournal%5D)">Survival benefit associated with early cyclosporine treatment for dermatomyositis-associated interstitial lung disease.</a> Rheumatol Int. 36 (2), 125-132 (2015).</li>
<li>Tsuji, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multicenter+prospective+study+of+the+efficacy+and+safety+of+combined+immunosuppressive+therapy+with+high-dose+glucocorticoid%2C+tacrolimus%2C+and+cyclophosphamide+in+interstitial+lung+diseases+accompanied+by+anti-melanoma+differentiation-associated+gene+5-positive+dermatomyositis%5BTitle%5D)+AND+%22Arthritis+Rheumatol%22%5BJournal%5D)">Multicenter prospective study of the efficacy and safety of combined immunosuppressive therapy with high-dose glucocorticoid, tacrolimus, and cyclophosphamide in interstitial lung diseases accompanied by anti-melanoma differentiation-associated gene 5-positive dermatomyositis.</a> Arthritis Rheumatol. 72 (3), 488-498 (2020).</li>
<li>Hozumi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+assessment+of+myositis-specific+autoantibodies+in+polymyositis%2Fdermatomyositis-associated+interstitial+lung+disease%5BTitle%5D)+AND+%22Respir+Med%22%5BJournal%5D)">Comprehensive assessment of myositis-specific autoantibodies in polymyositis/dermatomyositis-associated interstitial lung disease.</a> Respir Med. 122, 1-8 (2016).</li>
<li>Nakashima, R., Hosono, Y., Mimori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+significance+and+new+detection+system+of+autoantibodies+in+myositis+with+interstitial+lung+disease%5BTitle%5D)+AND+%22Lupus%22%5BJournal%5D)">Clinical significance and new detection system of autoantibodies in myositis with interstitial lung disease.</a> Lupus. 25 (8), 925-933 (2016).</li>
<li>Koga, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+diagnostic+utility+of+anti-melanoma+differentiation-associated+gene+5+antibody+testing+for+predicting+the+prognosis+of+Japanese+patients+with+DM%5BTitle%5D)+AND+%22Rheumatology%22%5BJournal%5D)">The diagnostic utility of anti-melanoma differentiation-associated gene 5 antibody testing for predicting the prognosis of Japanese patients with DM.</a> Rheumatology. 51 (7), 1278-1284 (2012).</li>
<li>Fuzzi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-MDA5+dermatomyositis%3A+an+update+from+bench+to+bedside%5BTitle%5D)+AND+%22Curr+Opin+Rheumatol%22%5BJournal%5D)">Anti-MDA5 dermatomyositis: an update from bench to bedside.</a> Curr Opin Rheumatol. 34 (6), 365-373 (2022).</li>
<li>Sato, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoantibodies+to+a+140-kd+polypeptide%2C+CADM-140%2C+in+Japanese+patients+with+clinically+amyopathic+dermatomyositis%5BTitle%5D)+AND+%22Arthritis+Rheum%22%5BJournal%5D)">Autoantibodies to a 140-kd polypeptide, CADM-140, in Japanese patients with clinically amyopathic dermatomyositis.</a> Arthritis Rheum. 52 (5), 1571-1576 (2005).</li>
<li>Tansley, S. L., Diederichsen, L. P., Zampeli, V. A., McHugh, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+reliability+of+immunoassays+to+detect+autoantibodies+in+patients+with+myositis+is+dependent+on+autoantibody+specificity%5BTitle%5D)+AND+%22Rheumatology%22%5BJournal%5D)">The reliability of immunoassays to detect autoantibodies in patients with myositis is dependent on autoantibody specificity.</a> Rheumatology. 59 (5), 1029-1034 (2020).</li>
<li>Chen, Y. L., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Positive+predictive+value+of+myositis+antibody+line+blot+testing+in+patients+with+suspected+idiopathic+inflammatory+myopathy%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">Positive predictive value of myositis antibody line blot testing in patients with suspected idiopathic inflammatory myopathy.</a> Muscle Nerve. 69 (3), 345-350 (2024).</li>
<li>Nombel, A., Pin, J. J., Fabien, N., Miossec, P., Coutant, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+performance+of+an+indirect+immunofluorescence+assay+for+the+detection+of+anti-MDA5+antibodies%5BTitle%5D)+AND+%22Biomedicines%22%5BJournal%5D)">Evaluation of the performance of an indirect immunofluorescence assay for the detection of anti-MDA5 antibodies.</a> Biomedicines. 10 (12), 3125(2022).</li>
<li>Nakashima, R., Hosono, Y., Mimori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+significance+and+new+detection+system+of+autoantibodies+in+myositis+with+interstitial+lung+disease%5BTitle%5D)+AND+%22Lupus%22%5BJournal%5D)">Clinical significance and new detection system of autoantibodies in myositis with interstitial lung disease.</a> Lupus. 25 (8), 925-933 (2016).</li>
<li>Sato, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoantibodies+to+a+140-kd+polypeptide%2C+CADM-140%2C+in+Japanese+patients+with+clinically+amyopathic+dermatomyositis%5BTitle%5D)+AND+%22Arthritis+Rheum%22%5BJournal%5D)">Autoantibodies to a 140-kd polypeptide, CADM-140, in Japanese patients with clinically amyopathic dermatomyositis.</a> Arthritis Rheum. 52 (5), 1571-1576 (2005).</li>
<li>Chang, Y. C., Yang, L., Budhram, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Positive+predictive+value+of+myositis+antibody+line+blot+testing+in+patients+with+suspected+idiopathic+inflammatory+myopathy%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">Positive predictive value of myositis antibody line blot testing in patients with suspected idiopathic inflammatory myopathy.</a> Muscle Nerve. 69 (5), 626-630 (2024).</li>
</ol>

The authors have no competing interests to declare. 
Portions of this manuscript were generated with the assistance of OpenAI's GPT-4. The authors reviewed and edited the content to ensure accuracy and originality.

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...

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 outcomes4,5,6.
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-ILD7,8,9. 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 autoantibodies12,13, 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 &#39;rapid death&#39;; 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.

<table><tbody><tr><td>24-well plates (1.9 cm&amp;sup2;/well)</td><td>Corning</td><td>354541</td><td></td></tr><tr><td>Antibiotic-antimycotic</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>beakers</td><td>PYREX</td><td>1000-600</td><td></td></tr><tr><td>Conical tubes (15 mL)</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>Conical tubes (50 mL)</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>Deionized distilled water</td><td></td><td></td><td>Our deionized distilled water is produced in-house using a Milli-Q system (Column 1: Quantum TEX, SN: F4EB53476; Column 2: Prepak, SN: F1CB11442)</td></tr><tr><td>Fetal Bovine Serum Characterized</td><td>Cytiva</td><td>SH30396.03</td><td></td></tr><tr><td>graduated cylinders (50 mL)</td><td>VITLAB</td><td>64804</td><td></td></tr><tr><td>Hematoxylin Gill II</td><td>Leica</td><td>3801522</td><td></td></tr><tr><td>Hyclone Dulbecco&amp;rsquo;s Modified Eagle Medium with high glucose</td><td>Cytiva</td><td>SH30243.02</td><td></td></tr><tr><td>Lipofectamin 2000 transfection reagent</td><td>Invitrogen</td><td>11668019</td><td>Transfection reagent</td></tr><tr><td>MDA-5 (D74E4) Rabbit mAb</td><td>Cell Signaling Technology</td><td>5321</td><td></td></tr><tr><td>Microcentrifuge tubes (1.5 mL Eppendorf tubes)</td><td>AXYGEN</td><td>142503</td><td></td></tr><tr><td>Opti-MEM I reduced serum medium</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>Orbital shaker (optional)</td><td>FIRSTEK</td><td>S300R</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td></td><td>4% in 1x PBS</td></tr><tr><td>PBS</td><td>PRO TECH</td><td>ME222605</td><td></td></tr><tr><td>pENTER-MDA5</td><td>Vigene Biosciences Inc.</td><td>CH863586</td><td></td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Human IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>109-035-003</td><td></td></tr><tr><td>pipette controller</td><td>Thermo Scientific</td><td>130165</td><td></td></tr><tr><td>Pipettes&amp;nbsp;</td><td>GILSON</td><td></td><td></td></tr><tr><td>Pipettes and pipette tips (10 &amp;micro;L)</td><td>QSP</td><td>104-Q</td><td></td></tr><tr><td>Pipettes and pipette tips (200 &amp;micro;L)</td><td>QSP</td><td>T090-Q</td><td></td></tr><tr><td>Pipettes and pipette tips (1000 &amp;micro;L)</td><td>QSP</td><td>111-Q</td><td></td></tr><tr><td>Serological pipettes (5 mL)</td><td>SPL</td><td>91005</td><td></td></tr><tr><td>Serological pipettes (10 mL)</td><td>SPL</td><td>91010</td><td></td></tr><tr><td>Serological pipettes (25 mL)</td><td>SPL</td><td>91025</td><td></td></tr><tr><td>SignalStain DAB substrate kit</td><td>Cellsignaling</td><td>8059</td><td>0.3% in PBS</td></tr></tbody></table>

detection of anti-mda5 autoantibodies using hela cells and immunocytochemistry with light microscopy

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

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.

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 ...

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.

Anti-MDA5 autoantibodies are critical biomarkers for dermatomyositis (DM), amyopathic dermatomyositis (CADM), and polymyositis (PM), particularly in ...

The main purpose of the following experiment is to use immunocytochemistry to screen for anti-MDA5 antibodies. This is done via using the HeLa cells and then transfect the HeLa cell with MDA5 construct. As such, the HeLa cells will produce MDA5 proteins that can be attached by MDA5 antibodies.After including HeLa cell with MDA5 construct, the Triton reagent is used to permeabilize the membrane of HeLa cells, which would allow the anti-MDA5 antibodies to pass through. After which, the patient serum which contains the anti-MDA5 antibodies is added. The anti-MDA5 antibodies would then attach to the MDA5 proteins inside the HeLa cells.After that, secondary antibodies, which horseradish peroxidase is attached to, are deployed into the cells, the secondary antibodies would then attach to the anti-MDA5 antibodies. Finally, the chromogen is added to the cells and oxidized in the presence of horseradish peroxidase-conjugated secondary antibodies, producing a visible color under an optical microscope. A positive standing result would show something like the picture above.In today's video demonstration, we flow into a critical area of medical research, the detection of anti-melanoma differentiation-associated gene five antibody, also called anti-MDA5 antibody. These autoantibody plays a critical role of simple diagnostic indicator for patients with interstitial lung disease, especially those with rapidly progressive The significance of this research lie in its potential impact on patient outcomes. Early and accurate detection of MDA5 autoantibody is vital for effectively managing this severe autoimmune disease.The primary advantage of the method we demonstrated has over the golden method of screening anti-MDA5 autoantibody is that it could be done faster and the save time and effort in comparison to the traditional radio-labeled immunovisitation assay. This increased efficiency allows us to process more samples and generate result rapidly. Furthermore, it's crucial to note that our method not only offers superior efficiency, but also itself in safety and cost effectiveness when compared to the transitional immunoprecipitation assay.Now, let us see how it's done. My research assistant, Teo Kai Fa will be leading the procedure. Maintain HeLa cells with Dulbecco's Modified Eagle's Medium containing 10%fetal bovine serum, and 1%antibiotic antimycotic at 37 degrees celsius in a humidified 5%CO2 atmosphere.Passage the cells every two to three days, or when the cells reach 90 to 100%confluency. Sit 70, 000 HeLa cells on each well off a 24 well plate. 24 hours before transfection, and net the cells, which approximately 90%confluency.Dilute 500 nanogram of plasmin and 1.5 microliter of transfection reagent each in a 25 microliter of reduced serum medium. Add diluted DNA, do diluted transfection reagent at a one-to-one ratio. Mix well and incubate for five minutes.Add mixture to the cells seated one day before and agitate to mix the DNA lipid complex immediately. Confirm that the cells are 80 to 90%confluent. Incubate the cells at 37 degrees Celsius in a humidified 5%CO2 hemisphere, 40 to four hours, and proceed to immunocytochemistry.We use a commercially available factor. More information can be found in the table of materials. Their transfection efficacy is 50%Success of transfection and expression of the target protein can be determined by transfecting the cells with frozen protein expressing plasmid and conducting western blood to visualize expression of the target protein After incubation, wash the cells twice with PBS to remove any remaining media.Washing can be performed by shaking the plate or an orbital shaker. Fix the cells with 4%paraformaldehyde in PBS Caution. Paraformaldehyde is toxic.Use appropriate handling guidelines. Leave the cells for 15 minutes at room temperature. Next, dispose of the fixative reagent.Use PBS to wash the cell twice. Apply Triton reagent and then let the cells sit for 10 minutes at room temperature. We used Triton X 400 in PBS at a concentration of 0.3%After 10 minutes, dispose of the Triton reagent.Add PBS to wash the cells once. Next step, perform blocking with 10%fetal bovine serum in PBS and leave the cells for an hour at room temperature, Remove the blocking reagent. Then, add PBS to wash the cells once.Add the diluted patient plasma to cells. Make sure to dilute patient plasma in a ratio of one to 5, 000 dilution in PBS before use. Adjust the dilution to enhance the signal or reduce the background as needed to ensure unbiased results.The staff conducted the test, had no knowledge of which samples belonged to patients and which were from healthy individuals. Incubate a sample at four degrees Celsius for 12 to 16 hours. Following the incubation period, remove the patient's sample and wash the cells three times with PBS.Treat the cells with diluted horseradish peroxidase-conjugated goat, anti-human IgG in a ratio of one to 250 dilution in PBS. Leave the cells for an hour at room temperature. An hour later, dispose of the secondary antibody.Rinse with PBS three times. After washing the cells, stain the cells with 300 microliter DAB working solution per well. The DAB working solution is prepared by missing DAB promotion concentrate, and DAB diluent following the manufacturer's instructions.After staining the cell for five minutes, remove the dye. After removing the dye, rinse with the ionized distilled water and shake for five minutes. Counterstain the cell nucleus with diluted hematoxin.We use hemotoxin in to like a, in 10 dilution fold. Wait for five minutes for the staining process. After five minutes, dispose of the hemotoxin, then wash with the analyzed distilled water twice for five minutes each.Final step, observe the staining results under an optical microscope. Let's interpret our immunocytochemistry assay results for anti-MDA5 autoantibodies. A positive result.A true positive shows strong brown staining inside HeLa expressing MDA5. This confirms anti-MDA5 autoantibodies in the patient's sample. A negative result.A negative result shows HeLa cells with no brown staining, indicating no anti-MDA5 autoantibodies. A false positive result. Critically, a false positive shows extensive brown staining in all cells, regardless of MDA5s expression.This non-specific staining seen in order autoimmune conditions must be distinguished from a true positive to avoid misdiagnosis. Our method offers a faster, safer, and more cost effective way to detect anti-MDA5 organ antibodies. This has the potential to significantly impact patient outcomes in the management of interstitial lung disease.After watching this video, you shall have learned how to perform the immunocytochemistry procedure and how to identify clinical relevant in the test results.

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Research

JoVE Journal

Immunology and Infection

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.

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

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

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.

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

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

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.

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

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

In Mycoplasma pneumoniae infection, serology tests can generate good results, yet with low specificity because of immunological cross-reaction. The in-house antigen-capture ELISA, described in this paper, guarantees high species specificity and has been shown to be a reliable screening test for accurate diagnosis of M. pneumoniae.

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

Learn immunocytochemistry to detect anti-MDA5 autoantibodies using transfected HeLa cells. This method enables rapid diagnosis of interstitial lung disease in myositis patients.

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

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