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

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.