Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

According to the recent international diagnostic criteria for neuromyelitis optica spectrum disorders, anti-AQP4 IgG is considered to be a core diagnostic biomarker. Anti-AQP4 IgG detection by cell-based assay can facilitate clinical diagnosis. So, cell-based assay is more sensitive and specific than other detection methods.

And it can be applied to both clinical diagnosis and the scientific studies. First, bring the patient's serum samples, the biochip slide, the PBS powder, and the TWEEN 20 from the refrigerated cell-based assay kit to the room temperature. Then, prepare more than 200 milliliters of PBS wash buffer containing 0.2 percent TWEEN 20 for each biochip slide.

Use the wash buffer to make 10-fold serial dilutions of each serum sample. Prepare positive and negative controls according to the manufacturer's instructions. Add 30 microliters of 10-fold serial dilutions of the sample, the positive control, and the negative control, to five individual reaction fields on the reagent tray.

Hold the biochip slide by its slides and remove its protective cover without touching its reaction fields. Put the slide on top of the reagent tray and incubate the setup at room temperature for 30 minutes. Gently rinse the biochip slide with the wash buffer.

Then immerse it in a 500 milliliter beaker filled with 100 milliliters of the wash buffer, and leave it at the room temperature for 10 minutes. Take out the biochip slide from the wash buffer. Use a paper towel to carefully wipe away any remaining buffer from outside of the reaction fields.

Let the slide air-dry for one to two minutes. In the dark room, add 25 microliters of fluorescent secondary antibody to each reaction field of the reagent tray. Put the slide on top of the reagent tray and incubate the setup at room temperature for 30 minutes.

Gently rinse the biochip slide with the wash buffer. Then immerse it in a 500 milliliter beaker filled with 100 milliliters of the fresh wash buffer, and leave it at the room temperature for 10 minutes. Take out the biochip slide from the wash buffer.

Use a paper towel to carefully wipe away any remaining buffer from outside of the reaction fields. Let the slide air-dry for one to two minutes. Carefully add one drop of the embedding medium to each reaction field on the biochip slide.

Finally, seal the biochip slide with a cover glass while avoiding air bubbles and proceed with imaging. The weak fluorescence intensity of the negative control observed in the infected area indicates the unspecific binding of the secondary antibody. The comparison between the detected fluorescence intensities of the positive control in the infected and uninfected areas indicates the specific binding of the anti-AQP4 IgG to AQP4M1 in the infected cells.

The anti-AQP4 IgG negative serum shows binding patterns similar to the negative control in both infected and uninfected areas. The positive serum shows binding patterns similar to the positive control in both infected and uninfected areas. Samples are considered anti-AQP4 IgG positive when fluorescence heterogeneously increases in the infected area.

The intensity of fluorescence is correlated to anti-AQP4 IgG titer in the serum. Samples are considered probably positive when only a small number of cells in the transfected area show detectable fluorescence intensities. A sample should be regarded as anti-AQP4 IgG negative when its intensity in the infected area is only homogeneously stronger than in the uninfected area.

Avoiding air bubbles is vital and difficult. To eliminate air bubbles, first use a pipette to avoid air bubbles in droplets. Second, while applying the biochip slide to the reagent tray, first let one side of the slide touch the droplets, and then level the slide slowly.

In addition, do not touch the reaction fields throughout the experiment.