I don't quite understand your experimental setup: Do you have any way to distinguish between the fluorescent signal of the free antibody from that of the antibody-antigen complex? If not, I would not expect a fluorescence change dependent on antigen concentration!

Dear Suchen!

You see in the article:

Antibody against envelope protein from human endogenous retrovirus activates neutrophils in systemic lupus erythematosus

https://www.biorxiv.org/content/10.1101/776468v1.full.pdf

IgG promoted phagocytosis of ERV-K102 envelope protein by neutrophils through immune complex formation. ERV immune complex phagocytosis resulted in subsequent NET formation consisting of DNA, neutrophil elastase, and citrullinated histone H3. Finally, analysis of anti-ERVK102 IgG in SLE patients showed that IgG2 likely mediates this effect. Together, we identified an immunostimulatory ERV-K envelope protein elevated in SLE that may be a target of SLE IgG and able to promote neutrophil activation.

Microscopy For NET analysis, 40K PMNs were plated on poly-L-lysine coverslips in a 24 well plate for 15 minutes at 37°C and unbound cells were washed off with PBS. Each well was incubated with 500ul of 0.1ug immune complex as described above for 2 to 3 hours at 37°C. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed in PBS, and blocked overnight in 2mM EDTA PBS containing 10% FBS, 1% BSA, 0.05% Tween 20 at 4°C. Cells were sequentially stained with the following antibodies: mouse anti-neutrophil elastase (MABS461, Millipore) at 1:250, Cy3 antimouse IgG (Jackson ImmunoResearch) at 1:1000, rabbit anti-histone H3 (Ab5103, Abcam) at 1:250, and Cy5 anti-rabbit IgG (Jackson ImmunoResearch) at 1:1000. Cells were then stained with Hoechst 33342 (Thermo Fisher) at 1:100 for 10 minutes at room temperature and ProLong Gold Antifade Mountant (Thermo Fisher) was added along with cover slips. Leica TCS SP8 confocal microscope using the 40x immersion lens was used to obtain images of NETs.

I didn't get your experiment set up properly.

Which instrument are you going to use for recording your fluorescence?

How would that distinguish between bound and unbounded antibody?

Dear Alexandr, Thank you for the paper. I did't find live recording or time-lapse Video in the paper.

Annemarie Honegger Jitendra Kumar Shandilya . I want to treat isolated human Neutrophil with PMA in 96 well plate. for the proof of concept, i want to capture the moment when Neutrophil release of Neutrophil elastase alongwith the DNA from the cell during the process of NETOSIS.

To achieve this, i had planned to use anti NE-FITC antibody in 96 well plate during continuous recording in fluorescence confocal microscope.

hope to get any modification/alternative or any suggestions.

Depending on the exact setup, and how much effort you are willing to invest in developing an assay, you might use FRET (Fluorescence resonance energy transfer) between two different antibodies binding to different but close together epitopes to detect your antigen. Look at this paper for an example of an assay for cell-bound elastase: Article Neutrophil adhesion is a prerequisite for antibody-mediated ...