I wanted to attach antigen protein to particles and use it for antibody detection. Previously, I attached protein to particles by treating them with edc/nhs, but for a new experiment, I wanted to attach it using the streptavidin-biotin method. First, I biotinylated the antigen protein using a biotinylation kit, and obtained biotinylated antigen by removing biotin using the column in the kit. Next, I added biotinylated antigen to streptavidin particles and incubated them to attach protein to the particles.

To check whether the protein was attached well to the particles, I reacted the primary antibody and fluorescently labeled secondary antibody respectively and measured the fluorescence intensity. However, the fluorescence intensity was lower than expected and showed significantly lower results than the particles treated with edc/nhs. Of course, it may not be comparable because it is a different attachment method, but I know that the streptavidin-biotin method can attach more proteins.

I expected that the particles with the streptavidin-biotin reaction would get a higher signal, but the results were not good, so I would like to know why. At first, I thought biotinylation was not done properly, but the HABA assay result included in the kit showed approximately 8 biotin molecules per protein molecule. Since quite a lot of biotin molecules were attached, I expected that they were well attached to the streptavidin particles. Another thought is that biotin was attached to the epitope of the antigen protein and interfered with the antigen-antibody reaction. I have searched several papers, but I have not found any data explaining the exact reason. I would really appreciate it if you could give me some advice.