Antibody Conjugated Nanoparticle-based Quantification Using Dark Field Microscopy: A Procedure to Capture and Quantify Specific Exosomes from Body Fluids
Transcript
Exosomes are small, membrane-enclosed structures that are secreted by the cells into the extracellular space. Exosomes are abundantly present in biofluids.
To identify and capture specific exosomes from body fluids, begin by taking a multi-well immunoassay slide. This slide is functionalized with proteins that act as antigens and bind to specific antibodies.
Supplement the slide with the desired primary antibody solution and incubate. These antibodies bind to the pre-coated antigens on the slide surface.
Aspirate the remaining solution to remove any unbound antibodies. Now, add a blocking buffer that blocks the free antigens, thereby preventing the nonspecific binding of exosomes.
Then, remove the blocking buffer, load the exosomes containing serum suspension onto the slide, and incubate. This allows the exclusive binding of target-specific exosomes to the captured primary antibodies.
Next, load a suspension of gold nanoparticle conjugated secondary antibodies onto the slide surface. These antibodies attach to the pre-bound exosomes and form a detection complex.
Finally, using a pipette, discard any unbound secondary antibodies. Visualize the slides under a dark-field microscope. Gold nanoparticles present on the surface of exosomes scatter light and appear as bright spots against the dark background.
