DETERMINATION of ANTIBODY BINDING TO PARTICLES
The amount of the antibody binding to the particles after coating can be determined by using the subtraction method as follows:
1. Measure the absorbance of antibody solution to be used for coating at 280 nm (OD280). The IgG has OD280 of ~1.4/mg.
2. Pellet the particles to be coated by centrifugation or magnetic separation.
3. Resuspend the particles in the antibody solution from step 1.
4. After coating the particles, aspirate and save the supernatant from the first centrifugation or magnetic separation.
5. Measure the OD280 of the supernatant which contains unbound antibody. Normalize the OD280 reading to the same volume as in Step 1.
6. The % reduction in OD280 reading from Step 5 as compared to Step 1 indicate the % of antibody bound to the particles.