Preparation and characterization of the antibody recognizing AMACR inside its catalytic center

6 wells of a 24-well plate were loaded with cover glasses (one 8-mm glass into each well) and seeded with 2x10^5 exponentially growing HeLa or T98G cells in DMEM containing 10% FCS. The following day, (confluency 70-80%), the cells were washed twice with PBS and the growth medium in wells was exchanged to 900µl of DME serum-free medium. 3 master mixes were used. The mixes were filtered and supplemented with 5 µl of PULSin reagent for 15 min RT. Each mixture was then divided in two and added to the wells with the different cells.

Alpha-methylacyl-CoA racemase (AMACR) catalyzes the beta-oxidation of fatty acids and is overexpressed in carcinomas in various organs, while its inactivation results in the inhibition of cancer growth. In the present study, we prepared and characterized 20 different mouse monoclonal antibodies against human AMACR. In the course of biopanning of a phage peptide commercial library against in-house prepared 6H9 and 2A5, and commercial 13H4 antibodies, 10 phage mimotopes recognized by each type of the antibody were selected. Using the program Pepitope and the crystal structure of AMACR from Mycobacterium tuberculosis, we reveal for the first time, at least to the best of our knowledge, that the epitopes recognizing the antibody against AMACR are composed of conformation sequences localized inside the AMACR catalytic center. When delivered into live HeLa cells using cationic lipid-based PULSin reagent, the specific antibodies against AMACR were co-localized with peroxisomes. The in-house made 6H9 antibody exhibited a low level of this co-localization compared to the commercially available 63340 antibody, and did not inhibit the growth rate of HeLa and T98G cells. The results obtained suggest that antibody against AMACR may possess anti-AMACR catalytic activity and needs to be further investigated as a potential drug for use in anticancer therapy. On the whole, in this study, we generated several clones of AMACR antibodies and demonstrated that these antibodies can be colonized into live cells. Currently, we are testing the growth inhibitory properties of these antibodies against AMACR.