PRODUCTION AND PURIFICATION OF RABBIT ANTI-CD166 POLYCLONAL ANTIBODY

CD166 is an Activated Leukocyte Cell Adhesion Molecule (ALCAM) which acts as a cell-to-cell adhesion molecule. CD166 has an important role in the growth, survival and invasion of the tumor cells. The aim of the present study was to produce polyclonal antibodies against CD166.

The selected peptide was synthesized from the CD166 molecule and after conjugation with KLH protein, in an emulsion with Freund's complete adjuvant, it was injected subcutaneously to two female New Zealand rabbits several times. Then the antibodies in rabbit serum were purified by FPLC method and their amounts were determined by indirect ELISA method. By CNBr gel purification method, specific antibodies against CD166 peptide bound to KLH were isolated, and the concentration of produced antibody against peptide or KLH-peptide was determined by ELISA method.

After repeated injection of CD166 peptide bound to KLH, antibody against CD166 peptide was produced, but it had a low titer. Most of the antibody produced was directed against the KLH-peptide binding site. Also, the tendency of produced antibodies to bind to KLH-peptide was higher compared to the peptide alone.

The produced antibodies were mostly against the antigenic index of KLH peptide. The synthetic and small size of the injected peptide can be the reason for the low titer and affinity of the antibody to it.