EPITOPE ANALYSIS OF ANTIBODIES RECOGNIZING THE CELL-PROLIFERATION ASSOCIATED NUCLEAR ANTIGEN PREVIOUSLY DEFINED BY THE ANTIBODY KI-67 (KI-67 PROTEIN)

Aims-To elucidate the fine specificities of the antibodies MIB 1 and M IB 3 and of additional monoclonal antibodies which also recognise the Ki-67 protein (MIB 5, IND.64, JG-67-2a). Methods-Different parts of th e Ki-67 protein cDNA were expressed in Escherichia coli. Bacterial lys ates were separated by sodium dodecyl sulphate-polyacrylamide gel elec trophoresis (SDS-PAGE) and blotted on to nitrocellulose. Additionally different peptides were synthesised on a membrane support (SPOT-Blot). The immunoreactivity of the antibodies with the recombinant proteins and the immobilised synthetic peptides, respectively, was analysed. A competition enzyme linked immunosorbent assay (ELISA) using a soluble synthetic peptide was also performed. Results-The epitopes of all anti bodies tested were contained within the same region of seven amino aci ds. The antibodies MIB 1 and MIB 3 required the five amino acid sequen ce FKELF for binding, whereas Ki-67, JG-67-2a, MIB 5 and IND.64 detect ed the sequence FKEL. Conclusions-It is concluded that the amino acid sequence FKELF represents an immunodominant area of the Ki-67 protein and that there is no correlation between the ability to detect the Ki- 67 protein in paraffin wax sections irradiated with microwaves and the epitopes recognised by the antibodies.