Abstract:

Objective To establish double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of ubiquitous mitochondrial creatine kinase(uMtCK) in human serum. Methods Seven strains of monoclonal antibodies against human uMtCK were prepared with prokaryotic expression recombinant proteins from rats, and the best combination of double antibodies was selected by checkerboard titration method. Double antibody sandwich ELISA for the determination of human uMtCK was established by using HRP-labeled McAb 19F11 as a detection antibody and McAb 3C1 as a capture antibody. The methodology performance was evaluated. The serum concentrations of uMtCK in 50 healthy subjects and 85 patients with positive tumor markers [alpha fetal protein (AFP) and carcinoembryonic antigen (CEA)] were determined. Results The double antibody sandwich ELISA for the determination of human uMtCK was established by selecting the combination of double antibodies. The detection limit was 28.8ng/mL, and the within-run and between-run coefficients of variation (CV) were 4.5%-7.7％ and 8.2%-13. 5％, respectively.The average recovery was 97.1％. The serum concentrations of uMtCK in 50 healthy subjects were negative. The 15 of 85 patients with positive tumor markers had positive results of uMtCK, and the concentration was 52.8-1 442.2 ng/mL. Conclusions The established double antibody sandwich ELISA can be used to determine uMtCK in human serum.

Key words: Mitochondrial creatine kinase, Ubiquitous subtype, Enzyme-linked immunosorbent assay