Detection of tilapia metallothionein using antibody-immobilized quartz crystal microbalance sensor

Abstract

Metallothionein (MT) was induced in tilapia by three subcutaneous injections of CdCl2 and the recovered MT protein was purified to homogeneity through DEAE-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration. For use as biological component, a polyclonal anti-tilapia MT antibody was prepared using the purified tilapia MT. During the preparation of the tilapia MT sensor, topological changes pertinent to immobilization of the thiolated antibody, subsequent blocking of the surface with BSA and final immune reaction were observed by AFM imaging. Specificity of the sensor was confirmed through comparison of the responses of defective and intact sensor chips, and the responses of the sensor chips that were immobilized with the antibody and BSA. Concentration-dependent responses of the sensor showed a linear relationship of Y (log10 sensor response)=0.3438X (log10 tilapia MT)+0.7368 (r=0.9606) in double-logarithmic plot with a limit of detection of 20ng/mL. When the serum from a tilapia induced with CdCl2 was compared with the control serum, induction of MT was detectable even at 20,000-fold dilution of tilapia serum.