Production of monoclonal antibodies against Chinemys reevesii turtle vitellogenin and their usage for comparison of biochemical and immunological characters of vitellogenins and yolk proteins of freshwater turtles

Abstract

Four hybridoma clones (ACV-1, -3, -4, and -5) were established for Chinemys reevesii (Reeves' turtle) vitellogenin (VTG) as a precursor protein of egg yolk and a biomarker of environmental pollution. Binding-inhibition experiments indicated that the epitopes of four mAbs were distinct. No binding of ACV-4 to C. reevesii VTG in the Western blot suggests that the epitope of ACV-4 would be dependent on the three-dimensional structure. ACV-1, -3, and -5 bound to C. reevesii VTG in the Western blot. The signal for ACV-1 and -5 disappeared by reduction of the VTG, suggesting that the construction of the epitopes for ACV-1 and -5 were dependent on the disulfide bridge in the VTG molecule. All four mAbs recognized Trachemys scripta and Mauremys japonica VTGs in the ELISA. The yolk proteins were tested for the binding of the mAbs in the Western blot. ACV-1 being capable to bind to the VTG in the reduced condition did not bind to any protein bands of the yolk. This indicates that ACV-1 recognizes a part of the VTG molecule that is not incorporated in the oocytes. Both ACV-3 and -5 bound to the 32- and 70-kDa yolk proteins. Since a mAb recognizes only one site (epitope) on a protein molecule, the 32-kDa protein originated from the 70-kDa one. An ELISA system using ACV-5 as the capture antibody and ACV-3 as the detecting antibody showed the lower detectable concentration (2 ng/mL) and a wide detectable range to 1000 ng/mL ( R 2 = 0.999). The system was used to determine serum VTG levels of juvenile turtles treated with estradiol-17β or vehicle (corn oil). By the use of the mAbs described in this paper, basic and applied studies for turtle VTGs would be improved.