The development, purification and characterization of anti- vitellogenin antibodies raised in hens (Gallus gallus domesticus) against Tilapia (Oreochromis niloticus) vitellogenin protein

Abstract

This study was conducted to induce, isolate and characterize and determine the role of Vitellogenin (Vtg) which is used as a biomarker of environmental quality and steroidogenesis in oviparous organisms. We have purified Vtg from male adult tilapia scientifically known as Oreochromis niloticus previously treated with estradiol 5 μg/g body weight (BW). By using ion exchange chromatography Vtg was purified from blood plasma yielding 1.13 mg Vtg/ml. Subsequently, Lohmann Brown hens were immunized at a 125 and 100 μg Vtg/ml, including three antigen treatments of booster doses of 75 and 50 μg Vtg/ml, respectively. At day 30 post-immunization, posture eggs were collected, we have successfully optimized the extraction of anti-VtgIgY antibody. The purified anti-VtgIgY was validated with SDS-PAGE, detecting heavy and light chains of 70 kDa and 30 kDa, respectively. Further, we have developed ELISA using anti-VtgIgY. To estimate the Vtg specificity, commercial anti-Vtg antibodies of dorada (Sparusaurata) were used and validated for tilapia by Western Blot. Detection of the Vtg monomer was at a dilution of 1/2000 IgY primary antibody. The antibodies thus obtained through the conventional techniques of production and validation of IgY antibodies for Vtg of tilapia demonstrate that they are viable under local conditions, can be successfully used for programs of environmental monitoring and reproduction control in oviparous species.