The development and validation of a homologous tilapia vitellogenin enzyme linked immunosorbent assay (t-VTG-ELISA) as biomarker of estrogenic exposure

Water is essential to all life but many freshwater resources are polluted through human activities. Humans and wildlife are exposed to a wide range of contaminants through their water, many of which pose a risk to health. Some of the contaminants released into the environment have been reported to have the capability to disrupt the endocrine functions in humans and wildlife and they can mimic or antagonise the action of estrogenic. These endocrine disrupting chemicals (EDCs) interact with physiological systems and cause alterations in development, growth and reproduction in wildlife and humans. To achieve some measure of assessing the potential harm that the contaminants pose, we need to know the environmental concentration of the chemical concerned and to monitor their effect on the organisms. The water supply sector need to include EDCs in standard systems of routine water source monitoring which include indicator bacteria and nutrient species but before the system can be incorporated, methods to measure the occurrence of EDCs in aquatic environment need to be developed and validated and a reliable guidelines data need to be in place. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) to quantify vitellogenin (VTG) in Oreochromis mossambicus (Mozambique tilapia) VTG has been used successfully as a biomarker for estrogenic contamination in different studies. For this study, VTG was isolated and purified from plasma of 17β-estradiol exposed tilapia by gel filtration chromatography. The purity of the VTG isolate was confirmed by polyacrylamide gel electrophoreses (SDS-PAGE). Polyclonal antibodies against t-VTG were raised in rabbits and the specificity of the anti-t-VTG was confirmed by western blot. Using purified t-VTG as a standard and anti-t-VTG antibody, a homologous competitive ELISA was developed and validated. The standard curves of the ELISA, which were generated on different days, were identical which indicate that the assay is reliable, reproducible and repeatable. The intra-assay and inter-assay coefficient variation was 2.41 (n = 4) and 8.71 (n = 10) respectively. The serial dilution of plasma VTG from exposed tilapia showed a good parallelism with the standard t-VTG within the working range of the assay. The serial dilution of the reference fish did not cover the whole range of the t-VTG standard curve. By using the standard curve and the dilution of the exposed plasma, we were able to demonstrate that the ELISA was able to quantify VTG. With good laboratory practise, this ELISA can be use to quantify VTG in chemically exposed fish. It will also be ideal to continue analyzing the antibody to determine the appropriate dilutions necessary to ensure that the assay work its optimal capabilities.Dr. I. Barnhoorn Prof. P. Jagals Prof. J.H.J. Van Vure