Production of Metallothionein Polyclonal Antibodies Using Chickens as Model

Abstract

The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.

IgY-Technology Applied to Studies of Toxoplasma gondii Infection

“…Sequential precipitation with 31% ammonium sulfate and 12% polyethylene glycol (PEG) produces IgY antibodies with above 95% purity and there is no loss in immunoreactivity [28]. The salt-based egg antibody precipitation combined with thiophilic acid chromatography provides a simple and efficient mean of IgY from egg yolk [29,30]. Association between AmS and ethanol to IgY precipitation results in a high quantity of antibodies with great purity [31].…”