Preparation and application of anti-ouabain IgY antibody / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To prepare highly specific anti-ouabain polyclonal antibody for detecting endogenous ouabain in tissues.</p><p><b>METHODS</b>Ouabain-BSA compound was used to immunize hens, and the eggs were collected one week after the first immunization. The IgY antibodies in the egg yolk were separated and purified by PEG-6000 Method, and analyzed by 12% SDS-PAGE and enzyme-linked immunosorbent assay (ELISA) for titration. The IgY antibodies obtained were applied subsequently in ELISA and immunohistochemistry.</p><p><b>RESULTS</b>The IgY titer increased rapidly after the second immunization, with the highest titer of 110240 that lasted for at least 4 weeks. Competitive ELISA for IgY detection showed an average intraassay coefficient of variation (CV) of 2.03% and an inter-assay CV of 2.34%. Immunohistochemistry visualized the location of the endogenous ouabain mainly in the cytoplasm of the zona reticularis of rat adrenal cortex.</p><p><b>CONCLUSION</b>Immunization of hens allows efficient preparation of IgY antibody which can be used in routine immunoassays.</p>