[Preparation and application of anti-ouabain IgY antibody].
OBJECTIVE
To prepare highly specific anti-ouabain polyclonal antibody for detecting endogenous ouabain in tissues.


METHODS
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.


RESULTS
The IgY titer increased rapidly after the second immunization, with the highest titer of 1:10240 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.


CONCLUSIONS
Immunization of hens allows efficient preparation of IgY antibody which can be used in routine immunoassays.
Zhang
Ming-juan
MJ
Department of Cardiology, Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710004, China.
Yang
Jun
J
Duan
Zong-ming
ZM
Qiang
Lei
L
D000818:Animals; D002138:Calibration; D002417:Cattle; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D007114:Immunization / Q000379:methods*; D007136:Immunoglobulins / Q000276:immunology*; D007150:Immunohistochemistry; D010042:Ouabain / Q000032:analysis / Q000276:immunology*; D051381:Rats
C016512:IgY; D007136:Immunoglobulins; D010042:Ouabain