Fast purification of trace vitellogenin from Chinese rare minnow using protein A-immobilized antibody.
Vitellogenin (Vtg) is a highly responsive biomarker for environmental exposure to various estrogenically active compounds. Here we present a simple, fast, mild, and stable immobilization of anti-Vtg antibody, and demonstrate its powerful applications for preconcentration and purification of fish Vtg proteins, allowing for the monitoring and screening of environmental exposure to estrogenically active compounds. In this immobilization method, rabbit antiserum containing a specific polyclonal antibody against Vtg was directly immobilized on an antibody-binding Staphylococcal protein A matrix (SpA) without the need for prior purification. Under the unique elution conditions, the Vtg protein can be eluted out alone without any leaked specific antibody. The developed method was further used to purify Vtg from whole-body homogenate of Chinese rare minnow. Compared with previous purification methods, the isolated Vtg fraction by this method displays higher purity and well-preserved structure integrity. Moreover, our method is eight times faster. The simple one-step protein A-based specific antibody immobilization and its associated elution strategy may be extended to a number of antibodies for various application purposes, highlighting the paramount advantages over traditional immunoprecipitation and covalent immobilization of antibodies, and suggesting a wide range of promising applications in environmental monitoring and proteome analysis.
Song
Maoyong
M
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing, 100085, China.
Lv
Xuefei
X
Wang
Hailin
H
Jiang
Guibin
G
D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western; D002681:China; D003530:Cyprinidae; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000737:chemistry; D013997:Time Factors; D014819:Vitellogenins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*
D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D014819:Vitellogenins