Detection of human asialo-alpha(1)-acid glycoprotein using a heterosandwich immunoassay in conjunction with the light addressable potentiometric sensor.
Highly specific detection of human alpha 1-acid glycoprotein (AGP) and asialo-alpha 1-acid glycoprotein (asialo-AGP) was made possible by use of a sandwich immunoassay. The glycoproteins were sandwiched between biotinylated and fluoresceinated polyclonal rabbit anti-human AGP antibodies. Additionally, asialo-AGP could be distinctly detected, apart from AGP, via the formation of a heterosandwich immunoassay using biotinylated polyclonal rabbit anti-human AGP and the lectin, fluoresceinated ricin toxin. Streptavidin was added to the formed immunocomplexes and the immunocomplexes captured on a biotinylated nitrocellulose membrane. The signal generator, urease conjugate of an anti-fluorescein antibody, was then bound to the complex on the membrane. The rate of pH change under microvolume conditions (0.6 microliters) was monitored using a silicon chip-based, light addressable potentiometer sensor. Results indicated that AGP and asialo-AGP can be detected to the 2 pg level when two antibodies are used to form the immunocomplex. Asialo-AGP can be detected down to 250 pg when the heterosandwich immunoassay is used; this assay exhibited no response up to 10 ng for native AGP or asialofetuin. Both immunoassays can be used to quantify the level of AGP and asialo-AGP in solution. Although the assay presented is very specific for AGP, asialo-AGP and terminal galactose, it is readily adaptable for the detection of any glycoprotein and terminal carbohydrate (or branched structure) by use of a protein-specific antibody and various lectins.
Dill
K
K
Molecular Devices Corporation, Sunnyvale, CA 94089, USA.
Bearden
D W
DW
D000818:Animals; D001212:Asialoglycoproteins / Q000032:analysis*; D015374:Biosensing Techniques; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008027:Light; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000032:analysis; D011817:Rabbits
D001212:Asialoglycoproteins; D009961:Orosomucoid; C017030:asialoorosomucoid