Picogram detection levels of asialofetuin via the carbohydrate moieties using the light addressable potentiometric sensor

Fetal calf serum asialofetuin was assayed in the sandwich format using biotinylated and fluoresceinated ricin toxin (B-RCA and F-RCA). The sandwiched species was captured on a biotin-BSA coated nitrocellulose membrane with streptavidin. Anti-fluorescein antibody-urease conjugate was bound to the complex, and detected and quantitated under microvolume conditions using the light addressable potentiometric sensor. As little as 250 pg of asialofetuin was detectable whereas fetuin gave no response at conditions as high as 32 ng. Using a competitive inhibition assay, we established that the binding constant for the asialofetuin-ricin complex was 3.6 x 10(8) M-1. This is in good agreement with data published using glycopeptides derived from asialofetuin, and RCA and the ricin agglutinin, RCA-120.