Immunochemical Studies in Mannolipids

A specific method for determining α-mannosidase activity was developed using an enzymelinked immunosorbent assay (ELISA) and a specific polyclonal antibody which recognizes theterminal Man β1-4 structure of the reaction product, mannosylglucosylceramide (Man β1-4 Glc β1-ceramide, MlOse2Cer). In the assay, the dimannosylglucosylceramide substrate (Man α1-3 Man β1-4 Glc β1-ceramide, MlOse3Cer) immobilized on the solid phase of a 96-wellmicrotiter plate was incubated with Canavalia ensiformis α-mannosidase in citrate buffer containing detergent. The optimum conditions for the enzyme assay were as follows : buffer solution, 0.05 M citrate buffer (pH 4.04.5); detergent, sodium taurodeoxycholate (40 μg); enzyme concentration, 1.0 μg (1 mU); substrate concentration, 300 ng (300 pmole) : total reaction volume, 200 μL; incubation time, 1 h. Three sequential additions with washes between each were applied as follows : polyclonal antibody against the exposed Man β1-4 groups (anti-MlOse2Cer), peroxidase-conjugated anti-rabbit IgG antiserum against anti-MlOse2Cer, and peroxidase substrate. By this method, it became possible to quantitate the amount of reaction product, MlOse2 Cer, when present as low as 25 pmole.