Glycan Analysis of CD52-Fc Isoforms

We have previously (4 (link)) used Maackia amurensis and Sambucus nigra lectins to distinguish CD52-Fc glycans containing, respectively, sialic acid in α-2,3 and α-2,6 linkage with galactose (8 (link), 9 (link)). Here we used Maackia amurensis (MAA-I/MAL-I; Vector Laboratories, Burlingame, USA) to identify the α-2,3 linkage. A 96-well flat-bottom plate was coated with 20 μg/mL of MAL-1 overnight at 4°C and subsequently blocked with 200 μl of 1 % BSA for 1 h. After washing with PBS, CD52-Fc I, CD52-Fc II, or CD52-Fc III (20 μg/mL) were added and incubated at RT for 1 h and washed twice with PBS. After washing with PBS, 50 μl of a 1:1,000 dilution of HRP-conjugated antibody to CD52 (Campath H1; 1 μg/mL) was added and incubated at RT for 1 h. 50 μl of 3,3′5,5′-tetramethylbenzidine (TMB) substrate was added and color development stopped by addition of 50 μl of 0.5 M H₂SO₄. Absorbance was measured at 450 nm in a Multiskan Ascent 354 microplate photometer (Thermo Labsystems, San Francisco, USA).