Figure 1. Binding of monoclonal antibodies against human E-selectin to CHO E-sel (transfected cells) and CHO (control cells). Cells (1.5x103 to 105 per well) were coated onto microtiter plates and incubated with the mAb (2 mg/ml). Binding was quantified with anti-mouse Ig-HRPO conjugate. The data points are average of triplicates and error bars indicate standard deviation.

Carbohydrate probes were synthesized as analytical tools for study of cellular lectins, i.e., Sug-polyacrylamide-³H, Sug-PAA-biotin, Sug-PAA-fluorescein (flu), and Sug-PAA-digoxigenin, where PAA is a soluble polyacrylamide carrier of ~30 kDa. Binding of all types of probes, where Sug is the sialyl Lewis X (SiaLe^x) tetrasaccharide or a blank saccharide, was assessed using Chinese hamster ovary (CHO) cells either transfected with the E-selectin cDNA or mock-transfected.
High binding of SiaLe^x-PAA-³H to E-selectin-transfected cells and absence of binding to control cells (both native and permeabilized) allowed the conclusion that the polyacrylamide carrier and the spacer arm do not contribute to the binding. The biotinylated probe showed a nonspecific binding in cell enzyme-linked assays. A similarly built digoxigenin-labeled probe was significantly better. In flow cytometry assays, the fluorescein probe demonstrated a specific binding to E-selectin-transfected cells of a similar level to that given by an anti-E-selectin antibody. It could be inhibited by the anti-E-selectin antibody, further demonstrating specificity. Tumors were obtained from nude mice by injection of CHO E-selectin or mock-transfected cells. The fluorescent SiaLe^x-PAA-flu probe could bind to tumor sections from E-selectin-positive CHO cells, but not from control CHO cells. These probes can thus be used to reveal specifically complex carbohydrate-binding sites on cells either in culture or on tissue sections.

Figure 2. (a) Binding of SiaLex-PAA-3H to native and permeabilized cells. Cells (1.5x103 to 2x105) were coated onto microtiter plates and incubated with the SiaLex-PAA-3H probe (11.5 mM of carbohydrate, 8.7x109 cpm/mmol SiaLex). The data points are average of triplicates and error bars indicate standard deviation. (b) Binding of SiaLex-PAA-biot to CHO E-sel (transfected cells) and CHO (control cells). Cells (1.5x103 to 105 per well) were coated onto microtiter plates and incubated with SiaLex-PAA-biot (6.6 mM of carbohydrate). Binding of biotinylated probe was quantified with streptavidin-HRPO conjugate. The data points are average of triplicates. (c) Binding of SiaLex-PAA-dig to CHO E-sel (transfected cells) and CHO (control cells). Cells (1.5x103 to 2x105 per well) were coated onto microtiter plates and incubated with SiaLex-PAA-dig (12.6 mM of carbohydrate). Binding of the probe was quantified with anti-digoxigenin-HRPO conjugate. The data points are average of the triplicates and error bars indicate standard deviation.

Figure 3. Binding of SiaLex-PAA-3H to native and permeabilized cells. Cells (1.5x103 to 2x105) were coated onto microtiter plates and incubated with the SiaLex-PAA-3H probe (11.5 mM of carbohydrate, 8.7x109 cpm/mmol SiaLex). The data points are average of triplicates and error bars indicate standard deviation. CHO E-sel, white bars; CHO, dashed bars. Figure 4. Direct binding of SiaLex-PAA-flu and Lex-PAA-flu to CHO E-sel (transfected cells) and CHO (control cells). Cells (2x106 per ml) were incubated with the probes and then analyzed by flow cytometry. Table 1 Inhibition of Binding of SiaLex-PAA-flu to CHO E-sel Cells Inhibitor Concentration (mM) Inhibition (%) Lac-PAA 730 7 Lea-PAA 300 3 SiaLex-PAA 340 50 SiaLea-PAA 230 55 SiaLex-O(CH2)3NH2 279 67 SiaLex-O(CH2)3NH2 139 55 SiaLex-O(CH2)3NH2 70 17 Anti-albumin CD 62E mAb 10 (mg/ml) 87 Note. Inhibition of probe binding to cells was determined by flow cytometry. The concentration of inhibitors correspond to carbohydrate concentrations of the neoglyconjugates. The percentage of inhibition was calculated as (MFA-MF1) x 100/(MFA) where MFA is the mean value of fluorescence in absence of inhibitor and MF1 is the mean value of fluorescence in presencee of inhibitor. See: O.E. Galanina, A.B.Tuzikov, E.Rapoport, J. Le Pendu, N.V. Bovin. Carbohydrate-based probes for detection of cellular lectins. Analyt. Biochem. 265, 282-289 (1998) [Abstract]

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Fluorescent particles

Fluorescent particles (commercial material coated with streptavidin) after instant "glycosylation" with SiaLe^a-PAA-biot specifically bind activated (E-selectin expressing) HUVECs, see fluorescent microscopy data, Figure 5.

Figure 5. Stimulated HUVECs + SiaLea-PAA-flu BEADS	Unstimulated HUVECs

Figure 6.

Magnetic beads

Magnetic beads coated with neoglycoconjugate were assessed for their ability to selectively isolate human cells with known anti-carbohydrate reactivity. Four lung cancer cell lines, NCI-H146, NCI-N417D, SKMES-I, EKVX; two acute lymphoblastic leukemia lines, MOLT-4 and CCRF-CEM; and the anti- Le^c hybridoma, LU-BGRU- G7, were tested. Sug-PAA bound uniformly to streptavidin coated magnetic beads as demonstrated by FITC labeled lectin. The anti- Le^c hybridoma cell line, LU BCRU-G7, demonstrated binding only to Le^c coated magnetic beads. Subsequent incubation in the presence of Le^c resulted in the release of the beads from the cell surface. Although there was some heterogeneity within the individual lung and leukemic cell lines, positive cells showed strong rosette formation with the coated beads. The A_di disaccharide coated beads showed binding in all four lung cancer cell lines, with the Le^c and the H (type1)-bead conjugates only reactive in the N417 and H146 SCLC lines. The range of L-selectin ligand-coated beads were all successful in binding to the acute lymphoblastic leukemia cell lines MOLT4 and CCRF-CEM. This approach provides a versatile model for the study of cell-surface carbohydrate interactions that should find application in many areas of cell biology.