Denong Wang et al Presented by Chao Ji

1. Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells Denong Wang et al Presented by Chao Ji

2. Carbohydrates play important role in microbe-host interaction (e.g. many epitopes are carbohydrates) • A carbohydrate-based microarray system sensitive enough to be able to detect a wide range of antibodies specificities • Is it feasible and good enough? • Whether carbohydrates can be immobilized on a glass surface without covalent bond • Whether immobilized carbohydrates preserve their immunological properties • Whether the proposed system reaches the sensitivity and capacity to detect a broad range of antibody specificities using only small quantities of sample • Whether it can be used to study carbohydrate interactions with high throughput

3. Structure of Dextran • Polymerization of glucose • Linear:α(1,6) • Branched:α(1,6), α(1,3) or α(1,2) • Representative of a variety of types of sugars • Recognizable by antibodies with different epitopespecificity • Model system to immobilize carbohydrate antigens and study their immunological property

4. Nitrocellulose-coated glass slides can be used to immobilize carbohydrates without covalent bonds • FITC-conjugated dextrans of different MW and insulin were printed on slides • Dextran with MW 20kDa-2,000kDa stably immobilized non-covalently • Larger dextran molecules were better retained

5. Immunological properties of dextrans are well-preserved when immobilized on nitrocellulose coated slides • Carbohydrate antigens: N279,both internal linear and terminal non-reducing end; B1299S, heavily branched; LD7, 100% internal linear • Antibodies: IgG3(4.3F1), groove-type, targets internal linear α(1,6) chain; IgA(16.4.12E), cavity-type targets terminal non-reducing end strucuture

6. The carbohydrate arrays is sensitive enough for the detection of a broad spectrum of antibody specificities • 48 structurally distinct carbohydrate-containing macromolecules • 20 serum specimens, 1μl from each for staining • 12/48 specificities of IgM and 35/48 of IgG • A speculation based on IgG > IgM: naturally occurring anti-carbohydrate antibodies may be of IgG type

7. 4 Classes 1 polysaccharide: 29 2 glycosaminoglycan: 3 3 glycoprotein: 11 4 semisyntheticglycoconjugate: 5

8. Characterizing previously unnkown specificity and cross-reactivity of carbohydrate/antibody interaction • 2 anti-dextranmAbs: 4.3.F1 and 16.4.12E applied on the same panel of carbohydrate • CS-B is recognized by 4.3.F1 (unexpectedly) because of its non-dominant structure rather than its dominant repeating disaccharide sequence of GalNAc(4S) • In vivo study showed the structure recognized by 4.3F1 is an endogenous cellular element • 4.3F1 staining is resistant to the pretreatment of tissue by dextranase • 45.21.1: another anti α(1-6) groove-type antibody that also reacted with CS-B

9. Conclusion • A sensitive and efficient carbohydrate microarray with large capacity • Only small quantity of specimen is necessary in the detection of antibody specificities • Broad applicability in a wide range of biomedical research involving carbohydrate-based molecular recognition