Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes

and host cells

Denong Wang et alPresented by Chao Ji

• 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

• 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 epitope specificity– Model system to immobilize carbohydrate antigens and study

their immunological property

• 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

• 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

• 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

4 Classes1 polysaccharide: 292 glycosaminoglycan: 33 glycoprotein: 114 semisynthetic glycoconjugate: 5

• Characterizing previously unnkown specificity and cross-reactivity of carbohydrate/antibody interaction– 2 anti-dextran mAbs: 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

4.3.F1 16.4.12E

BSA(E1),KLM(E2) N Y

CS-B(A6) Y N

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