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Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Denong Wang et al Presented by Chao Ji. Carbohydrates play important role in microbe-host interaction (e.g. many epitopes are carbohydrates)

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slide1

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

Denong Wang et al

Presented by Chao Ji

slide2

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
slide3

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
slide4

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
slide5

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
slide6

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
slide7

4 Classes

1 polysaccharide: 29

2 glycosaminoglycan: 3

3 glycoprotein: 11

4 semisyntheticglycoconjugate: 5

slide8

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
conclusion
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