Follow-up Application of Antibody-Based Glycans Detection

- ELISA Using Biotinylated Glycans
Enzyme-linked immunosorbent assay (ELISA) is a highly sensitive quantitative method for detecting specific molecules using antigen-antibody reactions. ELISAs are used in a wide range of fields including immunology, biomarker discovery and quality control. ELISAs can also be used to detect a wide range of molecules. The following are our ELISA protocols using anti-glyco antibodies, HSA glycan conjugates and biotinylated glycans.

- ELISA Using HSA-Glycoconjugate

- Glycosphingolipid ELISA

- Detection of Chondroitin Sulfate A by Flow Cytometry
Glycosaminoglycans (GAGs) are polysaccharides that comprise repeated disaccharide structures of amino sugar and either uronic acid or galactose found on the surface of cells and in the extracellular matrix. Chondroitin sulfates are one example of GAGs; they can be found in various tissues such as cartilage, corneal tissue, and the brain, and are involved in various phenomena including moisture regulation, cell adhesion, and nerve regeneration. Roles for chondroitin in diseases such as breast cancer have also been the object of studies. As the instrumental analysis of polysaccharides, including GAGs, is known to be difficult, specific antibodies against GAGs are extremely useful as a detection tool. Flow cytometry (FCM) makes the expression analysis of target antigens such as GAGs in target cells quick and easy through the use of fluorescence-labeled antibodies, allowing for the characterization of individual cells.

1. Follow-up Application of Antibody-Based Glycans Detection

2. ELISA Using Biotinylated Glycans ELISA Using HSA- Glycoconjugate Glycosphingolipid ELISA Detection of Chondroitin Sulfate A by Flow Cytometry Table of Contents

3. ELISA Using Biotinylated Glycans Enzyme-linked immunosorbent assay (ELISA) is a highly sensitive quantitative method for detecting specific molecules using antigen-antibody reactions. ELISAs are used in a wide range of fields including immunology, biomarker discovery and quality control. ELISAs can also be used to detect a wide range of molecules. The following are our ELISA protocols using anti- glyco antibodies, HSA glycan conjugates and biotinylated glycans .

4. Reagents and Materials Streptavidin from  Streptomyces avidinii  (Product No.  S0951 ) Biotinylated glycans : L2-L2- β- PEG3-biotin (Product No.  G0516 ), L4-L4- β- PEG3-biotin (Product No.  G0517 ) Coating buffer: 0.1M Carbonate Buffer (pH8.5) Blocking solution: 1% BSA/TBST Plate washing buffer: TBST ( Tris -buffered saline with 0.05% Tween20) HRP Chromogenic substrate solution: TMB solution (Product No.  T3854 ), etc Primary antibodies: Anti-6-sulfo LacNAc Monoclonal Antibody (AG105) (Product No.  A3251 ), Anti-6,6'-disulfo LacNAc Monoclonal Antibody (L4L4-8) (Product No.  A3252 ) Secondary antibody: Goat Anti-Mouse IgG+M Secondary Antibody

5. Experimental Example Related Products Product Number Product Name S0951 Streptavidin from Streptomyces avidinii G0516 L2-L2- β- PEG3-biotin G0517 L4-L4- β- PEG3-biotin T3854 TMB [for ELISA] (Ready-to-use solution) A3251 Anti-6-sulfo LacNAc Monoclonal Antibody (AG105) A3252 Anti-6,6'-disulfo LacNAc Monoclonal Antibody (L4L4-8)

6. ELISA Using HSA- Glycoconjugate Reagents and Materials Human Serum Albumin-Gb 5  (= HSA-Gb 5 ) (Product No.  H1777 ) HSA (Human Serum Albumin) Coating buffer: 0.1 M Carbonate Buffer (pH 8.5) Blocking solution: 1% BSA/TBST Plate washing buffer: TBST ( Tris -buffered saline with 0.05% Tween 20) HRP Chromogenic substrate solution: TMB solution (Product No.  T3854 ), etc. Primary antibodies: Anti-SSEA-3 (Gb 5 ) Monoclonal Antibody (Product No.  A3729 ) Secondary antibody: Goat Anti-Mouse IgG+M HRP Conjugate Chemical Structure of HSA-Gb 5  (Product No.  H1777 )

7. Experimental Example Related Products Product Number Product Name H1777 HSA-Gb5 T3854 TMB [for ELISA] (Ready-to-use solution) A3729 Anti-SSEA-3 (Gb5) Monoclonal Antibody

8. Reagents and Materials Glycosphingolipid ELISA Glycolipid solution (Dissolved at 1 μ g/mL in ethanol) Blocking solution: 1% BSA (bovine serum albumin) in PBS Plate washing buffer: PBS (phosphate-buffered saline) HRP Chromogenic substrate solution: TMB solution (Product No.  T3854 ), etc. Primary antibodies: Anti- Globo -H Monoclonal Antibody (Product No.  A3703 ), Anti-SSEA-4 (SialylGb 5 ) Monoclonal Antibody (Product No.  A3742 ), Anti-SSEA-3 (Gb 5 ) Monoclonal Antibody (Product No.  A3252 ), Anti-Gb 4  Monoclonal Antibody (Product No.  A3776 ), Anti-Gb 3  Monoclonal Antibody (Product No.  A2506 ) Secondary antibody: Goat Anti-Mouse IgG+M HRP Conjugate

9. Experimental Example Specificity analysis of antibodies using various glycolipids Related Products Product Number Product Name T3854 TMB [for ELISA] (Ready-to-use solution) A3703 Anti-Globo-H Monoclonal Antibody A3742 Anti-SSEA-4 (SialylGb5) Monoclonal Antibody A3729 Anti-SSEA-3 (Gb5) Monoclonal Antibody A3776 Anti-Gb4 Monoclonal Antibody A2506 Anti-Gb3 Monoclonal Antibody

10. Detection of Chondroitin Sulfate A by Flow Cytometry Glycosaminoglycans (GAGs) are polysaccharides that comprise repeated disaccharide structures of amino sugar and either uronic acid or galactose found on the surface of cells and in the extracellular matrix. Chondroitin sulfates are one example of GAGs; they can be found in various tissues such as cartilage, corneal tissue, and the brain, and are involved in various phenomena including moisture regulation, cell adhesion, and nerve regeneration. Roles for chondroitin in diseases such as breast cancer have also been the object of studies. As the instrumental analysis of polysaccharides, including GAGs, is known to be difficult, specific antibodies against GAGs are extremely useful as a detection tool. Flow cytometry (FCM) makes the expression analysis of target antigens such as GAGs in target cells quick and easy through the use of fluorescence-labeled antibodies, allowing for the characterization of individual cells.

11. Reagents and Materials MCF-7 cells (* MCF-7 cell line (JCRB0134, H. D. Soule  et.al. ) was obtained from JCRB Cell Bank.) Mouse IgM isotype control (10 μg /mL dilution) Anti-Chondroitin Sulfate A Monoclonal Antibody (LY111) (Product No.  A3143 ) (10 μg /mL dilution) Goat Anti-Mouse IgM R-PE Conjugate (2.5 μg /mL dilution) Phosphate-Buffered Saline (PBS) Trypsin/EDTA Cell culture medium for MCF-7 0.5% BSA-containing PBS

12. Experimental Example Flow Cytometric Analysis. MCF-7 cells were reacted with anti-Chondroitin Sulfate A antibody (Product No.  A3143 ) (red line) or isotype control (blue line), then stained with fluorescent secondary antibody and analyzed by flow cytometer. Related Product Product Number Product Name A3143 Anti-Chondroitin Sulfate A Monoclonal Antibody (LY111)

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