Application of anti-GAG antibody and biotinylated hyaluronan binding protein(bHABP) [2]

~ Determination of the concentration of heparan sulfate using ELISA

Authors：

Category

Glycosaminoglycans

Protocol Name

Application of anti-GAG antibody and biotinylated hyaluronan binding protein(bHABP) [2]

~ Determination of the concentration of heparan sulfate using ELISA

Authors

Yamada, Shuhei
Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University

KeyWords

biotinylation antibody enzyme-linked immunosorbent assay

Reagents

●	HS from bovine kidney (Seikagaku Corp., Tokyo, Japan) or porcine intestine (Sigma-Aldrich, St. Louis, MO) Biotinylated HS
●	0.1 M Phosphate-buffered saline (PBS)
●	PBS containing 0.05% Tween-20 (PBST)
●	25 mM Tris buffered saline (TBS)
●	TBS containing 0.05% Tween-20 (TBST)
●	Bovine serum albumin (BSA)
●	Anti-HS antibody, F58-10E4 or HepSS-1 (Seikagaku Corp.)
●	The secondary antibody, anti-mouse IgG+IgM (H+L) conjugated with alkaline phosphatase (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD)
●	2 mg/ml p-Nitrophenyl phosphate (pNPP) in 50 mM carbonate buffer, pH 9.8, containing 0.5 mM MgCl₂

Instruments

●	Streptavidin Plate C8 Transparent (Nunc, Roskilde, Denmark)
●	Heparin/GAG Binding Plates (Iduron Ltd., Manchester, UK)
●	Microplate reader (Bio-Rad Laboratories, Hercules, CA)
●	Aspirator

Methods

ELISA using Heparin/GAG Binding Plates

1)	Wash the wells with 200 μL of PBS.	Comment 0

2)	Add HS/50 μL of PBS to the wells and incubate at room temperature overnight.	Comment 0

3)	Wash the wells with 200 μL of PBS.	Comment 0

4)	Add 200 μL of 1% BSA/PBS for blocking and incubate at 37˚C for 1.5 h.	Comment 0

5)	Wash the wells with 200 μL of PBST three times.	Comment 0

6)	Add 50 μL of the anti-HS antibody (diluted 1:150 in PBS) and incubate at 37˚C for 1 h.	Comment 1

7)	Wash the wells with 200 μL of TBST three times.	Comment 0

8)	Add 50 μL of the secondary antibody (diluted 1:2,000 in TBS) and incubate at 37˚C for 1 h.	Comment 1

9)	Wash the wells with 200 μL of TBST three times.	Comment 0

10)	Add 50 μL of the pNPP solution and incubate at room temperature.	Comment 1

11)

Measure the absorbance at 415 nm with a microplate reader (Fig. 1).

Comment 0

Competition ELISA using streptavidin-coated plates

1)	Wash the wells with 200 μL of PBS.	Comment 0

2)	Add 2 μg each of biotinylated HS/50 μL of PBS to the wells and incubate at 4˚C overnight.	Comment 1

3)	Add 200 μL of 3% BSA/PBS for blocking and incubate at room temperature for 1 h.	Comment 0

4)	Incubate the anti-HS antibody (diluted 1:150 in PBS) and various concentration of HS in 50 μL of PBS in a test tube at room temperature for 30 min.	Comment 0

5)	Wash the wells with 200 μL of PBST three times.	Comment 0

6)	Add 50 μL of the anti-HS antibody or the mixture of the anti-HS antibody and HS to the wells.	Comment 1

7)	Incubate the plate at 37˚C for 1 h.	Comment 0

8)	Wash the wells with 200 μL of TBST three times.	Comment 0

9)	Add 50 μL of the secondary antibody (diluted 1:2,000 in TBS) and incubate at 37˚C for 1 h.	Comment 1

10)	Wash the wells with 200 μL of TBST three times.	Comment 0

11)	Add 50 μL of the pNPP solution and incubate at room temperature.	Comment 1

12)

Measure the absorbance at 415 nm with a microplate reader (Fig. 2).

Comment 0

Figure & Legends

Fig. 1. The anti-HS antibody F58-10E4 interacts with HS in a dose-dependent manner.

Various concentrations of bovine kidney HS were immobilized on a Heparin/GAG Binding Plate from Iduron, and the binding was detected using F58-10E4, and then alkaline phosphatase-conjugated anti-mouse IgG/IgM secondary antibody. pNPP was incubated for 60 min, and the values represent the mean (n = 3).

Fig. 2. The interaction of the anti-HS antibody F58-10E4 with biotinylated HS immobilized on a streptavidin-coated plate was inhibited by HS in a dose-dependent manner.

Biotinylated porcine intestinal HS was immobilized on the plate, and F58-10E4 preincubated with various concentrations of unlabeled porcine intestinal HS was added. The binding of the anti-HS antibody was detected with an alkaline phosphatase-conjugated anti-mouse IgG/IgM secondary antibody. pNPP was incubated for 60 min, and the values represent the mean (n = 2).

Copyrights

Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses

Date of registration：2017-01-23 15:52:31