JCGGDB TOP GlycoScience Protocol Online Database

Insert title here

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

~ Determination of the concentration of heparan sulfate using ELISA

Authors： Q&A(0) |  Feedback(0)
Introduction Protocol References Credit lines
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
 Shuhei Yamada
Laboratory of Proteoglycan Signaling and Therapeutics, Faculty of Advanced Life Science, Hokkaido University
Keywords

biotinylation

antibody

enzyme-linked immunosorbent assay

Reagents
●

HS from bovine kidney (Seikagaku Corp.) or porcine intestine (Sigma)

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.)

●

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)
●

Heparin/GAG Binding Plates (Iduron Co.)
●

Microplate reader (Bio-Rad)
●

Aspirator
Methods
1.

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 hours.

 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 hour.

 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 hour.

 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
2.

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 hour.

 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 hour.

 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 hour.

 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

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 Creative Commons License   Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
Date of registration：2012-02-14 10:50:55
©2010 - 2012 Ritsumeikan University, AIST & JCGGDB. All Rights Reserved