Application of anti-glycosaminoglycan antibody and biotinylated hyaluronan binding protein (bHABP) [2] ~ Determination of the concentration of heparan sulfate using enzyme-linked immunosorbent assay

HS from bovine kidney (Seikagaku Corp., Tokyo, Japan) or porcine intestine (Sigma-Aldrich, St. Louis, MO)

Biotinylated HS (Note 1)

0.01 M Phosphate-buffered saline, pH 7.4 (PBS)

PBS containing 0.05% Tween-20 (PBST)

25 mM of Tris buffered saline, pH 7.4 (TBS)

TBS containing 0.05% Tween-20 (TBST)

Bovine serum albumin (BSA)

Anti-HS antibody: F58-10E4 or HepSS-1 (Seikagaku Corp.) (Note 2)

The secondary antibody, anti-mouse IgG + IgM (H + L) conjugated with alkaline phosphatase (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD)

2 mg/mL of p-nitrophenyl phosphate (pNPP) in 50 mM of carbonate buffer, pH 9.8, containing 0.5 mM of MgCl₂

Streptavidin Plate C8 Transparent (Thermo Fisher Scientific)

Microplate reader iMark (Bio-Rad Laboratories, Hercules, CA)

Aspirator

Quantitative estimation of HS concentrations using competitive ELISA

a.

Wash the wells with 200 μL of PBS.

b.

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

c.

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

d.

Incubate the anti-HS antibody (diluted 1:150 in PBS containing 0.1% BSA) and various concentrations of HS in 50 μL of PBS in a test tube at room temperature for 30 min.

e.

Wash the wells with 200 μL of PBST three times.

f.

Add 50 μL of the anti-HS antibody or the mixture of the anti-HS antibody and HS to the wells.

g.

Incubate the plate at 37°C for 1 h.

h.

Wash the wells with 200 μL of TBST three times.

i.

Add 50 μL of the secondary antibody (diluted 1:2,000 in TBS) and incubate at 37°C for 1 h (Note 3).

j.

Wash the wells with 200 μL of TBST three times.

k.

Add 50 μL of the pNPP solution and incubate at room temperature (Note 4).

l.

Measure the absorbance at 415 nm with a microplate reader (Figure 1) (Note 5).

The biotinylation of HS preparations is conducted as follows. HS is dissolved in 100 mM of 2-(N-morpholino)ethanesulfonate buffer, pH 5.5, at a concentration of 2 mg/mL. The solution is mixed with a 50 mM solution of freshly prepared biotin-LC-hydrazide (Pierce) in dimethyl sulfoxide. The weight ratio of HS to biotin-LC-hydrazide is 20:1. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) hydrochloride, dissolved in the same buffer, is added to this mixture. The weight ratio of HS to EDAC hydrochloride is 8:1. The labeling reaction occurs overnight at room temperature by gently mixing the solution. The reaction mixture is dialyzed against PBS at room temperature for 24 h. Dialysis is performed in Spectra/Por molecular porous membrane tubing, with a molecular mass cutoff of 3,500 Da (Spectrum Medical Industries, Inc., Laguna Hills, CA). The hexuronic acid in the preparation is quantified by the carbazole method using glucuronate as a standard.

Seikagaku Corp. discontinued its research reagent business on Sept 30, 2011. Anti-HS antibodies are available from several companies, including Cosmo Bio Co., Ltd. Authentic HS preparations can be purchased from Sigma-Aldrich.

The antibody solution should be diluted just before use.

The pNPP stock solution (20 mg/mL) is diluted 20 times with 50 mM of carbonate buffer, pH 9.8, containing 0.5 mM of MgCl₂, just before use.

Although the wavelength of the maximum absorption for p-nitrophenol is 405 nm, the absorbance at 415 nm was measured because of the limitation of the filters available.