Application of anti-GAG antibody and biotinylated hyaluronan binding protein(bHABP) [1]

~ Characteristics and epitopes for anti-heparan sulfate antibodies

Authors：

Category

Glycosaminoglycans

Protocol Name

Application of anti-GAG antibody and biotinylated hyaluronan binding protein(bHABP) [1]

~ Characteristics and epitopes for anti-heparan sulfate antibodies

Authors

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

Kaneiwa, Tomoyuki
Laboratory of Proteoglycan Signaling and Therapeutics, Faculty of Advanced Life Science, Hokkaido University

*To whom correspondence should be addressed.

KeyWords

sulfation epitope enzyme-linked immunosorbent assay

Reagents

●	Hep and HS from various sources
●	Biotinylated Hep/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 antibodies (Seikagaku Biobusiness Corp., Tokyo, Japan)
●	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)
●	Microplate reader (Bio-Rad Laboratories, Hercules, CA)
●	Aspirator

Methods

ELISA using Streptavidin plates

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

2)	Add 0.5 μg each of biotinylated Hep/HS in 50 μL of PBS to the wells and incubate at 4˚C overnight.	Comment 1

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

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

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

6)	Add 50 μL of an appropriate concentration of the anti-HS antibody to the wells, and incubate the plate 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

Figure & Legends

Clone	Antigen	Subtype	Specificity	References
F58-10E4	HS-proteoglycans from human fetal lung fibroblasts	mouse IgM	Mixed HS domains containing both N-acetylated and N-sulfated disaccharide units HS saccharide sequence including an N-unsubstituted glucosamine	David et al., 1992; Leteux et al., 2001; van den Born et al. 2005
HepSS-1	Murine methylcholanthrene-induced　fibrosarcoma	mouse IgM	N-Sulfated HS domains	Kure and Yoshie, 1986; van den Born et al., 2005
JM403	Rat glomerular HS-proteoglycans	mouse IgM	HS saccharide sequence rich in glucuronate and N-unsubstituted glucosamine residues	Leteux et al., 2001; van den Born et al., 1992
NAH46	N-Acetyl-heparosan from Escherichia coli K5	mouse IgM	HS saccharide sequence containing nonsulfated disaccharide units	Suzuki et al., 2008
F69-3G10	Heparitinase-digested HS-proteoglycans from human fetal lung fibroblasts	mouse IgG2b	HS oligosaccharide containing an unsaturated uronate at the nonreducing end	David et al., 199

Table 1. Commercially available anti-HS antibodies

Fig. 1. Reactivity of the anti-HS antibodies, F58-10E4 and HepSS-1, toward various HS/Hep preparations (A) and chemically modified Hep preparations (B).

Closed and open columns show the reactivity of F58-10E4 and HepSS-1, respectively. Two separate experiments were carried out and results are expressed as means ± SDs. Liver 1M HS and Liver 2 M HS are bovine liver HS preparations purified by anion-exchange chromatography with 1 M and 2 M LiCl, respectively. EHS 1M HS and EHS 2 M HS are HS preparations purified from mouse EHS chondrosarcoma by anion-exchange chromatography with 1 M and 2 M LiCl, respectively. BKHS and PIHS stand for bovine kidney and porcine intestinal HS, respectively. The disaccharide composition of these Hep/HS preparations is shown in Table 2. CDNS, CDNA, NDNA, 2ODS, and 6ODS represent completely desulfated N-acetylated Hep, completely desulfated N-sulfated Hep, N-desulfated N-acetylated Hep, 2-O-desulfated Hep, and 6-O-desulfated Hep, respectively.

Disaccharide				Bovine liver^b		EHS chondrosarcoma
	BKHS^a	PIHS	Hep	1M HS	2 M HS	1M HS	2 M HS
	%
ΔHexA^d-GlcNAc	54	46	5	38	0	30	32
ΔHexA-GlcNAc(6S)	12	12	4	9	2	0	0
ΔHexA-GlcN(NS)	21	32	5	23	11	67	61
ΔHexA-GlcN(NS,6S)	5	3	15	6	10	1	7^e
ΔHexA(2S)-GlcN(NS)	4	2	4	3	4	2	7^e
ΔHexA(2S)-GlcNAc(6S)	0	0	0	0	6	0	0
ΔHexA(2S)- GlcN(NS,6S)	4	4	67	21	67	0	0
total	100	100	100	100	100	100	100

Table 2. Disaccharide composition of various HS and Hep preparations

^aBKHS, bovine kidney heparan sulfate; PIHS, porcine intestine heparan sulfate; ΔHexA, GlcNAc, and GlcN stand for unsaturated hexuronic acid, N-acetyl-D-glucosamine, and D-glucosamine, respectively. 2S, 6S, and NS represent 2-O-sulfate, 6-O-sulfate, and 2-N-sulfate, respectively.

^bBovine liver 1 M and 2 M HS were purified by anion-exchange chromatography with 1 M and 2 M LiCl, respectively.

^cEHS chondrosarcoma 1 M and 2 M HS were purified from mouse EHS chondrosarcoma by anion-exchange chromatography with 1 M and 2 M LiCl, respectively.

^dTreatment with bacterial eliminases converts the original uronic acid structure in the polysaccharides into an artificial structure, the 4,5-unsaturated uronic acid, 4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid.

^eThe combined proportion of disulfated disaccharide units, ΔHexA-GlcN(NS,6S) and ΔHexA(2S)-GlcN(NS).

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Date of registration：2017-01-23 15:51:46