Anti-chondroitin sulfate antibodies and their utility for enzyme-linked immunosorbent assay and western blotting

Tadahisa Mikami; Hiroshi Kitagawa

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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.

Anti-chondroitin sulfate antibodies and their utility for enzyme-linked immunosorbent assay and western blotting

Tadahisa Mikami, Ph.D.

Kobe Pharmaceutical Univ.

Email: pj.ca.u-amrahpebok@imakimt

Hiroshi Kitagawa, Ph.D.

Kobe Pharmaceutical Univ.

Email: pj.ca.u-amrahpebok@awagatik

Corresponding author.

Created: October 2, 2021; Last Revision: March 15, 2022.

Introduction

Chondroitin sulfate (CS) chains are representative sulfated glycosaminoglycan (GAG) polysaccharides comprising repeating glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) disaccharide units. In vivo, CS GAG chains are covalently linked to specific core proteins to form CS proteoglycans (CSPG). CSPGs are principal microenvironmental components that regulate various cellular events, such as cell adhesion, proliferation, differentiation, and cell fate decision (1–3). A wide spectrum of CSPG functions is mainly attributed to the sulfation pattern-based, structural divergence of the GAG moieties (1–3). This is because the precursor building blocks of CS chains could be enzymatically sulfated at various positions, thereby forming diverse structures with various combinations of the characteristic disaccharide units O, A, C, D, and E (Figure 1). Therefore, the characterization of CS side chains of CSPGs is an essential task for understanding the biological importance of CSPGs.

Protocol

To date, various anti-CS antibodies are commercially available (Table 1). In this section, the experimental utility of anti-CS antibodies 1) for enzyme-linked immunosorbent assay (ELISA) against immobilized CS polymers and 2) for western blotting to detect CSPGs is described.

Materials

1.: Biotinylated chondroitin sulfate A (whale cartilage, CS-A, PG Research, Tokyo, Japan) (Note 1)
2.: Biotinylated chondroitin sulfate C (shark cartilage, CS-C, PG Research)
3.: Biotinylated chondroitin sulfate D (shark cartilage, CS-D, PG Research)
4.: Biotinylated chondroitin sulfate E (squid cartilage, CS-E, PG Research)
5.: Anti-CS monoclonal antibody (2B6, mouse immunoglobulin G₁ [IgG₁] Cosmo Bio, Tokyo, Japan)
6.: Anti-CS monoclonal antibody (2H6, mouse IgM, Cosmo Bio)
7.: Anti-CS monoclonal antibody (LY111, mouse IgM, Tokyo Chemical Industry, Tokyo, Japan)
8.: Anti-CS monoclonal antibody (MO-225, mouse IgM, Tokyo Chemical Industry)
9.: Goat HRP-conjugated anti-mouse IgM (Sigma-Aldrich, St. Louis, MO)
10.: Goat HRP-conjugated anti-mouse IgG + IgM (Jackson ImmunoResearch, West Grove, PA)
11.: ABTS microwell peroxidase substrate (SeraCare Life Sciences, Milford, MA)
12.: Assay buffer (50 mM of sodium acetate, 100 mM of NaCl, 0.2% (v/v) Tween-20, pH 7.2)
13.: Bovine serum albumin (BSA, Fujifilm, Osaka, Japan)
14.: Blocking buffer (Assay buffer containing 1% (w/v) BSA)
15.: Chondroitinase ABC (Proteus vulgaris, Seikagaku, Tokyo, Japan) (Notes 2 and 3)
16.: 5× chondroitinase reaction buffer (250 mM of Tris, 300 mM of sodium acetate, pH 8.0)
17.: 4× Bolt™ lithium dodecyl sulfate (LDS) sample buffer (Thermo Fisher Scientific, Waltham, CA)
18.: 10× Bolt™ sample reducing agent (500 mM of dithiothreitol, Thermo Fisher Scientific).
19.: 20× Bolt™ MOPS sodium dodecyl sulfate (SDS) running buffer (Thermo Fisher Scientific).
20.: Precast polyacrylamide gel (Bolt™ 4%–12% Bis-Tris Plus, Thermo Fisher Scientific)
21.: Polyvinylidene fluoride (PVDF) blotting membrane (Hybond™, 0.45 µm, Cytiva, Tokyo, Japan)
22.: Blocking One (blocking buffer for Western blotting, Nacalai tesque, Kyoto, Japan)
23.: Dilution buffer (Signal enhancer HIKARI, Nacalai tesque)
24.: Enhanced luminol-based chemiluminescence substrate (Chemi‐Lumi One Ultra, Nacalai tesque)
25.: Transfer buffer (25 mM of Tris, 192 mM of glycine, and 20% (v/v) methanol)
26.: TBST (25 mM of Tris, 137 mM of NaCl, 2.68 mM of KCl, 0.1% (v/v) Tween-20, pH 7.4)

Instruments

1.: Avidin-coated 96-well plate (Sumitomo Bakelite, Tokyo, Japan)
2.: Microplate reader (iMark™, Bio-Rad, Hercules, CA)
3.: Vertical mini-gel electrophoresis apparatus (Mini gel tank, Thermo Fisher Scientific)
4.: Tank blotter (Mini-Protean II Cell, Bio-Rad)
5.: Power supply (PowerPac™ HC, Bio-Rad)
6.: Luminoimage analyzer (ImageQuant 800, Cytiva)

Methods

1.

ELISA against immobilized CS polymers

a.: Prepare biotinylated CS solution (0.01–100 µg/mL in assay buffer).
b.: Add 100 µL of CS solution to each well of an avidin-coated 96-well plate.
c.: Incubate overnight at 4°C.
d.: Wash the wells thrice with assay buffer.
e.: Add 200 µl/well of blocking buffer and incubate at room temperature for 1 h.
f.: Wash the wells thrice with assay buffer.
g.: Add 100 µl/well of an anti-CS antibody diluted with blocking buffer and incubate at room temperature for 1 h (Note 4).
h.: Wash the wells thrice with assay buffer.
i.: Add 100 µl/well of HRP-conjugated anti-mouse IgM diluted 5,000-fold with blocking buffer and incubate at room temperature for 30 min.
j.: Wash the wells thrice with assay buffer.
k.: Add 100 µl/well of ABTS microwell peroxidase substrate and incubate at room temperature for ≤ 30 min.
l.: Measure the absorbance at 410 nm with a microplate reader (Figure 2).

2.

Western blotting for detecting CSPGs

a.: Incubate CSPG samples at 37°C for 2 h with or without 20 mIU of chondroitinase ABC (Notes 5 and 6).
b.: Prepare 40 µL (total volume) of CSPG samples by mixing with 10 µL of 4× Bolt™ LDS sample buffer and 4 µL of 10× Bolt™ sample reducing agent (Note 7).
c.: Heat the samples at 70°C for 10 min to denature proteins.
d.: Prepare 1× Bolt™ MOPS SDS running buffer, and set a precast polyacrylamide gel to a vertical mini-gel electrophoresis apparatus.
e.: Apply the heat-treated CSPG sample (10 µl/lane) onto each lane of the precast gel.
f.: Electrophorese at constant voltage (200 V).
g.: Incubate the gel in transfer buffer for 5 min.
h.: Transfer the CSPGs from the gel to the PVDF blotting membrane at constant voltage (100 V) for 90 min using a tank blotter.
i.: Incubate the membrane in Blocking One at room temperature for 30 min.
j.: Wash the membrane twice with TBST.
k.: Incubate the membrane with anti-CS antibody diluted with dilution buffer at room temperature for 2 h (or overnight at 4°C) (Note 4).
l.: Wash the membrane thrice with PBST.
m.: Incubate the membrane with HRP-conjugated secondary antibody diluted 5,000–10,000-fold with dilution buffer at room temperature for 1 h.
n.: Wash the membrane thrice with TBST.
o.: Briefly immerse the membrane in an enhanced luminol-based chemiluminescence substrate and detect CSPGs and/or their GAG moieties using a luminoimage analyzer (Figure 3).

Notes

1.: CS-A, CS-C, CS-D, and CS-E correspond to CS polysaccharide preparations rich in disaccharide units, A, C, D, and E, respectively. Since all of them are derived from natural biological resources, the disaccharide composition of each polysaccharide preparation is heterogenous.
2.: Chondroitinase ABC is currently available from Cosmo Bio or Sigma-Aldrich.
3.: Chondroitinase ABC cleaves CS chains by eliminating the GalNAc-GlcA linkages, thereby generating unsaturated disaccharide neoepitopes (∆Di-0S, ∆Di-4S, or ∆Di-6S) at nonreducing terminal “CS stub” region of CSPG core protein (Figure 3).
4.: Optimum working dilution for each experimental condition should be determined by each researcher.
5.: Endogenous CSPGs purified from biological samples (tissues, cell culture supernatants, cell extracts, sera, etc.) and/or their soluble, epitope-tagged recombinant forms can be readily applied to such analysis. Although crude extracts containing CSPGs can also be used, be aware that some buffer components, such as SDS and chaotropic agents, may interfere with the following chondroitinase reaction to cleave CS moieties of CSPGs in the extracts.
6.: The reaction mixture should be adjusted to contain one-fifth volume of 5× chondroitinase reaction buffer.
7.: A conventional SDS sample buffer can be used in place of the LDS sample buffer. Here, the following heat treatment should be conducted under typical conditions (95°C–100°C for 5 min).

References

1.: Mikami T, Kitagawa H. Biosynthesis and function of chondroitin sulfate. Biochim Biophys Acta. 2013 Oct;1830(10):4719–33. [PubMed: 23774590] [CrossRef]
2.: Kitagawa H. Using sugar remodeling to study chondroitin sulfate function. Biol Pharm Bull. 2014;37(11):1705–12. [PubMed: 25366475] [CrossRef]
3.: Mikami T, Kitagawa H. Sulfated glycosaminoglycans: their distinct roles in stem cell biology. Glycoconj J. 2017 Dec;34(6):725–735. [PubMed: 27709407] [CrossRef]
4.: Avnur Z, Geiger B. Immunocytochemical localization of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody. Cell. 1984 Oct;38(3):811–22. [PubMed: 6435883] [CrossRef]
5.: Ito Y, Hikino M, Yajima Y, Mikami T, Sirko S, von Holst A, Faissner A, Fukui S, Sugahara K. Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library. Glycobiology. 2005 Jun;15(6):593–603. [PubMed: 15625183] [CrossRef]
6.: Yamagata M, Kimata K, Oike Y, Tani K, Maeda N, Yoshida K, Shimomura Y, Yoneda M, Suzuki S. A monoclonal antibody that specifically recognizes a glucuronic acid 2-sulfate-containing determinant in intact chondroitin sulfate chain. J Biol Chem. 1987 Mar 25;262(9):4146–52. [PubMed: 2435733]
7.: Yada T, Arai M, Suzuki S, Kimata K. Occurrence of collagen and proteoglycan forms of type IX collagen in chick embryo cartilage. Production and characterization of a collagen form-specific antibody. J Biol Chem. 1992 May 5;267(13):9391–7. [PubMed: 1577766]
8.: Sugiura N, Shioiri T, Chiba M, Sato T, Narimatsu H, Kimata K, Watanabe H. Construction of a chondroitin sulfate library with defined structures and analysis of molecular interactions. J Biol Chem. 2012 Dec 21;287(52):43390–400. [PMC free article: PMC3527926] [PubMed: 23129769] [CrossRef]
9.: Yamamoto Y, Atoji Y, Oohira A, Suzuki Y. Immunohistochemical localization of chondroitin sulfate in the forestomach of the sheep. Eur J Histochem. 1995;39(4):265–72. [PubMed: 8835180]
10.: Faissner A, Clement A, Lochter A, Streit A, Mandl C, Schachner M. Isolation of a neural chondroitin sulfate proteoglycan with neurite outgrowth promoting properties. J Cell Biol. 1994 Aug;126(3):783–99. [PMC free article: PMC2120143] [PubMed: 7519189] [CrossRef]
11.: Caterson B, Christner JE, Baker JR, Couchman JR. Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans. Fed Proc. 1985 Feb;44(2):386–93. [PubMed: 2578417]
12.: Caterson B. Fell-Muir Lecture: chondroitin sulphate glycosaminoglycans: fun for some and confusion for others. Int J Exp Pathol. 2012 Feb;93(1):1–10. [PMC free article: PMC3311016] [PubMed: 22264297] [CrossRef]
13.: Matsushita K, Nakata T, Takeda-Okuda N, Nadanaka S, Kitagawa H, Tamura JI. Synthesis of chondroitin sulfate CC and DD tetrasaccharides and interactions with 2H6 and LY111. Bioorg Med Chem. 2018 Mar 1;26(5):1016–1025. [PubMed: 29402610] [CrossRef]

Footnotes

: The authors declare no competing or financial interests.

Figures

Figure 1:

Schematic representation of structure of the repeating disaccharide units in glycosaminoglycan (GAG) chains of chondroitin sulfate (CS) proteoglycans (CSPGs). CS GAG chains are expressed as PGs, where individual GAG chains are covalently attached to a panel of core proteins, through a distinct tetrasaccharide linker (GlcA-Gal-Gal-Xyl), where Gal and Xyl represent galactose and xylose, respectively. The repeating disaccharide region could be modified by various CS-specific sulfotransferases, generating divergent disaccharide units, O, A, C, D, and E. 2S, 4S, and 6S stand for 2-O-, 4-O-, and 6-O-sulfate, respectively. Native CS moieties of CSPGs exhibit remarkable structural diversity generated by various combinations of the characteristic disaccharide units.

Figure 2:

Reactivity of anti-chondroitin sulfate (CS) antibodies, MO-225 (A, diluted 1:500) and 2H6 (B, diluted 1:1,000), toward biotinylated CS polysaccharide preparations. 2H6 shows almost the same reactivity preferences toward chemically synthetic biotinylated CS tetrasaccharides, A-A, C-C, and D-D (13).

Figure 3:

Immunoblots of chondroitin sulfate (CS) proteoglycans (CSPGs) using anti-CS antibodies, LY111 and 2B6, and an antibody specific to a CSPG core protein, decorin (Dcn). For detection of Dcn core protein and its PG form, anti-Dcn antibody (goat immunoglobulin G [IgG], 0.5 µg/ml, R&D systems, Minneapolis, MN) was used. LY111 (diluted 1:1,000) recognized a PG form of Dcn but not a deglycosylated form of Dcn pretreated with chondroitinase ABC (ChABC). Conversely, the 2B6 reactivity (diluted 1:400) was shown in an opposite fashion. Asterisks in each blot are nonspecific signals derived from ChABC reagent.

Tables

Table 1:

Commercially available anti-CS antibodies

Clone	Antigen	Isotype	Specificity	Refs
CS-56	Ventral membranes of chicken gizzard fibroblasts	Mouse IgM	CS-D > CS-A, CS-C	4,5
MO-225	Chick embryo limb bud proteoglycan (PG-M)	Mouse IgM	CS-D > CS-C, CS-E > CS-A	5,6
LY111	Chicken type IX collagen containing chondroitin-4-sulfate	Mouse IgM	CS-A > CS-D	7,8
2H6	CSPGs purified from 10-day-old rat brain	Mouse IgM	CS-A > CS-D	8,9
473HD	Purified tissue fraction corresponding to mouse DSD-1-PG (phosphacan)	Rat IgM	A distinct CS epitope (DSD-1 epitope) containing D unit.	5,10
1B5^a	CSPGs predigested with chondroitinase ABC	Mouse IgG₁	∆Di-0S (non-sulfated, unsaturated CS disaccharide neoepitope)	11,12
2B6^a	CSPGs predigested with chondroitinase ABC	Mouse IgG₁	∆Di-4S (4-sulfated, unsaturated CS disaccharide neoepitope)	11,12
3B3^a	CSPGs predigested with chondroitinase ABC	Mouse IgM	∆Di-6S (6-sulfated, unsaturated CS disaccharide neoepitope) non-reducing terminal, 6-sulfated CS disaccharide structure on native CS moieties of CSPGs	11,12

^a: These antibodies recognize unsaturated CS disaccharide neoepitopes generated at non-reducing terminal of CS moieties of CSPGs that have been predigested with chondroitinase ABC.

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