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<title>Application of anti-glycosaminoglycan antibody and biotinylated hyaluronan binding protein (bHABP) [2] ~ Determination of the concentration of heparan sulfate using enzyme-linked immunosorbent assay - Glycoscience Protocols (GlycoPODv2) - NCBI Bookshelf</title>
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK594045_"><span class="title" itemprop="name">Application of anti-glycosaminoglycan antibody and biotinylated hyaluronan binding protein (bHABP) [2] ~ Determination of the concentration of heparan sulfate using enzyme-linked immunosorbent assay</span></h1><div class="contrib half_rhythm"><span itemprop="author">Shuhei Yamada</span>, Ph.D.<div class="affiliation small">Meijo Univ.<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-ojiem@yiehuhs" class="oemail">pj.ca.u-ojiem@yiehuhs</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 5, 2021</span>; Last Revision: <span itemprop="dateModified">March 22, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g160-antiglycosamin2.Introduction"><h2 id="_g160-antiglycosamin2_Introduction_">Introduction</h2><p>The concentration of heparan sulfate (HS) can be determined using enzyme-linked immunosorbent assay (ELISA). Competitive ELISA is a practical method for the quantitative estimation of HS concentrations. An anti-HS antibody (<a class="bk_pop" href="#g160-antiglycosamin2.REF.1">1</a>–<a class="bk_pop" href="#g160-antiglycosamin2.REF.3">3</a>) is preincubated with increasing concentrations of standard HS, and the mixture is added to the biotinylated HS-immobilized plate. HS preparations derived from various sources differ in their reactivity to an anti-HS antibody depending on the epitope content along the HS chain. Only when the same HS preparation is used as a standard, the concentration of HS can be determined. To quantify uncharacterized HS preparations, ELISA methods might not be suitable, but high-performance liquid chromatography after digestion with heparin lyases appears to be effective.</p></div><div id="g160-antiglycosamin2.Protocol"><h2 id="_g160-antiglycosamin2_Protocol_">Protocol</h2><p>Quantitative estimation of HS concentrations by competitive ELISA using the biotinylated HS immobilized on streptavidin-coated plates is described here as an application of anti-HS antibody. However, this method might be applicable only to the measurement of the same HS preparation with that used as a standard because the reactivity of anti-HS may be different among the HS preparations depending on the sources that they are derived from.</p><div id="g160-antiglycosamin2.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">HS from bovine kidney (Seikagaku Corp., Tokyo, Japan) or porcine intestine (Sigma-Aldrich, St. Louis, MO)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Biotinylated HS (<b>Note 1</b>)</p></dd><dt>3.</dt><dd><p class="no_top_margin">0.01 M Phosphate-buffered saline, pH 7.4 (PBS)</p></dd><dt>4.</dt><dd><p class="no_top_margin">PBS containing 0.05% Tween-20 (PBST)</p></dd><dt>5.</dt><dd><p class="no_top_margin">25 mM of Tris buffered saline, pH 7.4 (TBS)</p></dd><dt>6.</dt><dd><p class="no_top_margin">TBS containing 0.05% Tween-20 (TBST)</p></dd><dt>7.</dt><dd><p class="no_top_margin">Bovine serum albumin (BSA)</p></dd><dt>8.</dt><dd><p class="no_top_margin">Anti-HS antibody: F58-10E4 or HepSS-1 (Seikagaku Corp.) (<b>Note 2</b>)</p></dd><dt>9.</dt><dd><p class="no_top_margin">The secondary antibody, anti-mouse IgG + IgM (H + L) conjugated with alkaline phosphatase (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD)</p></dd><dt>10.</dt><dd><p class="no_top_margin">2 mg/mL of <i>p</i>-nitrophenyl phosphate (<i>p</i>NPP) in 50 mM of carbonate buffer, pH 9.8, containing 0.5 mM of MgCl<sub>2</sub></p></dd></dl></div><div id="g160-antiglycosamin2.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Streptavidin Plate C8 Transparent (Thermo Fisher Scientific)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Microplate reader iMark (Bio-Rad Laboratories, Hercules, CA)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Aspirator</p></dd></dl></div><div id="g160-antiglycosamin2.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Quantitative estimation of HS concentrations using competitive ELISA</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Wash the wells with 200 μL of PBS.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Add 2 μg each of biotinylated HS/50 μL of PBS to the wells and incubate at 4°C overnight.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 200 μL of 3% BSA/PBS for blocking and incubate at room temperature for 1 h.</p></dd><dt>d.</dt><dd><p class="no_top_margin">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.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Wash the wells with 200 μL of PBST three times.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add 50 μL of the anti-HS antibody or the mixture of the anti-HS antibody and HS to the wells.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Incubate the plate at 37°C for 1 h.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Wash the wells with 200 μL of TBST three times.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Add 50 μL of the secondary antibody (diluted 1:2,000 in TBS) and incubate at 37°C for 1 h (<b>Note 3</b>).</p></dd><dt>j.</dt><dd><p class="no_top_margin">Wash the wells with 200 μL of TBST three times.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Add 50 μL of the <i>p</i>NPP solution and incubate at room temperature (<b>Note 4</b>).</p></dd><dt>l.</dt><dd><p class="no_top_margin">Measure the absorbance at 415 nm with a microplate reader (<a class="figpopup" href="/books/NBK594045/figure/g160-antiglycosamin2.F1/?report=objectonly" target="object" rid-figpopup="figg160antiglycosamin2F1" rid-ob="figobg160antiglycosamin2F1">Figure 1</a>) (<b>Note 5</b>).</p></dd></dl></dd></dl></div><div id="g160-antiglycosamin2.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">The biotinylation of HS preparations is conducted as follows. HS is dissolved in 100 mM of 2-(<i>N</i>-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.</p></dd><dt>2.</dt><dd><p class="no_top_margin">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.</p></dd><dt>3.</dt><dd><p class="no_top_margin">The antibody solution should be diluted just before use.</p></dd><dt>4.</dt><dd><p class="no_top_margin">The <i>p</i>NPP stock solution (20 mg/mL) is diluted 20 times with 50 mM of carbonate buffer, pH 9.8, containing 0.5 mM of MgCl<sub>2</sub>, just before use.</p></dd><dt>5.</dt><dd><p class="no_top_margin">Although the wavelength of the maximum absorption for <i>p</i>-nitrophenol is 405 nm, the absorbance at 415 nm was measured because of the limitation of the filters available.</p></dd></dl></div></div><div id="g160-antiglycosamin2.References"><h2 id="_g160-antiglycosamin2_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g160-antiglycosamin2.REF.1">Kure S, Yoshie O. A syngeneic monoclonal antibody to murine Meth-A sarcoma (HepSS-1) recognizes heparan sulfate glycosaminoglycan (HS-GAG): cell density and transformation dependent alteration in cell surface HS-GAG defined by HepSS-1. <span><span class="ref-journal">J Immunol. </span>1986 Dec 15;<span class="ref-vol">137</span>(12):3900–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/2431047" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2431047</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g160-antiglycosamin2.REF.2">David G, Bai XM, Van der Schueren B, Cassiman JJ, Van den Berghe H. Developmental changes in heparan sulfate expression: in situ detection with mAbs. <span><span class="ref-journal">J Cell Biol. </span>1992 Nov;<span class="ref-vol">119</span>(4):961–75.</span> [<a href="/pmc/articles/PMC2289686/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2289686</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/1385449" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1385449</span></a>] [<a href="http://dx.crossref.org/10.1083/jcb.119.4.961" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g160-antiglycosamin2.REF.3">van den Born J, Salmivirta K, Henttinen T, Ostman N, Ishimaru T, Miyaura S, Yoshida K, Salmivirta M. Novel heparan sulfate structures revealed by monoclonal antibodies. <span><span class="ref-journal">J Biol Chem. </span>2005 May 27;<span class="ref-vol">280</span>(21):20516–23.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15778504" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15778504</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M502065200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594045_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g160-antiglycosamin2.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g160-antiglycosamin2.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK594045/bin/g160-antiglycosamin2-Image001.jpg" alt="Figure 1: . The interaction of the anti–heparan sulfate (HS) antibody F58-10E4 with biotinylated HS immobilized on a streptavidin-coated plate was inhibited by HS in a dose-dependent manner." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>The interaction of the anti–heparan sulfate (HS) antibody F58-10E4 with biotinylated HS immobilized on a streptavidin-coated plate was inhibited by HS in a dose-dependent manner.</p><p>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. <i>p</i>NPP was incubated for 60 min, and the values represent the mean (n = 2).</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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