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<title>Quantification of keratan sulfate in blood by enzyme-linked immunosorbent assay inhibition assay - Glycoscience Protocols (GlycoPODv2) - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Glycoscience Protocols (GlycoPODv2) [Internet]" /><meta name="citation_title" content="Quantification of keratan sulfate in blood by enzyme-linked immunosorbent assay inhibition assay" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/23" /><meta name="citation_author" content="Tomoya Akama, O" /><meta name="citation_pmid" content="37590740" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK594016/" /><meta name="citation_keywords" content="keratan sulfate" /><meta name="citation_keywords" content="5D4 monoclonal antibody" /><meta name="citation_keywords" content="enzyme-linked immunosorbent assay (ELISA)" /><meta name="citation_keywords" content="serum" /><meta name="citation_keywords" content="diagnostic marker" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Quantification of keratan sulfate in blood by enzyme-linked immunosorbent assay inhibition assay" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Tomoya Akama, O" /><meta name="DC.Date" content="2022/03/23" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK594016/" /><meta name="description" content="Quantification of keratan sulfate glycosaminoglycans (KS-GAGs) in body fluids, such as blood serum, can be achieved using a specific monoclonal antibody, 5D4, and purified keratan sulfate as a standard (1). This method is based on two-step immunoreaction of 5D4 antibody, which specifically recognizes highly sulfated KS-GAG. The first step is the reaction between KS-GAG in sample or in standard solution and 5D4. At this step, a certain amount of 5D4 is consumed by KS-GAG in the sample. The second step is the reaction between the remaining unoccupied 5D4 and solid-phase KS-GAG on enzyme-linked immunosorbent assay (ELISA) plate. At this step, all unoccupied 5D4 antibodys are absorbed on ELISA plate. Using horseradish peroxide (HRP)-labeled secondary antibody, the amount of 5D4 antibody is determined, and thus, the amount of KS-GAG that reacted with 5D4 is calculated. Since dynamic range of ELISA is not large, it is important to optimize dilution degree of primary antibody (5D4) and secondary antibody (HRP-labeled anti-mouse immunoglobulin G [IgG] antibody)." /><meta name="og:title" content="Quantification of keratan sulfate in blood by enzyme-linked immunosorbent assay inhibition assay" /><meta name="og:type" content="book" /><meta name="og:description" content="Quantification of keratan sulfate glycosaminoglycans (KS-GAGs) in body fluids, such as blood serum, can be achieved using a specific monoclonal antibody, 5D4, and purified keratan sulfate as a standard (1). This method is based on two-step immunoreaction of 5D4 antibody, which specifically recognizes highly sulfated KS-GAG. The first step is the reaction between KS-GAG in sample or in standard solution and 5D4. At this step, a certain amount of 5D4 is consumed by KS-GAG in the sample. The second step is the reaction between the remaining unoccupied 5D4 and solid-phase KS-GAG on enzyme-linked immunosorbent assay (ELISA) plate. At this step, all unoccupied 5D4 antibodys are absorbed on ELISA plate. Using horseradish peroxide (HRP)-labeled secondary antibody, the amount of 5D4 antibody is determined, and thus, the amount of KS-GAG that reacted with 5D4 is calculated. 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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="_NBK594016_"><span class="title" itemprop="name">Quantification of keratan sulfate in blood by enzyme-linked immunosorbent assay inhibition assay</span></h1><div class="contrib half_rhythm"><span itemprop="author">Tomoya Akama, O</span>, Dr<div class="affiliation small">Kansai Medical University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.umk.atakarih@tamaka" class="oemail">pj.ca.umk.atakarih@tamaka</a></div></div></div><p class="small">Created: <span itemprop="datePublished">September 30, 2021</span>; Last Revision: <span itemprop="dateModified">March 23, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g167-kerataninhibit.Introduction"><h2 id="_g167-kerataninhibit_Introduction_">Introduction</h2><p>Quantification of keratan sulfate glycosaminoglycans (KS-GAGs) in body fluids, such as blood serum, can be achieved using a specific monoclonal antibody, 5D4, and purified keratan sulfate as a standard (<a class="bk_pop" href="#g167-kerataninhibit.REF.1">1</a>). This method is based on two-step immunoreaction of 5D4 antibody, which specifically recognizes highly sulfated KS-GAG. The first step is the reaction between KS-GAG in sample or in standard solution and 5D4. At this step, a certain amount of 5D4 is consumed by KS-GAG in the sample. The second step is the reaction between the remaining unoccupied 5D4 and solid-phase KS-GAG on enzyme-linked immunosorbent assay (ELISA) plate. At this step, all unoccupied 5D4 antibodys are absorbed on ELISA plate. Using horseradish peroxide (HRP)-labeled secondary antibody, the amount of 5D4 antibody is determined, and thus, the amount of KS-GAG that reacted with 5D4 is calculated. Since dynamic range of ELISA is not large, it is important to optimize dilution degree of primary antibody (5D4) and secondary antibody (HRP-labeled anti-mouse immunoglobulin G [IgG] antibody).</p></div><div id="g167-kerataninhibit.Protocol"><h2 id="_g167-kerataninhibit_Protocol_">Protocol</h2><p>In Method 1, we prepare a 96-well plate coated with KS-GAG for later analysis. This plate can be prepared ahead of the analysis and stored at a freezer. In Method 2, serially diluted samples and standards are reacted with 5D4 antibody in a new 96-well plate (or tubes) (<a class="figpopup" href="/books/NBK594016/figure/g167-kerataninhibit.F1/?report=objectonly" target="object" rid-figpopup="figg167kerataninhibitF1" rid-ob="figobg167kerataninhibitF1">Figure 1</a>). At this point, 5D4 antibodies are consumed by KS-GAG containing in the samples, and the remaining 5D4 molecules are captured on the KS-GAG-coated plate and detected at step 3 (<a class="figpopup" href="/books/NBK594016/figure/g167-kerataninhibit.F1/?report=objectonly" target="object" rid-figpopup="figg167kerataninhibitF1" rid-ob="figobg167kerataninhibitF1">Figure 1</a>).</p><div id="g167-kerataninhibit.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">96-well ELISA plate (Nunc MaxiSorp flat-bottom, Thermo Scientific, MA, USA)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Cartilage powder (from bovine, 10128850, MP Biomedicals, CA, USA)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Extraction buffer (4 M guanidine-HCl, 50 mM of Tris/HCl, and 10 mM of ethylenediaminetetraacetic acid [EDTA], pH 8.0)</p></dd><dt>4.</dt><dd><p class="no_top_margin">Chondroitinase ABC (C2905, SIGMA-Aldrich, MO, USA)</p></dd><dt>5.</dt><dd><p class="no_top_margin">10× chondroitinase ABC buffer (1 M Tris/HCl and 1 M NaOAc, pH7.3)</p></dd><dt>6.</dt><dd><p class="no_top_margin">10× coating buffer (0.2 M Na<sub>2</sub>CO<sub>3</sub> and 0.2 M NaHCO<sub>3</sub>, pH 9.2)</p></dd><dt>7.</dt><dd><p class="no_top_margin">Phosphate-buffered saline containing 0.1% Tween-20, pH5.3 adjusted by HCl (PBST5.3)</p></dd><dt>8.</dt><dd><p class="no_top_margin">Bovine serum albumin (Fraction-V, A7906, SIGMA-Aldrich)</p></dd><dt>9.</dt><dd><p class="no_top_margin">5D4 monoclonal antibody (from Dr. Bruse Caterson, Cardiff University, UK or MABN2483, Merck Millipore, MA, USA)</p></dd><dt>10.</dt><dd><p class="no_top_margin">Keratan sulfate (from Dr. Eugene Thonar at Rush University, USA)</p></dd><dt>11.</dt><dd><p class="no_top_margin">HRP-labeled goat anti-mouse IgG antibody (HAF007, R&D systems, MN, USA)</p></dd><dt>12.</dt><dd><p class="no_top_margin">HRP substrate solution (DY999, R&D Systems)</p></dd><dt>13.</dt><dd><p class="no_top_margin">2N sulfuric acid</p></dd></dl></div><div id="g167-kerataninhibit.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Multi-channel pipette (Pipetman P8X20L and P8X200L, Gilson, Villiers-le-Bel, France)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Microtiter-plate reader (Multiskan FC, Thermo Scientific)</p></dd></dl></div><div id="g167-kerataninhibit.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Preparation of ELISA plate</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Mix 5 g of cartilage powder with 30 mL of extraction buffer in a 50-mL Falcon tube.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Rotate the mixture at 4°C for 2 d.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Centrifuge the mixture at 9,000 ×<i>g</i> for 5 min and recover the supernatant.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Dialyze against water at 4°C for 1–2 d to remove the salt.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Lyophilize the dialyzed solution.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Dissolve in a small volume of water and measure the protein concentration (<b>Note 1</b>).</p></dd><dt>g.</dt><dd><p class="no_top_margin">To coat one 96-well plate, aliquot 120 µg of the reconstituted cartilage extract into a 50-mL tube (<b>Note 2</b>).</p></dd><dt>h.</dt><dd><p class="no_top_margin">Add 10× Chondroitinase ABC buffer and adjust to 1× concentration.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Add 0.024 unit of Chondroitinase ABC to the tube and incubate at 37°C for 2 h (<b>Note 3</b>).</p></dd><dt>j.</dt><dd><p class="no_top_margin">Add water to adjust to 18 mL and add 2 mL of 10× coating buffer.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Using multi-channel pipette, apply 200 µL of the cartilage extract in coating the buffer in each well.</p></dd><dt>l.</dt><dd><p class="no_top_margin">Cover the plate with a sealing tape and incubate at room temperature for at least 2 h (<b>Note 4</b>).</p></dd><dt>m.</dt><dd><p class="no_top_margin">Discard the coating buffer and wash wells with PBST5.3 five times.</p></dd><dt>n.</dt><dd><p class="no_top_margin">Use the plate for ELISA detection immediately or cover the plate with a sealing tape and store at −20°C until use.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">First immunoreaction between 5D4 and the sample (or standard)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">On a new 96-well plate, make serially diluted solutions of a sample with PBST5.3 containing 1% BSA (original sample, 1/2 diluted solution, 1/4 diluted solution, 1/8 diluted solution, and so on).</p></dd><dt>b.</dt><dd><p class="no_top_margin">For making a standard curve, prepare 1 µg/mL of keratan sulfate solution in PBST5.3 containing 1% BSA and make serially diluted samples (<b>Note 5</b>).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Make 1/1,000 diluted 5D4 antibody in PBST5.3 containing 1% BSA and apply 110 µL in each well of a new 96-well plate (<b>Note 6</b>).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Using a multi-channel pipette, add 110 µL of diluted sample in each well containing diluted 5D4 and mix by pipetting.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Cover the plate with a sealing tape and incubate at room temperature for an hour or at 4°C overnight.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Second immunoreaction between unreacted 5D4 and coated ELISA plate</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Place the plate containing 5D4 and samples and the coated ELISA plate at room temperature.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Using multi-channel pipette, transfer 200 µL of 5D4-sample mixture to the coated ELISA plate.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Cover the ELISA plate with a sealing tape and incubate at room temperature for an hour.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Remove the mixture from the ELISA plate and wash the plate with PBST5.3 for 3 times (5-min interval).</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add 1/2,000 diluted HRP-labeled secondary antibody in PBST5.3 containing 1% BSA into each well of the plate (<b>Note 7</b>).</p></dd><dt>f.</dt><dd><p class="no_top_margin">Cover the ELISA plate with a sealing tape and incubate at room temperature for an hour.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Remove the antibody solution and wash the plate with PBST5.3 three times (5-min interval).</p></dd><dt>h.</dt><dd><p class="no_top_margin">Prepare HRP substrate solution, and add 100 µL of the solution to each well of the plate using a multi-channel pipette (color reaction may be observed quickly).</p></dd><dt>i.</dt><dd><p class="no_top_margin">Stop color development by adding 100 µL of 2N sulfuric acid and measure 450-nm absorbance using a multi-channel plate reader.</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">Calculation of keratan sulfate concentration from the standard curve</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">To draw the standard curve, plot absorbance on the Y-axis versus log (keratan sulfate concentration) on the X-axis (<a class="figpopup" href="/books/NBK594016/figure/g167-kerataninhibit.F2/?report=objectonly" target="object" rid-figpopup="figg167kerataninhibitF2" rid-ob="figobg167kerataninhibitF2">Figure 2</a>). It usually shows a sigmoid curve. Use the linear region of the curve for the working range (usually 20–500 ng keratan sulfate in case of standard).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Calculate a half inhibition point on the standard curve, as it is the most accurate point for the measurement. For the calculation of the sample keratan sulfate concentration, confirm the plotted pattern (absorbance versus log [dilution degree]) shows a sigmoid curve, and select the point that is on the linear region and is the closest absorbance to the absorbance of the half inhibition point on the standard curve.</p></dd></dl></dd></dl></div><div id="g167-kerataninhibit.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">A total of 2–5 mL of water will be sufficient to dissolve the lyophilized material. Remove insoluble material by centrifugation.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Final protein concentration of the coating solution is 6 µg/mL.</p></dd><dt>3.</dt><dd><p class="no_top_margin">Enzyme/protein ratio is 0.2 unit/mg of cartilage protein.</p></dd><dt>4.</dt><dd><p class="no_top_margin">This plate can be stored at 4°C overnight.</p></dd><dt>5.</dt><dd><p class="no_top_margin">Calculated keratan sulfate concentration in samples is a relative value over the standard. Therefore, different keratan sulfate standard may give different values of keratan sulfate concentration to a sample. Indeed, keratan sulfate concentration in normal human serum was determined to be 4.78 ± 1.49 µg/mL based on commercially available corneal keratan sulfate (Sigma-Aldrich) as a standard (<a class="bk_pop" href="#g167-kerataninhibit.REF.2">2</a>), whereas it was measured as 251 ± 78 ng/mL by their own keratan sulfate as a standard in another paper (<a class="bk_pop" href="#g167-kerataninhibit.REF.3">3</a>). It is essential to obtain the same standard to compare keratan sulfate concentration in different samples.</p></dd><dt>6.</dt><dd><p class="no_top_margin">Depending on concentration of keratan sulfate in the samples, 1/1,000 dilution of 5D4 may show very high or very low signals. If it is the case, dilution degree should be optimized.</p></dd><dt>7.</dt><dd><p class="no_top_margin">Same as 5D4, dilution degree of secondary antibody may need to be optimized.</p></dd></dl></div></div><div id="g167-kerataninhibit.References"><h2 id="_g167-kerataninhibit_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g167-kerataninhibit.REF.1">Thonar EJ, Lenz ME, Klintworth GK, Caterson B, Pachman LM, Glickman P, Katz R, Huff J, Kuettner KE. Quantification of keratan sulfate in blood as a marker of cartilage catabolism. <span><span class="ref-journal">Arthritis Rheum. </span>1985 Dec;<span class="ref-vol">28</span>(12):1367–76.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/2935158" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2935158</span></a>] [<a href="http://dx.crossref.org/10.1002/art.1780281209" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g167-kerataninhibit.REF.2">Akama TO, Nishida K, Nakayama J, Watanabe H, Ozaki K, Nakamura T, Dota A, Kawasaki S, Inoue Y, Maeda N, Yamamoto S, Fujiwara T, Thonar EJ, Shimomura Y, Kinoshita S, Tanigami A, Fukuda MN. Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene. <span><span class="ref-journal">Nat Genet. </span>2000 Oct;<span class="ref-vol">26</span>(2):237–41.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11017086" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11017086</span></a>] [<a href="http://dx.crossref.org/10.1038/79987" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g167-kerataninhibit.REF.3">Klintworth GK, Meyer R, Hewitt AT, Stock EL, Lenz ME, Hassel JR, Stark WJ, Kuettner KE, Thonar EJ. Macular corneal dystrophy. Lack of keratan sulfate in serum and cornea. <span><span class="ref-journal">Ophthalmic Paediatr Genet. </span>1986 Dec;<span class="ref-vol">7</span>(3):139–43.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/2951638" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2951638</span></a>] [<a href="http://dx.crossref.org/10.3109/13816818609004130" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594016_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g167-kerataninhibit.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="g167-kerataninhibit.F1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201%3A%20.%20Enzyme-linked%20immunosorbent%20assay%20(ELISA)%20inhibition%20assay%20for%20keratan%20sulfate%20glycosaminoglycans.&p=BOOKS&id=594016_g167-kerataninhibit-Image001.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK594016/bin/g167-kerataninhibit-Image001.jpg" alt="Figure 1: . Enzyme-linked immunosorbent assay (ELISA) inhibition assay for keratan sulfate glycosaminoglycans." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Enzyme-linked immunosorbent assay (ELISA) inhibition assay for keratan sulfate glycosaminoglycans.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g167-kerataninhibit.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK594016/bin/g167-kerataninhibit-Image002.jpg" alt="Figure 2: . Standard curve by SIGMA bovine corneal keratan sulfate." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Standard curve by SIGMA bovine corneal keratan sulfate. The X-axis is log10 (KS concentration) and the Y-axis is the absorbance of enzyme-linked immunosorbent assay plate. The concentration between 15 and 500 ng KS/mL is on a linear standard line.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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