Chondroitin sulfates are unbranched sulfated polysaccharides composed of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid. Due to differential sulfation of hydroxyl groups and C5 epimerization of glucuronic acid (Fig. 1), chondroitin sulfate shows highly diverse structures depending on the developmental stage, aging, pathological states and tissue sources. There are several monoclonal antibodies that recognize chondroitin sulfate epitopes. The levels of these epitopes in the test samples can be measured by competitive ELISA. It should be noted that the epitopes of these antibodies may be expressed only under some specific conditions such as pathological states. Thus, the competitive ELISA method is not suitable for measurement of the total amounts of chondroitin sulfate. Instead, it may be useful for the monitoring of certain pathological conditions. In this section, measurement of the levels of MO-225 epitopes, D unit-containing sequences, is described (Fig. 1). |
| Category | Glycosaminoglycans |
| Protocol Name | Measurement of chondroitin sulfate epitope levels with competitive ELISA |
Authors
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Maeda, Nobuaki
Department of Brain Development and Neural Regeneration, Tokyo Metropolitan Institute of Medical Science
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| KeyWords |
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Reagents
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Chondroitin sulfate D (shark cartilage, Seikagaku Corp., Tokyo, Japan) |
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MO-225 (mouse IgM, Seikagaku Corp.) |
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Goat HRP-conjugated anti-mouse IgM (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) |
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BM Blue POD Substrate, soluble (Roche Applied Science, Penzberg, Germany) |
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MaxiSorp 96 well plate (Nunc, Roskilde, Denmark) |
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Instruments
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| Methods |
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Serially dilute the chondroitin sulfate D stock solution and the test solutions in the washing buffer (0.05% Tween 20/ PBS). |
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Mix 200 μL of the serially diluted samples with the same volume of MO-225 solution (5000-fold diluted in the washing buffer). Final concentrations of chondroitin sulfate D : 800, 160, 32, 6.4, 1.3, 0.26, and 0 ng/mL. Final dilution of MO-225: 1/10000. |
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Incubate the samples overnight at 20°C. |
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Add 100 μL of 10 μg/mL chondroitin sulfate D, which is diluted in 50 mM sodium carbonate-bicarbonate buffer, pH 9.3, into each well of 96-well plate. |
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Shake the plate for 2 h at room temperature. |
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Store the plate overnight at 4°C. |
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Wash the plate three times with washing buffer. |
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Add 250 μL of 2% BSA/ PBS into each well, and shake the plate for 1 h at room temperature. |
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Wash the plate three times with washing buffer. |
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Add 100 μL/well of samples (in triplicate). Blank wells should be prepared, in which only the washing buffer is added. |
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Shake the plate for 1 h at room temperature. |
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Wash the plate three times with washing buffer. |
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Add 100 μL/well of HRP-conjugated anti-mouse IgM diluted 500-fold in the washing buffer. |
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Shake the plate for 1 h at room temperature. |
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Wash the plate three times with washing buffer. |
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Add 100 μL/well of BM Blue POD Substrate. |
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Shake the plate for 5–30 min at room temperature. |
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Add 10 μL/well of 1 M sulfuric acid. |
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Measure the absorbance at 450 nm using multiplate reader. |
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Convert the absorbance values to percentage inhibition, by the formula: % inhibition = 100 × [1– (ODs – ODb)/(ODo-ODb)], where ODs and ODb are absorbance values of sample and blank, respectively, and ODo is the absorbance in the absence of competitor. |
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| Discussion | Figure 2 shows the standard curve. MO-225 epitope levels corresponding to 0.5–50 ng/mL chondroitin sulfate D can be measured. Optimization of the dilutions of MO-225 and secondary antibody may improve the sensitivity. Similar assay systems can be made using the other monoclonal antibodies against chondroitin sulfate. An assay kit for measurement of the serum 3B3(−) epitope (a terminal 6-sulfated chondroitin sulfate) levels in human rheumatic diseases can be commercially obtained (Chan et al., 2001). |
| Figure & Legends |
Figure & Legends
Fig. 1 Structures of disaccharide units of chondroitin sulfate
Fig. 2 Competitive ELISA of MO-225 epitopes |
| Copyrights |
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This work is released underCreative Commons licenses
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| Date of registration：2015-10-13 11:41:43 |
- Yamagata, M., Kimata, K., Oike, Y., Tani, K., Maeda, N., Yoshida, K., Shimomura, Y., Yoneda, M., and Suzuki, S. (1987) A monoclonal antibody that specifically recognizes a glucuronic acid 2-sulfate-containing determinant in intact chondroitin sulfate chain. J. Biol. Chem. 262, 4146–4152 [PMID : 2435733]
- Chan, S.S., Kent, G.N., and Will, R.K. (2001) A sensitive assay for the measurement of serum chondroitin sulfate 3B3(-) epitope levels in human rheumatic diseases. Clin. Exp. Rheumatol 19, 533–540 [PMID : 11579712]
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