Monoclonal antibodies against chondroitin sulfate
Chondroitin sulfates are unbranched sulfated polysaccharides composed of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid. Due to differential sulfation of hydroxyl groups in the disaccharides and C5 epimerization of glucuronic acid, chondroitin sulfate chains show highly diverse structures (Fig. 1). There are several monoclonal antibodies which recognize various chondroitin sulfate epitopes. It has been considered that certain sequences of the disaccharide units constitute the epitopes of these monoclonal antibodies. Although it is difficult to determine the strict epitope structures, one may obtain rough estimation by the ELISA assay described below.
Chondroitin sulfate A (whale cartilage CS-A, Seikagaku Kogyo)
Chondroitin sulfate B (pig skin CS-B, Seikagaku Kogyo)
Chondroitin sulfate C (shark cartilage CS-C, Seikagaku Kogyo)
Chondroitin sulfate D (shark cartilage CS-D, Seikagaku Kogyo)
Chondroitin sulfate E (squid cartilage CS-E, Seikagaku Kogyo)
CS-56 (mouse IgM, Sigma)
MO-225 (mouse IgM, Seikagaku Kogyo)
Goat HRP-conjugated anti-mouse IgM (Santa Cruz)
BM Blue POD Substrate, soluble (Roche)
MaxiSorp 96 well plate (Nunc)
Add 100 µl of 10 µg/ml chondroitin sulfate (CS-A, -B, -C, -D, or -E), which are diluted in 50 mM sodium carbonate-bicarbobate buffer, pH 9.3, into each well of 96-well plate.
Shake the plate for 2 hours at room temperature.
Store the plate overnight at 4ºC.
Wash the plate three times with washing buffer (0.05% Tween 20/ PBS).
Add 250 μl of 2% BSA/ PBS into each well, and shake the plate for 1 hour at room temperature.
Wash the plate three times with washing buffer.
Add 100 μl/well of antibody samples, which are serially diluted in the washing buffer (1/200, 1/600, 1/1800, 1/5400, 1/16200, 1/48600, 1/145800). Blank wells should be prepared, in which only the washing buffer is added.
Shake the plate for 1 hour at room temperature.
Add 100 μl/well of HRP-conjugated anti-mouse IgM diluted 500-fold in the washing buffer.
Add 100 μl/well of BM Blue POD Substrate.
Shake the plate for 5-30 min at room temperature.
Add 10 μl/well of 1M sulfuric acid to stop the reaction.
Measure the absorbance at 450 nm using multiplate reader.
Figure 2 shows the results of ELISA assays using monoclonal antibodies, MO-225 and CS-56. MO-225 exclusively bound with chondroitin sulfate D (CS-D), and the other chondroitin sulfate preparations were only slightly recognized by this antibody. On the other hand, CS-56 widely reacted with various chondroitin sulfate preparations, although it preferred CS-D. CS-A, -B, -C, -D, and -E are chondroitin sulfate preparations enriched with A, iA, C, D and E units, respectively (Fig. 1). Thus, it is supposed that MO-225 strictly recognizes D unit-containing epitopes (Yamagata et al., 1987). Although CS-56 also reacted selectively with CS-D, the specificity was low. Rather, this antibody seems to bind to various chondroitin sulfate epitopes (Avnur & Geiger, 1984; Ito et al., 2005). Strict epitope structures may be revealed by competitive ELISA using structurally defined chondroitin sulfate oligosaccharides.
Figure & Legends
Fig.1. Structures of disaccharide units of chondroitin sulfate.
Fig. 2. Reactivity of MO-225 and CS-56 with various chondroitin sulfate preparations.