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Monoclonal antibodies against chondroitin sulfate
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Monoclonal antibodies against chondroitin sulfate

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Introduction Protocol References Credit lines
Category
Glycosaminoglycans
Protocol Name

Monoclonal antibodies against chondroitin sulfate

Authors
Maeda, Nobuaki
Department of Brain Development and Neural Regeneration, Tokyo Metropolitan Institute of Medical Science
KeyWords
IgM   epitope   ELISA  
Reagents

Chondroitin sulfate A (whale cartilage CS-A, Seikagaku Corp., Tokyo, Japan)

Chondroitin sulfate B (pig skin CS-B, Seikagaku Corp.)

Chondroitin sulfate C (shark cartilage CS-C, Seikagaku Corp.)

Chondroitin sulfate D (shark cartilage CS-D, Seikagaku Corp.)

Chondroitin sulfate E (squid cartilage CS-E, Seikagaku Corp.)

CS-56 (mouse IgM, Sigma-Aldrich, St. Louis, MO)

MO-225 (mouse IgM, Seikagaku Corp.)

Goat HRP-conjugated anti-mouse IgM (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)

BM Blue POD Substrate, soluble (Roche Applied Science, Penzberg, Germany)

MaxiSorp 96 well plate (Nunc, Roskilde, Denmark)

Instruments

Multichannel Pipette

Microplate shaker

Microplate reader

Methods
1.

Monoclonal antibodies against chondroitin sulfate

1) 

 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.

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2) 

 Shake the plate for 2 h at room temperature.

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3) 

 Store the plate overnight at 4ºC.

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4) 

 Wash the plate three times with washing buffer (0.05% Tween 20/ PBS).

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5) 

 Add 250 μL of 2% BSA/ PBS into each well, and shake the plate for 1 h at room temperature.

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6) 

 Wash the plate three times with washing buffer.

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7) 

 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.

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8) 

 Shake the plate for 1 h at room temperature.

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9) 

 Wash the plate three times with washing buffer.

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10) 

 Add 100 μL/well of HRP-conjugated anti-mouse IgM diluted 500-fold in the washing buffer.

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11) 

 Shake the plate for 1 h at room temperature.

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12) 

 Wash the plate three times with washing buffer.

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13) 

 Add 100 μL/well of BM Blue POD Substrate.

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14) 

 Shake the plate for 5–30 min at room temperature.

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15) 

 Add 10 μL/well of 1 M sulfuric acid to stop the reaction.

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16) 

 Measure the absorbance at 450 nm using multiplate reader.

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Discussion

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

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.

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