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Quantification of keratan sulfate in blood by ELISA inhibition assay
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Quantification of keratan sulfate in blood by ELISA inhibition assay

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Introduction Protocol References Credit lines
Category
Glycosaminoglycans
Protocol Name

Quantification of keratan sulfate in blood by ELISA inhibition assay

Authors
Akama O., Tomoya
Department of Pharmacology, Kansai Medical University
KeyWords
Reagents

Cartilage powder (from bovine, MP Biomedicals, Santa Ana, CA)

Extraction buffer (4 M Guanidine-HCl, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0)

Chondroitinase-ABC (Sigma-Aldrich, St. Louis, MO)

10 x Chondroitinase ABC buffer (1 M Tris/HCl, 1 M NaOAc, pH 7.3)

10 x Coating buffer (0.2 M Na2CO3, 0.2 M NaHCO3, pH 9.2)

Phosphate buffered saline containing 0.1 % Tween-20, pH 5.3 adjusted by HCl (PBST5.3)

Bovine serum albumin (Fraction-V, Sigma-Aldrich)

5D4 monoclonal antibody (Cosmo Bio Co., Ltd. Tokyo, Japan)

Keratan sulfate (from Dr. Eugene Thonar at Rush University, Chicago, IL)

HRP-label goat anti-mouse IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA)

HRP substrate solution (Substrate Reagent Pack DY999, R&D Systems Minneapolis, MN)

2N sulfuric acid

Instruments

96-well ELISA plate

Multi-channel pipette

Microtiter-plate reader

Methods
1.

Preparation of ELISA plate

1) 

 Mix 5 g of cartilage powder with 30 mL of extraction buffer in a 50 mL Falcon tube.

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2) 

 Rotate the mixture at 4ºC for 2 days.

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3) 

 Centrifuge the mixture at 9,000 × g for 5 min and recover supernatant.

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4) 

 Dialyze against water at 4ºC for 1–2 days to remove salt.

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5) 

 Lyophilize dialyzed solution.

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6) 

 Dissolve in a small volume of water and measure protein concentration.

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7) 

 To coat one 96-well plate, aliquot 120 μg of the reconstituted cartilage extract into a 50 mL tube.

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8) 

 Add 10 × Chondroitinase ABC buffer and adjust to 1 × concentration.

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9) 

 Add 0.24 unit of Chondroitinase ABC to the tube and incubate at 37ºC for 2 h.

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10) 

 Add water to adjust to 18 mL and also add 2 mL of 10 × coating buffer.

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11) 

 Using multi-channel pipette, apply 200 μL of the cartilage extract in coating buffer in each well.

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12) 

 Cover the plate with a sealing tape and incubate at room temperature for at least 2 h.

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13) 

 Discard the coating buffer and wash wells with PBST5.3 for 5 times.

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14) 

 Use the plate for ELISA detection immediately, or cover the plate with a sealing tape and store at −20ºC until use.

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2.

First immunoreaction between 5D4 and sample (or standard)

1) 

 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).

For making a standard curve, prepare 1 μg/mL of keratan sulfate solution in PBST5.3 containing 1% BSA, and make serially diluted samples.

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2) 

 Make 1/1000 diluted 5D4 antibody in PBST5.3 containing 1% BSA and apply 110 μL in each well of a new 96-well plate.

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3) 

 Using a multi-channel pipette, add 110 μL of diluted sample in each well containing diluted 5D4 and mix by pipetting.

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4) 

 Cover the plate with a sealing tape and incubate at room temperature for an hour or at 4ºC for overnight.

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3.

Second immunoreaction between unreacted 5D4 and coated ELISA plate

1) 

 Place the plate containing 5D4 and samples, and the coated ELISA plate at room temperature.

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2) 

 Using multi-channel pipette, transfer 200 μL of 5D4-sample mixture to the coated ELISA plate.

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3) 

 Cover the ELISA plate with a sealing tape and incubate at room temperature for an hour.

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4) 

 Remove the mixture from the ELISA plate and wash the plate with PBST5.3 for 3 times (5 min interval).

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5) 

 Add 1/2000 diluted HRP-labeled secondary antibody in PBST5.3 containing 1% BSA into each well of the plate.

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6) 

 Cover the ELISA plate with a sealing tape and incubate at room temperature for an hour.

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7) 

 Remove the antibody solution and wash the plate with PBST5.3 for 3 times (5 min interval).

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8) 

 Prepare HRP substrate solution and add 100 μL of the solution to each well of the plate by multi-channel pipette (color reaction may be observed quickly).

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9) 

 Stop color development by adding 100 μL of 2N sulfuric acid and measure 450 nm absorbance by a multi-channel plate reader.

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Discussion

To draw the standard curve, plot absorbance on Y-axis versus log (keratan sulfate concentration) on X-axis. It usually shows a sigmoid curve. Use linear region of the curve for the working range (usually 20–500 ng keratan sulfate in case of standard) (Fig. 1). Calculate a half inhibition point on the standard curve, as it is the most accurate point for the measurement. For calculation of the sample keratan sulfate concentration, confirm the plotted pattern (absorbance versus log (dilution degree)) shows 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.

Calculated keratan sulfate concentration in samples is relative value over the standard. Therefore, different keratan sulfate standard may give different value 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 standard2), whereas it was measured as 251 ± 78 ng/mL by their own keratan sulfate as a standard in other paper 3). It is essential to obtain the same standard to compare keratan sulfate concentration in different samples.

Figure & Legends

Figure & Legends

 

 

Fig. 1. Standard curve of corneal keratan sulfate (Sigma-Aldrich).

Half inhibition point is 1.388 of absorbance at 88.4 ng/mL keratan sulfate concentration (1.946 at log scale).

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