Quantification of keratan sulfate in blood by enzyme-linked immunosorbent assay inhibition assay

96-well ELISA plate (Nunc MaxiSorp flat-bottom, Thermo Scientific, MA, USA)

Cartilage powder (from bovine, 10128850, MP Biomedicals, CA, USA)

Extraction buffer (4 M guanidine-HCl, 50 mM of Tris/HCl, and 10 mM of ethylenediaminetetraacetic acid [EDTA], pH 8.0)

Chondroitinase ABC (C2905, SIGMA-Aldrich, MO, USA)

10× chondroitinase ABC buffer (1 M Tris/HCl and 1 M NaOAc, pH7.3)

10× coating buffer (0.2 M Na₂CO₃ and 0.2 M NaHCO₃, pH 9.2)

Phosphate-buffered saline containing 0.1% Tween-20, pH5.3 adjusted by HCl (PBST5.3)

Bovine serum albumin (Fraction-V, A7906, SIGMA-Aldrich)

5D4 monoclonal antibody (from Dr. Bruse Caterson, Cardiff University, UK or MABN2483, Merck Millipore, MA, USA)

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

HRP-labeled goat anti-mouse IgG antibody (HAF007, R&D systems, MN, USA)

HRP substrate solution (DY999, R&D Systems)

2N sulfuric acid

Multi-channel pipette (Pipetman P8X20L and P8X200L, Gilson, Villiers-le-Bel, France)

Microtiter-plate reader (Multiskan FC, Thermo Scientific)

Preparation of ELISA plate

a.

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

b.

Rotate the mixture at 4°C for 2 d.

c.

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

d.

Dialyze against water at 4°C for 1–2 d to remove the salt.

e.

Lyophilize the dialyzed solution.

f.

Dissolve in a small volume of water and measure the protein concentration (Note 1).

g.

To coat one 96-well plate, aliquot 120 µg of the reconstituted cartilage extract into a 50-mL tube (Note 2).

h.

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

i.

Add 0.024 unit of Chondroitinase ABC to the tube and incubate at 37°C for 2 h (Note 3).

j.

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

k.

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

l.

Cover the plate with a sealing tape and incubate at room temperature for at least 2 h (Note 4).

m.

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

n.

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

First immunoreaction between 5D4 and the sample (or standard)

a.

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).

b.

For making a standard curve, prepare 1 µg/mL of keratan sulfate solution in PBST5.3 containing 1% BSA and make serially diluted samples (Note 5).

c.

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 (Note 6).

d.

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

e.

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

Second immunoreaction between unreacted 5D4 and coated ELISA plate

a.

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

b.

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

c.

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

d.

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

e.

Add 1/2,000 diluted HRP-labeled secondary antibody in PBST5.3 containing 1% BSA into each well of the plate (Note 7).

f.

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

g.

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

h.

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).

i.

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

Calculation of keratan sulfate concentration from the standard curve

a.

To draw the standard curve, plot absorbance on the Y-axis versus log (keratan sulfate concentration) on the X-axis (Figure 2). 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).

b.

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.

A total of 2–5 mL of water will be sufficient to dissolve the lyophilized material. Remove insoluble material by centrifugation.

Final protein concentration of the coating solution is 6 µg/mL.

Enzyme/protein ratio is 0.2 unit/mg of cartilage protein.

This plate can be stored at 4°C overnight.

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 (2), whereas it was measured as 251 ± 78 ng/mL by their own keratan sulfate as a standard in another paper (3). It is essential to obtain the same standard to compare keratan sulfate concentration in different samples.

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.

Same as 5D4, dilution degree of secondary antibody may need to be optimized.