KDO Assay

Objective:

This is a test for lipopolysaccharide (LPS), an outer membrane marker. The 8 carbon saccharide 2-keto-3-deoxyoctonate is exclusively found in LPS.

Reagents:

• Periodate H₅IO₆: 2.28 g in 100 ml H₂O
• Sodium Arsenite NaAsO₂: 2.0 g in 50 ml 0.5 N HCl
• Thiobarbituric acid: 150 mg in 25 ml H₂O (warm to dissolve, make up fresh daily)
• Sulphuric acid H₂SO₄ : 0.5 N
• Butanol reagent: 5.0 ml of conc. HCl added to 95 ml n-butanol

Standard:

KDO 200 µg/ml, use 0, 10, 20, 30, 40 µl for standards. Use dH₂O to make up each sample to a total volume of 50 µl. Omit the heating Step 3 for the standards.

Method:

1. 50 µl sample
2. 50 µl 0.5 N H₂SO₄. Vortex.
3. Place in 100^oC heating block for 8 minutes. Cool to room temperature. (This step is omitted for pure KDO.)
4. 50 µl H₅IO₆. Vortex.
5. Let stand for 10 minutes at room temperature.
6. 200 µl arsenite reagent. Vortex.
7. 800 µl thiobarbituric acid reagent. Vortex.
8. Place in 100^oC heating block for 10 minutes.
9. Cool to room temperature. Add 1.5 ml butanol reagent. Vortex.
10. Centrifuge at 2,000 rpm for 5 minutes in clinical centrifuge.
11. Aspirate about 800 µl of upper butanol layer. Measure optical density at 552 and 509 nm.
12. Extinction coefficient. An OD difference OD₅₅₂ – OD₅₀₉ of 19 = 1µM KDO.

Note:

Do not rinse cuvettes with water between readings.

Pre-clean cuvettes with ethanol and dry under nitrogen.

Read samples as soon as possible. Color development is unstable.

Note in Step 3, the hydrolysis times can be changed to obtain maximal release of KDO. Eight min. should be OK for Pseudomonas and E. coli; 12-15 min. is required for more refractory LPS samples (e.g. ß-cepacia). Test this by trial and error for a new bacterium.