Endotoxin Assay

This assay measures the shift in the absorbance maximum of the dye due to binding to endotoxin. The dye is thought to bind to lipid A, causing metachromasia. Sensitivity is approximately 1ug of LPS. Polymixin B (and probably aminogylcosides) interfere with the assay causing quenching. Serum components can also interfere. LPS O-antigen (eg., acid hydrolysed LPS) may not react.

Reference:

Keler and Nowotny. (1986). Analytical Biochemistry. 156:189.

Materials:

• 1.6mg 1,9-dimethylmethylene blue (Serva)
• 0.43g glycine
• 0.33g NaCl
• 4.7ml 1.0M NaOH
• 0.5ml 80% ethanol

Make up to 100ml with distilled water. Stir the above ingredients at room temperature for 3 to 5 hours until the DMB is dissolved. Store the dye solution in a dark bottle at room temperature.

Method:

1. Prepare a standard curve of eg., Shigella LPS. The curve is not linear at high concentrations of LPS.
2. Prepare dilutions of the test LPS at about 10 to 100ug LPS.
3. Add 1ml of dye solution to 50ul of LPS standard or test solution.
4. Mix and read immeadiately at 535nm against a reagent blank (ie., 1ml of dye solution with 50ul of water).

NOTES:

1. Rinse the cuvettes with acetone, then water after every 5 readings to remove the red film.
2. The pH of the dye solution is important (better up than down).
3. Nucleic acids may contribute to the values, but nuclease-treated samples can be tested if this is suspected.