Quantitative determination of bacterial endotoxins by the chromogenic limulus method: critical analysis and study of interactions between 3 divalent cations]

The reproducibility of the limulus amoebocyte lysate (LAL) test with the use of a chromogenic substrate was evaluated in the presence of 5 bacterial endotoxins (2 from Escherichia coli, 1 from Salmonella, 1 from Serratia marcescens and 1 from Vibrio cholerae) and the FDA reference endotoxin EC5. Endotoxins from E. coli 055B5, S. marcescens and V. cholerae were significantly more active than the reference endotoxin (p less than 0.01). Addition of Mg2+ activated the LAL chromogenic reaction (optimum = 160 mmol/l added), whereas Ca2+ in concentrations above 5 mmol/l inhibited the reaction. As little as 0.3 mmol/l Zn2+ strongly inhibited the reaction. Inhibition by Zn2+ was partly suppressed by addition of 160 mmol/l Mg2+. These divalent cations modified the LAL chromogenic reaction when added in the first step of the reaction (incubation with endotoxin and LAL reagent). The LAL chromogenic reaction was not modified by divalent cations when these were added in the second step of the reaction (with the chromogenic substrate).