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A Quantitative Microanalysis of Bacterial Endotoxin Using [ 3 H]‐Labeled L ‐ Glycero ‐ D ‐ mann oheptitol as a Marker
Author(s) -
Haishima Yuji,
Tanamoto Kenichi
Publication year - 1993
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.1993.tb01710.x
Subject(s) - microanalysis , chromatography , high performance liquid chromatography , heptose , biology , microbiology and biotechnology , bacteria , biochemistry , chemistry , organic chemistry , mutant , gene , genetics
Quantitative microanalysis of bacterial endotoxin was performed using [ 3 H]‐labeled L ‐ glycero‐ D ‐ manno heptitol ( LD ‐Heptitol) as a marker. Several different amounts of authentic L ‐ glycero‐ D ‐manno heptose ( LD ‐Heptose) were reduced with 20 μg of cold NaBH 4 containing 2 μg of NaB 3 H 4 (40 Ci/mmol) in 20 μ1 of 1 mM NaOH at 4 C for 48 hr. The product, [1‐3H]‐labeled LD ‐Heptitol, has high specific activity, and was purified by HPLC and detected using a liquid‐scintillation counter. As little as 50 pg of LD ‐Heptose was detectable, and the radioactivity increased dose‐dependently in the 100 pg to 80 ng range tested. More than 2 ng of Salmonella abortus equi endotoxin could be accurately determined by this method. It is possible to detect 50 pg of endotoxin by this method, if 100% hot material (NaB 3 H 4 ) is used for [ 3 H]‐labeling.