title: The Limulus Amebocyte Lysate assay may be unsuitable for detecting endotoxin in blood of healthy female subjects.
abstract: We examined the factors that may influence the outcome of the Limulus Amebocyte Lysate (LAL) assay, when it is used for quantifying Gram-negative bacterial endotoxin, also referred to as lipopolysaccharide (LPS), in samples of human blood. We found that the method recommended by the manufacturers, based on the reaction time, was inaccurate with any type of serum samples due to the slowing of the initial phase of reaction, likely by serum proteins. We describe an alternative method that is more accurate for use with heated serum samples. Further, we found that components of fresh serum irreversibly sequester endotoxin but that this action may be largely prevented by dilution and heating, but only if this occurs prior to the addition of endotoxin. The tests also indicated that a number of types of proprietary plastic vacutainers appeared to contain significant amounts of endotoxin. However, even when appropriate blood collection containers and calculation methods were used, the levels of endotoxin in serum samples detected by LAL assay were unlikely to reflect the total quantities of endotoxin in that sample and more likely to reflect the capacity of a given serum sample to sequester endotoxin.
authors.affiliation: Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand.
authors.forename: Anne
authors.initials: A
authors.lastname: Gnauck
authors.affiliation: Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand. Electronic address: R.G.Lentle@massey.ac.nz.
authors.forename: Roger G
authors.initials: RG
authors.lastname: Lentle
authors.affiliation: Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand.
authors.forename: Marlena C
authors.initials: MC
authors.lastname: Kruger
mesh_terms: D000328:Adult; D001681:Biological Assay / Q000379:methods*; D001798:Blood Proteins / Q000737:chemistry; D004731:Endotoxins / Q000737:chemistry*; D005260:Female; D006801:Humans; D008033:Limulus Test / Q000379:methods*; D008070:Lipopolysaccharides / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D044967:Serum / Q000737:chemistry*
chemical_list: D001798:Blood Proteins; D004731:Endotoxins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C025357:endotoxin binding proteins
keywords: Endotoxin; LAL assay; Limulus Amebocyte Lysate assay; Serum