title: How pH, Temperature, and Time of Incubation Affect False-Positive Responses and Uncertainty of the LAL Gel-Clot Test.
abstract: The limulus amebocyte lysate (LAL) test is the simplest and most widely used procedure for detection of endotoxin in parenteral drugs. The LAL test demands optimal pH, ionic strength, temperature, and time of incubation. Slight changes in these parameters may increase the frequency of false-positive responses and the estimated uncertainty of the LAL test. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. LAL tests were performed in nominal conditions (37 °C, 60 min, and pH 7) and in different conditions of temperature (36 °C and 38 °C), time of incubation (58 and 62 min), and pH (6 and 8). Slight differences in pH increase the frequency of false-positive responses 5-fold (relative risk 5.0), resulting in an estimated of uncertainty 7.6%. Temperature and time of incubation affect the LAL test less, showing relative risks of 1.5 and 1.0, respectively. Estimated uncertainties in 36 °C or 38 °C temperatures and 58 or 62 min of incubation were found to be 2.0% and 1.0%, respectively. Simultaneous differences in these parameters significantly increase the frequency of false-positive responses.
  The limulus amebocyte lysate (LAL) gel-clot test is a simple test for detection of endotoxin from Gram-negative bacteria. The test is based on a gel formation when a certain amount of endotoxin is present; it is a pass/fail test. The LAL test requires optimal pH, ionic strength, temperature, and time of incubation. Slight difference in these parameters may increase the frequency of false-positive responses. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. We find that slight differences in pH increase the frequency of false-positive responses 5-fold. Temperature and time of incubation affect the LAL test less. Simultaneous differences in these parameters significantly increase the frequency of false-positive responses.
authors.affiliation: Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Felipe Rebello
authors.initials: FR
authors.lastname: Lourenço
authors.forename: Túlia De Souza
authors.initials: Tde S
authors.lastname: Botelho
authors.forename: Terezinha De Jesus Andreoli
authors.initials: Tde J
authors.lastname: Pinto
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins; D006737:Horseshoe Crabs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008033:Limulus Test; D013696:Temperature; D035501:Uncertainty
chemical_list: D004731:Endotoxins