The Chromogenic Limulus Amoebocyte Lysate (LAL) Assay for Endotoxin: Developments and Applications

Piotrowicz, Boguslawa I (1986) The Chromogenic Limulus Amoebocyte Lysate (LAL) Assay for Endotoxin: Developments and Applications. PhD thesis, University of Glasgow.

Abstract

Endotoxin (lipopolysaccharide, LPS) is a part of the envelope of Gram-negative bacteria. Among the different methods for the detection and measurement of endotoxin, the Limulus amoebocyte lysate (LAL) assay is the most widely used. The reagent used in this assay is the lysate of blood cells (amoebocytes) of one of the species of horseshoe crab, usually Limulus polyphemus. The most recent development among the objective and quantitative LAL assay methods is a chromogenic method, which uses a synthetic chromogenic peptide substrate. This method is based on the principle that endotoxin-activated lysate releases from the colourless substrate yellow p-nitroaniline (pNA), which can be measured spectrophotometrically. In this study, the original two-stage assay, as recommended by the LAL manufacturer, was optimized. This resulted in a ten-fold increase in sensitivity of the assay and a two-fold decrease in the volumes of expensive reagents. The processing time of large numbers of samples was also significantly decreased by using microplates for spectrophotometric readings in a MICROELISA MiniReader. Subsequently, the two-stage assay was further modified and a one-stage method was developed. This resulted in a further five-fold increase in sensitivity of the assay, to 0.2 pg endotoxin/ml of pyrogen-free water, which is the equivalent of 0. 002 Endotoxin Units (EU)/ml of pyrogen-free water, with the linearity of the standard curve in the range of 0-2pg/ml or 0-0.23 EU/ml. In plasma, the threshold of sensitivity was 1 pg./ml (0.012 EU/ml) and the standard curve was linear in the range 0-30 pg/ml (0-0.345 EU/ml). Other advantages of the one-stage assay were: a further decrease in reagent volumes and reduction of the time required for processing large numbers of samples. In this study, the chromogenic LAL assay was used for detection of endotoxin in clinical and non-clinical studies. Endotoxin is thought to be an important factor in the pathogenesis of Gram-negative infections, although its exact role remains unknown. However,difficulties arise when the LAL assay is used for the detection of endotoxin in human blood. This is the presence in blood of inhibitors and non-specific activators of the assay. The clinical studies involved three trials: septic shock, cyclic neutropenia and acute pancreatitis. The septic shock trial consisted of two groups of patients: Group 1:ten critically ill, but not necessarily septic shock patients with daily LAL assays and simultaneous blood cultures; Group 2 - eight acute septic shock patients, who had four-hourly LAL assays for the first 48 hours, and thereafter daily. Blood for blood cultures and fibronectin was taken simultaneously with blood for LAL assays. Cardiovascular and respiratory functions were also measured at the same time. The septic shock trial demonstrated the need for frequent monitoring of endotoxin levels in acute conditions. In Group 2, an inverse correlation between endotoxin levels and plasma fibronectin was shown. In most cases, endotoxin levels correlated with cardiovascular and respiratory indices. However, there seemed to be no correlation between the severity of septic shock and absolute levels of endotoxin. Several hgpotheses are discussed for future verification. The cyclic neutropenia trial involved one patient. In the acute pancreatitis trial 16 patients were examined The results of both studies were unsatisfactory. This was most likely because of inadequate sampling. Therefore no conclusions could be made about the role of endotoxin in these conditions. A different approach is suggested for future studies. In the non-clinical studies, the LAL assay was used to examine two practical problems. One was the removal of endotoxin from human blood and the other was investigation of the role of endotoxin as an occupational hazard. Thus the performance of an extra-corporeal filter (DEP-1) for removal of endotoxin from human blood was tested. The filter appeared to be incapable of extracting clinically significant amounts of endotoxin from human plasma. Endotoxin levels were measured in baffle plate material from the hamidification system of a semi-conductor factory and in a dust sample from books in an archive library. Endotoxin, which was found in both, could have been at least partially responsible for the clinical symptoms reported by the factory workers and library staff.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Medicine
• Date of Award: 1986
• Depositing User: Enlighten Team
• Unique ID: glathesis:1986-77395
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jan 2020 11:53
• Last Modified: 14 Jan 2020 11:53
• URI: https://theses.gla.ac.uk/id/eprint/77395