The Limulus Amoebocyte Lysate Test: Detection of Endotoxin in Plasma of Swine and Cattle

Abstract

Endotoxin added in vitro to plasma of swine and cattle was detected by the Limulus Amoebocyte Lysate (LAL) test in order to evaluate the possible utility of the test for detection of endotoxemia in the veterinary clinic and in research.

The inhibitory effect of plasma on the test was diminished in porcine plasma by dilution with saline, by heat-extraction, or by the pH-shift method. The last-mentioned method was not applicable to porcine plasma, whereas the heat-extraction method followed by serial 2-fold dilutions in water admitted the detection of endotoxin at low concentrations in porcine as well as in bovine plasma. The inhibition of the LAL test was more pronounced by bovine than by porcine plasma in that, when the same lysate sensitivity was used, the detection limit of endotoxin was highest in bovine plasma. The detection limit could be lowered by increasing the lysate sensitivity either by prolongation of the incubation or by the use of water instead of saline as diluent for plasma. However, an essential increase in lysate sensitivity demands extreme precautions, and a comparatively greater increase in the clinical applicability of the LAL test would be obtained if the inhibitory effect, that is present at low concentrations of endotoxin even in heat-extracted plasma, could be eliminated.

It is concluded, that to get full benefit of the LAL test for detection of endotoxemia in the veterinary clinic and in research, a functional LAL laboratory must be made available and new techniques for the elimination of plasma inhibitors at low concentrations of endotoxin must be developed.

Sammendrag

Med henblik på at vurdere den mulige anvendelse af LAL testen til påvisning af endotoksinæmi i den veterinære klinik og i forskningsøjemed blev in vitro tilsat endotoksin søgt påvist i plasma fra svin og kvæg.

Plasmaets evne til at inhibere testen blev formindsket i svineplasma ved fortynding med saltvand, ved varme-ekstraktion eller ved pH-skifte. Den sidstnævnte metode havde ingen effekt på svineplasma, hvorimod varme-ekstraktion efterfulgt af en 2-folds fortyndingsrække i vand åbnede mulighed for påvisning af endotoksin i lave koncentrationer i både svine- og kvægplasma. Kvægplasmaets inhibition af LAL testen var mere udtalt end svineplasmaets, da påvisningsgrænsen for endotoksin var højest i kvægplasma, når lysat med samme følsomhed blev anvendt. Påvisningsgrænsen kunne nedsættes ved at øge lysatets følsomhed enten ved forlængelse af inkubationstiden eller ved at bruge vand til fortynding af plasma i stedet for saltvand. En væsentlig øgning af lysatets følsomhed kræver imidlertid, at der tages ekstreme forsigtighedsforanstaltninger, og en forholdsvis større forbedring af LAL testens kliniske anvendelighed ville opnås, hvis man helt kunne eliminere den inhibition, der til trods for varme-ekstraktion af plasmaet er til stede ved lave koncentrationer af endotoksin.

Det konkluderes, at for at få fuldt udbytte af LAL testen til påvisning af endotoksinæmi i den veterinære klinik og i forskningsøjemed må et laboratorium med rutine i anvendelsen af testen være tilgængeligt, og samtidig må nye metoder til elimination af plasmaets inhibition af testen ved lave endotoksinkoncentrationer udvikles og afprøves.