Improved chromogenic method of detecting endotoxins in blood

Abstract

Abstract An improved method is provided for detecting endotoxins in blood serum and/or plasma, particularly human blood fractions. The method employs king crab amebocyte lysate, preferably Limulus amebocyte lysate, in the presence of a substrate which has a selected colorimetric indicator bound to it. The indicator is capable of being split from the substrate by an enzyme which can be generated in the lysate by endotoxins in the blood. Thus, the endotoxins convert proenzyme in the lysate to the enzyme which effects the splitting off of the colorimetric indicator from the substrate. The endotoxin concentration in the blood can thus be determined colorimetrically, that concentration being proportional to the concentration of color indicator split from the substrate. The blood sample need not be extracted, as is required in prior methods, with a solvent such as chloroform to remove inhibitors therein which would interfere with a lysate gelation reaction. Heparin is utilized in the present method in a concentration sufficient to stabilize the lysate against loss of potency but insufficientto inhibit the reaction whereby endotoxin generates the described splitting enzyme from proenzyme in the lysate and the reaction of the enzyme to cause the split. The blood fraction need not be diluted with water and the lysate may be one which has been reconstituted from dry powder, if desired. The method is simple, highly effective, reproducible, inexpensive and rapid.

Inventors

• 👤 Gene Lindsay
• 👤 Andrew J. O'beirne

Bibliographic Details