Gene K Lindsay, 83Saint Peters, MO

4279774, Jul 21, 1981

Nov 13, 1979

6/093403

Gene K. Lindsay - Middletown MD
Andrew J. O'Beirne - Walkersville MD

Dynasciences Corporation - Los Angeles CA

G01N 3100
G01N 2104
G01N 3348

252408

The improved method of the present invention involves the removal of lipid inhibitors from Limulus amebocyte lysate to increase the sensitivity and quality of the lysate. The method comprises intimately contacting, as by mixing and stirring, Limulus amebocyte lysate with a selected binary liquid solvent system for a time sufficient to draw water into the solvent system from the lysate and to effectively extract and denature the lipid inhibitors in the lysate. The ability of the binary solvent to draw in water from the lysate and form, in effect, a new tertiary system is the important factor in this methodology. The amount of water drawn into the solvent is controlled by the amount of polar solvent in the solvent system. After this extraction and denaturation, the lysate is then separated from the solvent system and recovered in purified form of increased sensitivity.

4301245, Nov 17, 1981

May 29, 1980

6/154360

Gene Lindsay - Middletown MD
Andrew J. O'Beirne - Walkersville MD

Dynasciences Corporation - Los Angeles CA

C12Q 144

435 4

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 insufficient to 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.