Kinetic assay for endotoxin using limulus amebocyte lysate and chromogenic substrate

• US 5,310,657 A
• Filed: 07/13/1992
• Issued: 05/10/1994
• Est. Priority Date: 10/30/1989
• Status: Expired due to Term

• Current Assignee: Biowhittaker Technologies, Inc. (Lonza Group AG)
• Original Assignee: Whittaker Bioproducts Incorporated
• Inventors: Berzofsky, Ronald N.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): REARDON, TIMOTHY
• Application Number:
  US07/911,375
• Time in Patent Office: 666 Days
• Field of Search: 435/4, 435/13, 435/18, 435/23, 435/34, 435/38, 930/DIG. 785
• US Class Current: 435/34
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 2337/12 Para-Nitroanilides p-NA
• CPC Class Codes: G01N 2333/948 acting on peptide bonds (3.4)
• CPC Class Codes: G01N 33/579 involving limulus lysate

Abstract

A kinetic assay procedure is provided which permits measurement of endotoxin concentration in the range from 0.005 EU/ml to 50 EU/ml. The assay procedure relies on a single reagent substrate composition comprising Limulus amebocyte lysate and a chromogenic substrate. This assay procedure is easily automated and it provides substantial improvement in both range and sensitivity over previously available endotoxin assays.

Claims

1. A kinetic method for determination of endotoxin in a test fluid using a kinetic assay substrate composition comprising the steps of:

(a) mixing a sample of the test fluid with a kinetic assay substrate composition containing a chromogenic substrate of the clotting enzyme of Limulus amebocyte lysate and a Limulus amebocyte lysate comprising a pro-clotting enzyme and having sensitivity to endotoxin in a vessel containing a spectrophotometric path under conditions suitable for endotoxin assay to form a sample-kinetic assay substrate composition mixture, wherein both said chromogenic substrate and said lysate are present in an endotoxin determining amount;

(b) placing said vessel in a spectrophotometer so that the spectrophotometer beam passes through the vessel and the sample-kinetic assay substrate composition mixture;

(c) monitoring the change in optical absorbance of this sample-kinetic assay substrate composition mixture over time;

(d) determining the time required for a predetermined absorbance change to occur, wherein said predetermined absorbance change optimizes assay sensitivity and assay duration;

(e) calculating endotoxin concentration in the sample by comparison of said time to a standard curve obtained by correlating the concentration of endotoxin in standard endotoxin solutions with the time required to reach said predetermined absorbance change for each standard endotoxin solution, wherein said standard endotoxin solutions are mixed with said kinetic assay substrate composition under conditions suitable for endotoxin assay before determining the time required for the predetermined absorbance change to occur.

2. The method of claim 1 wherein said chromogenic substrate has the formula:

space="preserve" listing-type="equation">R.sub.1 --A.sub.1 --A.sub.2 --A.sub.3 --A.sub.4 --B--R.sub.2

wherein R₁ represents hydrogen, a blocking aromatic hydrocarbon or acyl;

A₁ represents an L or D-amino acid selected from Ileu, Val or Leu;

A₂ represents Glu or Asp;

A₃ represents Ala or Cyst;

A₄ represents Arg;

B represents a linkage selected from ester and amide; and

R₂ represents a chromogenic or fluorogenic group which is covalently attached to the C-carboxyl terminal of arginine through the B linkage, the fluorogenic or chromogenic moiety being capable of being enzymatically cleaved from the remainder of the chromogenic substrate in the presence of endotoxin and pro-clotting enzyme to form a chromogen or a fluorogen.

3. The method of claim 1 wherein the Limulus amebocyte lysate has decreased sensitivity due to the presence of an endogenous inhibitor and said kinetic assay substrate composition contains a sensitivity enhancing amount of an inactivating surfactant for the inhibitor.

4. The method of claim 2 wherein the Limulus amebocyte lysate has decreased sensitivity due to the presence of an endogenous inhibitor and said kinetic assay substrate composition contains a sensitivity enhancing amount of an inactivating surfactant for the inhibitor.

5. The method of claim 3 where the vessel is a microtiter plate wherein multiple sample-kinetic assay substrate composition mixtures are present in multiple wells and the spectrophotometer is a microtiter plate reader which monitors each well sequentially in a repeating pattern.