Biological extracts and method for making same

• US 4,376,819 A
• Filed: 02/23/1981
• Issued: 03/15/1983
• Est. Priority Date: 09/14/1979
• Status: Expired due to Term

• Current Assignee: Baxter Travenol Laboratories Incorporated (Baxter International Inc.)
• Original Assignee: Baxter Travenol Laboratories Incorporated (Baxter International Inc.)
• Inventors: Cotter, Richard, Young, Susan K., Brown, R. Clark
• Primary Examiner(s): Hruskoci, Peter A.
• Application Number:
  US06/236,963
• Time in Patent Office: 750 Days
• Field of Search: 23/230 B, 252/408 R, 424/2, 424/94, 435/23, 435/24, 435/184, 435/212, 435/219, 435/226, 435/4, 435/7, 435/29
• US Class Current: 435/4
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 2334/10 p-Nitrophenol derivatives
• CPC Class Codes: G01N 33/579 involving limulus lysate
• CPC Class Codes: G01N 33/96 involving blood or serum control standard
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/967 Standards, controls, materials, e.g. validation studies, buffer systems

Abstract

A p-nitrophenyl hydrolase contaminant present in Limulus coagulase sources interferes in, i.e., inhibits, coagulase-based assays for endotoxin. The contaminant inhibitor enzyme may be removed from coagulase by physical separation or by selective denaturation, for example by adjusting the pH of a coagulase solution to about from 8.5 to 11. Inhibitor-free coagulase is a novel composition. The coagulase activity of the composition is adjusted to a predetermined level to provide a standardized coagulase reagent for use in endotoxin assays.

Claims

1. A method for inactivating inhibitor in coagulase-containing amebocyte extracts which comprises adjusting the pH of the extract above about 8.5 to inactivate the inhibitor, followed by lowering the pH to a level at which coagulase is active.

2. A method for inactivating inhibitor in coagulase-containing amebocyte extracts which comprises increasing the pH of the extract until the desired degree of inactivation has occurred and then lowering the lysate pH to about 7.

3. A method comprising removing p-nitrophenyl phosphate hydrolase activity from coagulase-containing amebocyte extracts by increasing the pH of the extract until the desired degree of inactivation has occurred and then lowering the lysate pH to a level at which coagulase is active.

4. The method of claim 3 wherein the hydrolase is denatured by adjusting the pH of the extract above about 8.5, followed by lowering the pH to a level at which coagulase is active.

5. A method for preparing a standardized amebocyte extract from inhibitor- and coagulase-containing amebocyte extract which comprises:

(a) adjusting the pH of the extract above about 8.5 to inactivate the inhibitor, followed by lowering the pH to a level at which coagulase is active;

(b) determining the coagulase activity of the extract;

(c) determining the coagulogen content of the extract and, if the coagulogen content is not in excess of a predetermined level, adjusting said content so that it is in excess of said level.

6. The method of claim 5 wherein the coagulogen content is adjusted by adding coagulogen.

7. The method of claim 5 wherein the extract is separated into portions by dispensing volumes of extract into containers so that each container holds a predetermined coagulase activity.

8. The method of claim 7 wherein the extract is lyophilized after being dispensed into the containers.