US2012003662A1PendingUtilityA1

Methods and Kits for Detecting Mastitis

48
Assignee: MADURA KIRANPriority: Oct 18, 2007Filed: Jul 26, 2011Published: Jan 5, 2012
Est. expiryOct 18, 2027(~1.2 yrs left)· nominal 20-yr term from priority
Inventors:Kiran Madura
G01N 2800/365C12Q 1/6888C12Q 1/37G01N 33/573
48
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Claims

Abstract

Methods and kits for determining if one or more animals have mastitis and for monitoring animals and the quality of the milk they produce are disclosed. Kits and test assays disclosed are used to determine the quantity of proteasomes and proteins thereof, the activity of proteasome enzymes, the quantity of proteasome bound and regulating proteins, and the quantity of ubiquinated protein. Components and reagents for use in the kits and assays are also disclosed.

Claims

exact text as granted — not AI-modified
1 . A method for determining if one or more animals has mastitis comprising
 (a) performing a test assay for determining the level of proteasomes present in a test sample of milk collected from one or more animals, and   (b) comparing the level of proteasomes present in the test sample with a standard level of proteasomes in a control sample of milk, wherein an increase in the level of proteasomes present in the test sample relative to the standard level of proteasomes present in the control sample of milk indicates one or more animals has mastitis.   
     
     
         2 . The method of  claim 1 , wherein the test assay for determining the level of proteasomes present in the test sample detects proteasome activity in the sample. 
     
     
         3 . The method of  claim 2 , wherein the proteasome activity is measured by measuring the level of conversion of a proteasome enzyme substrate to a product. 
     
     
         4 . The method of  claim 3 , wherein the proteasome enzyme substrate comprises Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr (SEQ ID NO:8); Z-Gly-Gly-Leu; Suc-Leu-Leu-Val-Tyr (SEQ ID NO:9); Suc-Leu-Tyr; Ac-Arg-Leu-Arg; Boc-Leu-Arg-Arg; Bz-Val-Gly-Arg; Ac-Gly-Pro-Leu-Asp (SEQ ID NO:7); Z-Leu-Leu-Glu; or Ac-nLeu-Pro-nLeu-Asp (SEQ ID NO:6). 
     
     
         5 . The method of  claim 4 , wherein the substrate comprises a fluorogenic label, luminescent label or a chromogenic label. 
     
     
         6 . The method of  claim 2 , wherein the proteasome enzyme substrate is an ATPase substrate. 
     
     
         7 . The method of  claim 2 , wherein the proteasome enzyme substrate is a de-ubiquitinase substrate. 
     
     
         8 . The method of  claim 1 , wherein the test assay for determining the level of proteasomes present in the test sample is an assay comprising protein detection or detection of nucleic acid molecules encoding proteins. 
     
     
         9 . The method of  claim 8 , wherein the test assay detects one or more molecules comprising one or more proteasome proteins, one or more proteasome binding proteins, one or more proteasome regulating proteins, a nucleic acid molecule that encodes a proteasome protein, a proteasome regulating protein or a proteasome binding protein. 
     
     
         10 . The method of  claim 8 , wherein the test assay for determining the level of proteasomes present in the test sample is an assay to determine the level of immunoproteasomes present in the test sample. 
     
     
         11 . The method of  claim 8 , wherein the test assay for determining the level of proteasomes present in the test sample is an assay to detect the level of ubiquinated proteins in a sample or copper complex binding to proteasomes. 
     
     
         12 . A kit for detecting proteasome enzyme activity in a sample of fluid comprising:
 a) a ubiquitin-like domain affinity matrix, and   b) a container comprising a cap, wherein said container or cap comprises a proteasome enzyme substrate.   
     
     
         13 . The kit of  claim 12 , wherein the proteasome enzyme substrate comprises Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr (SEQ ID NO:8); Z-Gly-Gly-Leu; Suc-Leu-Leu-Val-Tyr (SEQ ID NO:9); Suc-Leu-Tyr; Ac-Arg-Leu-Arg; Boc-Leu-Arg-Arg; Bz-Val-Gly-Arg; Ac-Gly-Pro-Leu-Asp (SEQ ID NO:7); Z-Leu-Leu-Glu; or Ac-nLeu-Pro-nLeu-Asp (SEQ ID NO:6). 
     
     
         14 . The kit of  claim 13 , wherein the substrate comprises a fluorogenic label, luminescent label or a chromogenic label. 
     
     
         15 . The kit of  claim 12 , wherein the proteasome enzyme substrate is an ATPase substrate. 
     
     
         16 . The kit of  claim 12 , wherein the proteasome enzyme substrate is a de-ubiquitinase substrate. 
     
     
         17 . The kit of  claim 12 , further comprising a reagent for a test assay for determining the level of proteasomes present in the test sample, wherein said assay comprises protein detection or detection of nucleic acid molecules encoding proteins. 
     
     
         18 . The kit of  claim 17 , wherein the test assay detects one or more molecules comprising one or more proteasome proteins, one or more proteasome binding proteins, one or more proteasome regulating proteins, a nucleic acid molecule that encodes a proteasome protein, a proteasome regulating protein or a proteasome binding protein. 
     
     
         19 . The kit of  claim 17 , wherein the test assay is an assay to determine the level of immunoproteasomes present in the test sample. 
     
     
         20 . The kit of  claim 17 , wherein the test assay is an assay to detect the level of ubiquinated proteins in a sample or copper complex binding to proteasomes. 
     
     
         21 . A method for determining milk quality comprising
 (a) collecting a test sample of milk,   (b) performing a test assay for determining the level of proteasomes present in the test sample, and   (c) comparing the level of proteasomes present in the test sample with a standard level of proteasomes in a control sample of high quality milk or comparing the level of proteasomes present in the test sample with a standard level of proteasomes in a control sample of low quality milk;   wherein an increase in the level of proteasomes present in the test sample relative to the standard level of proteasomes present in the control sample of high quality milk indicates the milk is of lower quality;   an increase in the level of proteasomes present in the test sample relative to the standard level of proteasomes present in the control sample of low quality milk indicates the milk is of higher quality;   a substantially similar level of proteasomes present in the test sample relative to the standard level of proteasomes present in the control sample of high quality milk indicates the milk is of high quality; and   a substantially similar level of proteasomes present in the test sample relative to the standard level of proteasomes present in the control sample of low quality milk indicates the milk is of lower quality.   
     
     
         22 . A kit for determining the level of proteasomes present in a test sample comprising a first antibody and second antibody that each specifically bind one or more proteasomal subunits. 
     
     
         23 . The kit of  claim 22 , wherein the first and second antibodies bind the same proteasomal subunit at distinct epitopes. 
     
     
         24 . The kit of  claim 22 , wherein the first and second antibodies bind two distinct proteasomal subunits. 
     
     
         25 . The kit of  claim 22 , wherein the first antibody is bound to a solid support and the second antibody comprises a detectable label.

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