Methods of detecting mastitis by levels of proteasomes
View Patent ↗Method and systems for determining if one or more animals has mastitis and for monitoring animals and the quality of the milk they produce are disclosed. Systems 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 systems and assays are also disclosed.
1. A method for determining if one or more animals has mastitis, the method comprising
(a) performing a test assay and thereby determining a level of proteasomes present in a test sample of milk collected from the one or more animals;
(b) comparing the level of proteasomes present in the test sample with a standard level of proteasomes in a control sample of milk from an uninfected animal; and
(c) correlating an increased level of proteasomes present in the test sample relative to the standard level of proteasomes present in the control sample as being indicative of the one or more animals having mastitis.
2. The method of claim 1 , wherein the test assay detects proteasome activity in the test sample.
3. The method of claim 2 , wherein the proteasome activity is measured by measuring a level of conversion of a proteasome enzyme substrate to a product.
4. The method of claim 3 , wherein the proteasome enzyme substrate is selected from the group consisting of Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-AMC (SEQ ID NO:1); Z-Gly-Gly-Leu-betaNA; Z-Gly-Gly-Leu-AMC; Suc-Leu-Leu-Val-Tyr-AMC (SEQ ID NO:2); Suc-Leu-Tyr-AMC; Ac-Arg-Leu-Arg-AMC; Boc-Leu-Arg-Arg-AMC; Bz-Val-Gly-Arg-AMC; Ac-Gly-Pro-Leu-Glu-AMC (SEQ ID NO:3); Z-Leu-Leu-Asp-AMC; Z-Leu-Leu-Asp-betaNA; and Ac-nLeu-Pro-nLeu-Glu-AMC (SEQ ID NO:4); wherein Suc refers to succinimide; AMC refers to 7-amino-4-methylcoumarylamide; Z refers to benzyloxycarbonyl; betaNA refers to beta-naphthylamide; Boc refers to tert-butyloxycarbonyl; Bz refers to benzoyl; Ac refers to acetyl; and nLeu refers to norleucinal.
5. The method of claim 3 , which can distinguish eukaryotic proteasomes from prokaryotic proteasomes.
6. The method of claim 3 , wherein the proteasome enzyme substrate is an ATPase substrate.
7. The method of claim 3 , wherein the proteasome enzyme substrate is a de-ubiquitinase substrate.
8. The method of claim 1 , wherein the test assay comprises an assay selected from the group consisting of protein detection and detection of nucleic acid molecules that encode proteins.
9. The method of claim 8 , wherein the test assay detects one or more molecules selected from the group consisting of one or more proteasome proteins; one or more proteasome-binding proteins; one or more proteasome-regulating proteins; and 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 comprises an assay to determine a level of immunoproteasomes present in the test sample.
11. The method of claim 8 , wherein the test assay comprises an assay to detect a level of ubiquinated proteins in a sample or an assay to detect copper-complex binding to proteasomes.
12. A method for determining milk quality, the method comprising
(a) collecting a test sample of milk;
(b) performing a test assay and thereby determining a level of proteasomes present in the test sample;
(c) comparing the level of proteasomes present in the test sample with a first standard level of proteasomes in a first control sample of high quality milk, or comparing the level of proteasomes present in the test sample with a second standard level of proteasomes in a second control sample of low quality milk; and
(d) making at least one of the following correlation steps:
(i) correlating an increased level of proteasomes present in the test sample relative to the first standard level of proteasomes as being indicative of the test sample being milk of lower quality than the first control sample of high quality milk;
(ii) correlating an increased level of proteasomes present in the test sample relative to the second standard level of proteasomes as being indicative of the test sample being milk of higher quality than the second control sample of low quality milk;
(iii) correlating a substantially similar level of proteasomes present in the test sample relative to the first standard level of proteasomes as being indicative of the test sample being milk of high quality; and
(iv) correlating a substantially similar level of proteasomes present in the test sample relative to the second standard level of proteasomes as being indicative of the test sample being milk of low quality.