LONG SHELF-LIFE KITS AND METHODS FOR STANDARDIZING, VERIFYING, CALIBRATING OR RECALIBRATING DETECTION OF LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2
Long shelf-life kits, value-assigned solutions, and methods for standardizing, verifying, calibrating or recalibrating detection of lipoprotein-associated phospholipase A2 having using them are described herein. In particular, described herein are methods of using solutions of rLp-PLA2 that are stable for an extended period of time to standardize, verify, calibrate or recalibrate assays for Lp-PLA2.
1 . A method of recalibrating a calibration curve for detection of lipoprotein-associated phospholipase A2 (Lp-PLA2) from a biological sample using a value-assigned solution of recombinant Lp-PLA2 having a long shelf life, the method comprising:
detecting a first signal from a value-assigned solution having a first predetermined concentration of a recombinant Lp-PLA2 in a buffer solution, wherein the buffer solution comprises a detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2; and
transforming the calibration curve using the first signal.
2 . The method of claim 1 , wherein transforming comprises shifting, scaling or shifting and scaling the calibration curve based on the first signal.
3 . The method of claim 1 , wherein the calibration curve relates signal intensity to concentration of Lp-PLA2.
4 . The method of claim 1 , further comprising detecting a second signal from a second value-assigned solution having a second predetermined concentration of a recombinant Lp-PLA2 in the buffer solution, wherein the buffer solution comprises the detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2, and wherein transforming the calibration curve comprises using the first and second signals.
5 . The method of claim 1 , further comprising combining the value-assigned solution with an agent that interacts with Lp-PLA2 to produce a detectable signal before detecting the first signal.
6 . The method of claim 5 , wherein the agent that interacts with Lp-PLA2 comprises an antibody directed against Lp-PLA2 or a substrate for Lp-PLA2.
7 . The method of claim 5 , wherein the agent that interacts with Lp-PLA2 comprises a labeled antibody directed against Lp-PLA2 or a labeled substrate for Lp-PLA2.
8 . The method of claim 1 , wherein detecting the signal comprises detecting a complex of Lp-PLA2 and an antibody or detecting enzymatic activity Lp-PLA2.
9 . The method of claim 1 , wherein the detergent of the buffer solution forming the plurality of micelles comprises CHAPS.
10 . The method of claim 1 , wherein detecting comprises detecting the first signal from the value-assigned solution having the first predetermined concentration of a recombinant Lp-PLA2 in the buffer solution, wherein the buffer solution is a low-salt buffer solution having a salt concentration below about 1 M and comprising a detergent forming the plurality of micelles and a second detergent to prevent aggregation of the recombinant Lp-PLA2, further wherein the detergent forming the plurality of micelles is different from the second detergent.
11 . The method of claim 1 , wherein detecting comprises detecting the first signal from the value-assigned solution having the first predetermined concentration of a recombinant Lp-PLA2 in the buffer solution, wherein the buffer solution further comprises a protein buffered matrix.
12 . A method of recalibrating a calibration curve for detection of lipoprotein-associated phospholipase A2 (Lp-PLA2) from a biological sample using a value-assigned solution of recombinant Lp-PLA2 having a long shelf life, the method comprising:
combining a value-assigned solution comprising a first predetermined concentration of a recombinant Lp-PLA2 in a buffer solution with an agent that interacts with Lp-PLA2 to produce a detectable first signal, wherein the buffer solution comprises a detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2;
detecting the first signal; and
transforming a calibration curve by shifting, scaling or shifting and scaling the calibration curve based on the first signal.
13 . A method of producing a calibration curve for detection of lipoprotein-associated phospholipase A2 (Lp-PLA2) from a biological sample by using value-assigned solutions of recombinant Lp-PLA2 that have a long shelf life, the method comprising:
combining an agent that interacts with Lp-PLA2 to produce a detectable signal with a plurality of value-assigned solutions, wherein each value-assigned solution has a predetermined concentration of the recombinant Lp-PLA2 in a buffer solution, the buffer solution comprising a detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2;
detecting Lp-PLA2 signals from the value-assigned solutions; and
creating a calibration curved based on the relationship between the detected signals and the predetermined concentrations of the recombinant Lp-PLA2.
14 . The method of claim 13 , wherein detecting Lp-PLA2 signals comprises detecting Lp-PLA2 signals from at least four value-assigned solutions having different predetermined concentrations of the recombinant Lp-PLA2.
15 . The method of claim 13 , wherein detecting Lp-PLA2 signals comprises detecting Lp-PLA2 signals from between about four to 10 value-assigned solutions having different predetermined concentrations of the recombinant Lp-PLA2.
16 . The method of claim 13 , wherein the calibration curve relates a signal intensity of the signals to the predetermined concentrations of the recombination Lp-PLA2.
17 . The method of claim 13 , wherein the agent that interacts with Lp-PLA2 comprises an antibody directed against Lp-PLA2 or a substrate for Lp-PLA2.
18 . The method of claim 17 , wherein the agent that interacts with Lp-PLA2 comprises a labeled antibody directed against Lp-PLA2 or a labeled substrate for Lp-PLA2.
19 . The method of claim 13 , wherein detecting LpPLA2 signals comprises detecting a complex of Lp-PLA2 and an antibody or detecting enzymatic activity Lp-PLA2 on a substrate.
20 . The method of claim 13 , wherein combining comprises combining the agent with each of the plurality of value-assigned solutions, wherein the buffer solution of the value-assigned solutions comprises a plurality of micelles of CHAPS that stabilize the recombinant Lp-PLA2.
21 . The method of claim 13 , wherein combining comprises combining the agent with each of the plurality of value-assigned solutions, wherein the buffer solution of the value-assigned solutions comprises a low-salt buffer solution having a salt concentration below about 1 M and a second detergent to prevent aggregation of the recombinant Lp-PLA2, further wherein the detergent forming the plurality of micelles that stabilize the recombinant Lp-PLA2 is different from the second detergent.
22 . The method of claim 13 , wherein combining comprises combining the agent with each of the plurality of value-assigned solutions, wherein the buffer solution of the value-assigned solutions comprises a protein buffered matrix.
23 . The method of claim 13 , wherein creating the calibration curve comprises arranging the signals comprises and the predetermined concentrations of the recombinant Lp-PLA2.
24 . A method of producing a calibration curve for detection of lipoprotein-associated phospholipase A2 (Lp-PLA2) from a biological sample by using value-assigned solutions of recombinant Lp-PLA2 that have a long shelf life, the method comprising:
combining an agent that interacts with Lp-PLA2 to produce a detectable signal with a plurality of value-assigned solutions, wherein each value-assigned solution has a different predetermined concentration of the recombinant Lp-PLA2 in a buffer solution, the buffer solution comprising a detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2, a pH buffer, a protein buffered matrix and a non-chaotropic salt;
detecting Lp-PLA2 signals from the value-assigned solutions; and
creating a calibration curved based on the relationship between the detected signals and the predetermined concentrations of the recombinant Lp-PLA2.
25 . A lipoprotein-associated phospholipase A2 (Lp-PLA2) kit for use with an Lp-PLA2 assay, the kit having a shelf-life of greater than 4 months, the kit comprising:
a first value-assigned solution comprising a first predetermined concentration of a recombinant Lp-PLA2 in a first buffer solution, wherein the first buffer solution comprises a first detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2; and
a second value-assigned solution comprising a second predetermined concentration of the recombinant Lp-PLA2 in a second buffer solution, wherein the second buffer solution comprises a second detergent forming plurality of micelles that stabilize the recombinant Lp-PLA2.
26 . The kit of claim 25 , further comprising a third value-assigned solution comprising a third predetermined concentration of the recombinant Lp-PLA2 in a third buffer solution, wherein the third buffer solution comprises a third detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2.
27 . The kit of claim 25 , wherein the first and second detergents comprise a cholate detergent.
28 . The kit of claim 25 , wherein the first and second detergent comprise CHAPS.
29 . The kit of claim 25 , wherein the first buffer solution and the second buffer solution comprise a low-salt buffer solution.
30 . The kit of claim 25 , wherein the first buffer solution and the second buffer solution have a salt concentration of less than 1 M.
31 . The kit of claim 25 , wherein the second predetermined concentration of the recombinant Lp-PLA2 is zero.
32 . The kit of claim 25 , wherein the first buffer solution and the second buffer solution comprises a low-salt buffer solution comprising a non-chaotropic salt.
33 . The kit of claim 25 , wherein the first buffer solution and the second buffer solution comprises a low-salt buffer solution comprising one or more of: NaCl and an acetate salt.
34 . The kit of claim 25 , wherein the first buffer solution and the second buffer solution comprise a protein buffered matrix.
35 . The kit of claim 25 , wherein the first buffer solution and the second buffer solution comprise bovine serum albumin (BSA).
36 . The kit of claim 25 , wherein the first buffer solution and the second buffer solution include Tris as a pH buffer.
37 . A lipoprotein-associated phospholipase A2 (Lp-PLA2) kit for use with an Lp-PLA2 assay, the kit having a shelf-life of greater than 4 months, the kit comprising:
a first value-assigned solution comprising a first predetermined concentration of a recombinant Lp-PLA2 in a first buffer solution, wherein the first buffer solution comprises a cholate detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2, a protein buffered matrix, a pH buffer and a preservative; and
a second value-assigned solution comprising a second predetermined concentration of the recombinant Lp-PLA2 in a second buffer solution, wherein the second buffer solution comprises a cholate detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2.
38 . The kit of claim 37 , wherein the cholate detergent of the first and second buffer solution comprises CHAPS.
39 . The kit of claim 37 , wherein the preservative of the first and second buffer solution comprises sodium azide.
40 . The kit of claim 37 , wherein the protein buffered matrix of the first and second buffer solution comprises bovine serum albumin (BSA).
41 . A lipoprotein-associated phospholipase A2 (Lp-PLA2) assay utilizing a value-assigned solution having a long shelf-life for use as a standard, control, calibrator or re-calibrator, the assay comprising:
a value-assigned solution comprising a predetermined concentration of a recombinant Lp-PLA2 in a buffer solution, wherein the buffer solution comprises a cholate detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2;
a wash buffer;
a solid phase support configured to bind Lp-PLA2; and
a report antibody specific to Lp-PLA2.
42 . The assay of claim 41 , wherein the cholate detergent comprises CHAPS.
43 . The assay of claim 41 , wherein the buffer solution comprises a low-salt buffer solution having a salt concentration of less than 1 M.
44 . The assay of claim 41 , wherein the buffer solution comprises a low-salt buffer solution of a non-chaotropic salt.
45 . The assay of claim 41 , wherein the buffer solution comprises a low-salt buffer solution of one or more of: NaCl and an acetate salt.
46 . The assay of claim 41 , wherein the buffer solution includes a preservative.
47 . The assay of claim 41 , wherein the buffer solution includes bovine serum albumin (BSA) as a protein buffered matrix.
48 . The assay of claim 41 , wherein the buffer solution includes a pH buffer.
49 . The assay of claim 41 , wherein the buffer solution includes Tris as a pH buffer.
50 . A method of estimating the amount, activity or amount and activity of lipoprotein-associated phospholipase A2 (Lp-PLA2) from a patient sample, the method comprising:
combining a value-assigned solution comprising a first predetermined concentration of a recombinant Lp-PLA2 in a buffer solution with an agent that interacts with Lp-PLA2 to produce a detectable first signal, wherein the buffer solution comprises a detergent forming a plurality of micelles that stabilize the recombinant Lp-PLA2;
detecting the first signal;
combining at least a portion of the patient sample with the agent that interacts with Lp-PLA2 to produce a detectable second signal;
detecting the second signal; and
assigning a value for activity, concentration or activity and concentration of Lp-PLA2 from the patient sample using the second signal.
51 . The method of claim 50 , wherein assigning the value for an activity, concentration or activity and concentration of Lp-PLA2 from the patient sample comprises calibrating the second signal based on the first signal.
52 . The method of claim 50 , further comprising determining the validity of the assigned value by comparing the value of the first signal to a predetermined value or a predetermined range of values.
53 . The method of claim 50 , further comprising combining a second value-assigned solution comprising a second predetermined concentration of a recombinant Lp-PLA2 in a second buffer solution with the agent that interacts with Lp-PLA2 to produce a detectable third signal, wherein the second buffer solution comprises a plurality of micelles of a detergent stabilizing the recombinant Lp-PLA2; and detecting the third signal.
54 . The method of claim 50 , wherein combining the value-assigned solution with the solution comprising the agent that interacts with Lp-PLA2 comprises using a value-assigned solution that has a shelf-life of greater than 4 months.
55 . The method of claim 50 , wherein combining the value-assigned solution with the solution comprising the agent that interacts with Lp-PLA2 comprises combining the value-assigned solution with the solution comprising an antibody that binds to Lp-PLA2.
56 . The method of claim 50 , wherein combining the value-assigned solution with the solution comprising the agent that interacts with Lp-PLA2 comprises combining the value-assigned solution with the solution comprising a substrate to Lp-PLA2.