Methods Of Detecting Lp-PLA2 Activity
This invention relates to a method for measuring enzymatically active Lipoprotein Phospholipase A2 (Lp-PLA2) in a sample. Further, this invention relates to a Hybrid Immunocapture method for measuring enzymatically active Lp-PLA2 in a sample. Specifically, this invention relates to a Hybrid Immunocapture method for measuring enzymatically active Lp-PLA2 in a sample utilizing an enzymatically active Lp-PLA2 standard. In addition, this invention relates to a kit for measuring enzymatically active Lp-PLA2 in a sample. Specifically, this invention relates to a kit for measuring enzymatically active Lp-PLA2 in a sample containing an enzymatically active Lp-PLA2 standard.
1 . A method for measuring enzymatically active Lipoprotein-associated Phospholipase A2 (Lp-PLA2) in a sample comprising:
(a) contacting an immobilized binder, which specifically binds Lp-PLA2, with the sample;
(b) washing the immobilized binder to remove an enzymatically active unbound material or an interfering substance(s);
(c) contacting the bound Lp-PLA2 with a substrate converted to a detectable product in the presence of Lp-PLA2; and
(d) measuring detectable product indicative of enzymatically active Lp-PLA2 in the sample.
2 . The method of claim 1 , wherein the sample is a serum sample, a plasma sample, or an EDTA treated plasma sample.
3 . The method of claim 1 , wherein the immobilized binder is an antibody.
4 . The method of claim 3 , wherein the antibody is a monoclonal antibody, a phage display antibody, or a polyclonal antibody.
5 - 7 . (canceled)
8 . The method of claim 1 , wherein the substrate is selected from the group consisting of
wherein,
X is selected from the group consisting of O, S, and —O(CO)—;
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 , and (CH 2 ) 7 CH═CH (CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO and CH 2 ;
wherein,
X is selected from the group consisting of O, S, and —O(CO)—;
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 and (CH 2 ) 7 CH═CH(CH 2 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO and CH 2 ;
1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine (MNP);
2-thio PAF; and
wherein
X is selected from the group consisting of O, S, and —O(CO)—;
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 , and (CH 2 ) 7 CH═CH (CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO or CH 2 .
9 . The method of claim 8 where in the substrate is an oxidized derivative of (a), (b), (c), (d) or (e).
10 - 11 . (canceled)
12 . The method of claim 1 which further comprises comparing the measured detectable product of step (d) to detectable product in a control comprising an enzymatically active Lp-PLA2 standard.
13 - 16 . (canceled)
17 . A method for detecting vascular disease in an individual comprising utilizing the method of claim 1 to determine the individual's Lp-PLA2 activity in a sample wherein increased activity of Lp-PLA2 in the sample is indicative of vascular disease.
18 . (canceled)
19 . A method for selecting an individual for therapy to treat vascular disease comprising utilizing the method of claim 1 to determine the individual's Lp-PLA2 activity in a sample wherein increased activity of Lp-PLA2 in the sample is indicative of an individual who will benefit from therapy to treat vascular disease.
20 - 21 . (canceled)
22 . A method for monitoring an individual's response to therapy to treat vascular disease comprising utilizing the method of claim 1 to determine the individual's Lp-PLA2 activity in a sample wherein decreased activity of Lp-PLA2 in the sample is indicative of an individual who is responding favorably to therapy to treat vascular disease.
23 - 24 . (canceled)
25 . A method for measuring enzymatically active Lipoprotein-associated Phospholipase A2 (Lp-PLA2) in a sample comprising:
(a) contacting a binder, which specifically binds Lp-PLA2, with the sample to form a binder-Lp-PLA2 complex;
(b) immobilizing the binder-Lp-PLA2 complex;
(c) washing the immobilized binder-Lp-PLA2 complex to remove an enzymatically active unbound material or an interfering substance(s);
(d) contacting the immobilized bound Lp-PLA2 with a substrate converted to a detectable product in the presence of Lp-PLA2; and
(e) measuring detectable product indicative of enzymatically active Lp-PLA2 in the sample.
26 . The method of claim 25 , wherein the sample is a serum sample, a plasma sample or an EDTA treated plasma sample.
27 . The method of claim 25 , wherein the binder is an antibody.
28 - 29 . (canceled)
30 . The method of claim 25 wherein the binder-Lp-PLA2 complex is immobilized by binding to an immobilized compound, said immobilized compound comprising an antibody, protein or compound capable of binding the binder-Lp-PLA2 complex.
31 - 34 . (canceled)
35 . The method of claim 25 wherein the binder is conjugated to an immobilizing agent.
36 . The method of claim 35 , wherein the binder conjugated to an immobilizing agent is an antibody.
37 - 38 . (canceled)
39 . The method of claim 35 wherein the immobilizing agent is an antibody, protein or compound capable of binding an immobilized compound.
40 - 41 . (canceled)
42 . The method of claim 35 wherein the immobilizing agent is biotin.
43 . The method of claim 35 wherein the immobilizing agent, conjugated to the binder-Lp-PLA2 complex, binds to an immobilized compound.
44 . The method of claim 43 wherein the immobilized compound is bound to a multi-well plate, a magnetic bead, or a latex bead.
45 . The method of claim 44 wherein the bound compound is an antibody, protein or compound capable of binding the conjugated immobilizing agent.
46 - 47 . (canceled)
48 . The method of claim 45 wherein the bound substance is streptavidin.
49 - 50 . (canceled)
51 . The method of claim 25 , wherein the substrate is selected from the group consisting of
wherein,
X is selected from the group consisting of O, S, and —O(CO)—;
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 , and (CH 2 ) 7 CH═CH (CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO and CH 2 ;
wherein,
X is selected from the group consisting of O, S, and —O(CO)—;
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 and (CH 2 ) 7 CH═CH(CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO and CH 2 ;
1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine (MNP);
2-thio PAF; and
wherein
X is selected from the group consisting of O, S, and —O(CO)—;
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 , and (CH 2 ) 7 CH═CH (CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO or CH 2 .
52 . The method of claim 51 where in the substrate is an oxidized derivative of (a), (b), (c), (d) or (e).
53 - 54 . (canceled)
55 . The method of claim 25 further comprising comparing the measured detectable product of step (e) to detectable product in a control comprising an enzymatically active Lp-PLA2 standard.
56 - 58 . (canceled)
59 . A method for detecting vascular disease in an individual comprising utilizing the method of claim 55 to determine the individual's Lp-PLA2 activity in a sample wherein increased activity of Lp-PLA2 in the sample is indicative of vascular disease.
60 . (canceled)
61 . A method for selecting an individual for therapy to treat vascular disease comprising utilizing the method of claim 55 to determine the individual's Lp-PLA2 activity in a sample wherein increased activity of Lp-PLA2 in the sample is indicative of an individual who will benefit from therapy to treat vascular disease.
62 - 63 . (canceled)
64 . A method for monitoring an individual's response to therapy to treat vascular disease comprising utilizing the method of claim 55 to determine the individual's Lp-PLA2 activity in a sample wherein decreased activity of Lp-PLA2 in the sample is indicative of an individual who is responding favorably to therapy to treat vascular disease.
65 - 66 . (canceled)
67 . A kit for measuring enzymatically active Lipoprotein-associated Phospholipase A2 (Lp-PLA2) in a sample comprising a binder which specifically binds Lp-PLA2 and a substrate converted to a detectable product in the presence of Lp-PLA2.
68 . The kit of claim 67 wherein the substrate is selected from the group consisting of
wherein,
X is selected from the group consisting of O, S, and —O(CO)—;
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 , and (CH 2 ) 7 CH═CH (CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO and CH 2 ;
wherein,
X is selected from the group consisting of O, S, and —O(CO)—;
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 and (CH 2 ) 7 CH═CH(CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO and CH 2 ;
1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine (MNP);
2-thio PAF; and
wherein
X is selected from the group consisting of O, S, and —O(CO)—;
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 , and (CH 2 ) 7 CH═CH (CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO or CH 2 .
69 . The kit of claim 68 wherein the substrate is an oxidized derivative of (a), (b), (c), (d) or (e).
70 . The kit of claim 67 further comprising an enzymatically active Lp-PLA2 standard.
71 - 72 . (canceled)
73 . A method for measuring enzymatically active Lipoprotein-associated Phospholipase A2 (Lp-PLA2) in a sample comprising:
(a) incubating the sample with a compound which reduces active thiol(s) in the sample;
(b) contacting the incubated sample with a substrate converted to a free thiol product in the presence of enzymatically active Lp-PLA2; and
(c) measuring free thiol product indicative of enzymatically active Lp-PLA2 in the sample.
74 . The method of claim 73 , wherein the sample is a serum sample, a plasma sample or an EDTA treated plasma sample.
75 . (canceled)
76 . The method of claim 73 wherein the sample is incubated at room temperature or at 37° C.
77 . (canceled)
78 . The method of claim 73 wherein the sample is incubated from about 2 to about 120 minutes.
79 . (canceled)
80 . The method of claim 73 wherein the substrate is selected from the group consisting of
2-thio PAF; and
wherein,
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 and (CH 2 ) 7 CH═CH(CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO and CH 2 .
81 . The method of claim 80 where in the substrate is an oxidized derivative of (a) or (b).
82 . The method of claim 73 further comprising comparing measured free thiol product of step (c) to free thiol product in a control comprising an enzymatically active Lp-PLA2 standard.
83 - 85 . (canceled)
86 . A kit for measuring enzymatically active Lipoprotein-associated Phospholipase A2 (Lp-PLA2) in a sample comprising a compound which reduces active thiol(s) and a substrate converted to a detectable product in the presence of Lp-PLA2.
87 . The kit of claim 86 wherein the substrate is selected from the group consisting of
2-thio PAF; and
wherein,
R is selected from the group consisting of (CH 2 ) 4 CH 3 , (CH 2 ) 6 CH 3 , (CH 2 ) 8 CH 3 , (CH 2 ) 10 CH 3 , (CH 2 ) 12 CH 3 , (CH 2 ) 14 CH 3 and (CH 2 ) 7 CH═CH(CH 2 ) 2 CH 3 ;
Y 1 is selected from the group consisting of (CO) 1-2 and (CH 2 ) 2-7 ; and
Y 2 is selected from the group consisting of CO and CH 2 .
88 . The kit of claim 87 where in the substrate is an oxidized derivative of (a) or (b).
89 . The kit of claim 86 further comprising an enzymatically active Lp-PLA2 standard.
90 - 91 . (canceled)