Lp(a) subform size identification by capillary isotachophoresis electrophoresis with laser-induced-fluorescence
View Patent ↗The application describes methods for determining the concentration and/or particle number of a lipoprotein(a) subform in a biological sample using capillary isotachophoresis laser induced fluorescence (CE-ITP-LIF) and compositional analysis of lipoprotein(a) particles. The ability to measure the concentration and/or particle number of a lipoprotein(a) subform in a biological sample provides a useful diagnostic tool for assessing cardiovascular risk in a subject.
1. A method for determining the concentration of lipoprotein(a) (Lp(a)) subforms in a biological sample, wherein the lipoprotein(a) subforms comprise a apolipoprotein(a) (apo(a)) domain, the method comprising:
(a) contacting the biological sample with a signal-producing moiety under conditions suitable for the signal-producing moiety to bind to lipoprotein(a) subforms in the biological sample to form moiety-bound Lp(a) subforms,
wherein the signal-producing moiety is an anti-apo(a) antibody or antibody fragment thereof that specifically binds the kringle 4 type 2 (KIV 2 ) domain;
(b) depositing a fraction of the moiety-bound Lp(a) in a capillary isotachophoresis system;
(c) separating components of the fraction via capillary isotachophoresis;
(d) detecting signals produced by the signal-producing moiety bound Lp(a); and
(e) determining concentrations of the Lp(a) subforms in the biological sample by the detected signals, wherein the detected signals are proportional to the molar concentrations of the lipoprotein(a) subforms in the fraction.
2. The method of claim 1 , wherein the fraction is separated along a common capillary of the capillary isotachophoresis system such that the components of the fraction are separated from one another along the common capillary.
3. The method of claim 1 , wherein the signal-producing moiety is selected from the group consisting of a radiolabel, an enzyme, a luminophore and a fluorophore.
4. The method of claim 3 , wherein the enzyme is selected from the group consisting of peroxidase, alkaline phosphatase and β galactosidase.
5. The method of claim 1 , wherein the capillary isotachophoresis system is a multiplex capillary isotachophoresis laser induced fluorescence (MPCE-ITP-LIF) system.
6. A method for determining the particle number of lipoprotein(a) (Lp(a)) subforms in a biological sample, wherein the lipoprotein(a) subforms comprise a apolipoprotein(a) (apo(a)) domain, the method comprising:
(a) contacting the biological sample with a signal-producing moiety under conditions suitable for the signal-producing moiety to bind to lipoprotein(a) subforms in the biological sample to form moiety bound Lp(a) subforms,
wherein the signal-producing moiety is an anti-apo(a) antibody or antibody fragment thereof that specifically binds the kringle 4 type 2 (KIV 2 ) domain;
(b) depositing a fraction of the moiety-bound Lp(a) in a capillary isotachophoresis system;
(c) separating components of the fraction via capillary isotachophoresis;
(d) detecting signals produced by the signal-producing moiety bound Lp(a); and
(e) determining the particle number of the Lp(a) subforms in the biological sample by the detected signals, wherein the detected signals are proportional to the particle number of the Lp(a) subforms in the fraction.
7. The method of claim 6 , wherein the fraction is separated along a common capillary of the capillary isotachophoresis system such that components of the fraction are separated from one another along the common capillary.
8. The method of claim 6 , wherein the signal-producing moiety is selected from the group consisting of a radiolabel, an enzyme, a luminophore, and a fluorophore.
9. The method of claim 8 , wherein the enzyme is selected from the group consisting of peroxidase, alkaline phosphatase and β galactosidase.
10. The method of claim 6 , wherein the capillary isotachophoresis system is a multiplex capillary isotachophoresis laser induced fluorescence (MPCE-ITP-LIF) system.
11. The method of claim 1 , wherein the fraction of the sample containing the moiety-bound Lp(a) further comprises a known concentration of a signal-producing calibrator Lp(a) subform,
wherein the calibrator Lp(a) subform comprises a known molar mass and/or a known number of kringle KIV 2 domain repeats and/or a known concentration of apo(a) and/or a known concentration of apolipoprotein B (apo(B)).
12. The method of claim 11 , wherein the signal produced by the signal-producing calibrator Lp(a) is measured and compared with the signals produced from the signal producing moiety bound Lp(a) subforms and the molar mass of the Lp(a) subforms are determined based on the following formula:
[
LP
(
a
)
-
P
]
=
[
Cal
LP
(
a
)
-
P
]
×
(
LP
(
a
)
-
P
signal
)
(
Cal
LP
(
a
)
-
P
signal
)
wherein:
[Lp(a)−P] is the molar mass of a targeted Lp(a) subform,
[Cal LP(a)−P] is the molar mass of the calibrator Lp(a) subform, (LP(a)−P), and
(Cal LP (a)−P signal) is the signal intensity of the calibrator Lp(a) subform (Cal LP(a)−P).
13. The method of claim 11 , wherein the particle numbers of the Lp(a) subforms are determined as molar concentration in nmol/L, wherein the particle numbers of the Lp(a) subforms are quantified based on apo(b) concentration of the Lp(a) subforms.
14. A method of assessing cardiovascular risk in a subject, comprising
(i) determining molar mass of Lp(a) subforms in a biological sample from the subject by performing the method of claim 1 ; and
(ii) assessing cardiovascular risk of the subject based on the results of step (i); wherein assessing comprises assigning the subject to a low, moderate or high cardiovascular risk category based on the results of step (i),
wherein the subject is assigned to the low risk category when the molar mass of Lp(a) subforms is less than about 600 kD,
wherein the subject is assigned to the moderate risk category when the molar mass of the Lp(a) subforms is between about 600 kD and 700 kD,
and wherein the subject is assigned to the high risk category when the molar mass of the Lp(a) subforms is greater than about 700 kD; and
(iii) treating the subject based on the assignment to the low, moderate or high risk, wherein the treatment is selected from the group consisting of selecting a therapeutic regimen for the subject and modifying or changing an existing therapeutic regimen,
wherein the therapeutic regimen consists of administering to the subject a drug and/or supplement,
wherein the drug and/or supplement is selected from the group consisting of niacin, statin, ezetimibe, fenofibrate, estrogen, raloxifene and any combination thereof.
15. The method of claim 14 , wherein the selected therapeutic regimen further comprises giving recommendations on making or maintaining lifestyle choices based on the results of the cardiovascular risk determination.
16. The method of claim 15 , wherein the lifestyle choices are selected from the group consisting of changes in diet, changes in exercise, reducing or eliminating smoking, and a combination thereof.
17. The method of claim 1 , wherein the biological sample is selected from the group consisting of blood, plasma, urine and saliva.