Lp(a) subform size identification using elisa
View Patent ↗The application describes methods for determining the particle number and/or molar mass of lipoprotein(a) subform(s) in a biological sample using enzyme linked immunosorbent assay (ELISA). The methods described herein significantly improve lipoprotein ELISA methods and devices capable of quantifying particle numbers and population mass of Lp(a) particles. This technology offers a method for the efficient and cost-effective measurement of specific Lp(a) in a rapid, low-cost format, rather than limited measurement of Lp(a) concentration in patient tissues. The ability to measure the particle number and/or molar mass of lipoprotein(a) subform(s) in a biological sample also provides a useful diagnostic tool for assessing cardiovascular risk in a subject.
1. A method for simultaneously determining particle number and molar mass of lipoprotein(a) subforms (Lp(a)-P) in a biological sample, wherein the lipoprotein(a) subforms comprise an apolipoprotein(a) (apo(a)) domain, the method comprising:
(a) providing a solid phase support with a first antibody immobilized on the support, wherein the first antibody is an anti-Lp(a)-capture antibody specific to a region of lipoprotein(a) other than a Kringle IV type 2 (KIV2) domain;
(b) contacting the solid phase support with a biological sample comprising Lp(a)-P subforms to permit binding of the Lp(a)-P subforms to the first antibody and with a known concentration of signal-producing calibrator lipoprotein comprising a known number of KIV2 domains;
(c) contacting the solid phase support with second and third antibodies, each bound to fluorescence signal-producing molecules that are distinguishable from each other, wherein the second antibody is an anti-KIV2 antibody and the third antibody is an anti-apolipoproteinB (anti-apoB) antibody, and further wherein the signal producing calibrator lipoprotein is distinguishable from the fluorescence signal producing molecules bound to the first and second antibodies;
(d) detecting fluorescence signals produced by the second and third antibodies and detecting signal produced by the signal producing calibrator lipoprotein;
(e) quantifying the concentration of KIV2 domains based on the detected signal of the second antibody and quantifying the concentration of apoB based on the detected signal of the third antibody; and
(f) determining the particle number and the molar mass of the Lp(a)-P subforms present in the sample,
wherein (i) the particle number of the Lp(a)-P subforms present in the sample is determined by the following formula for concentration of the Lp(a)-P subforms:
[
Lp
(
a
)
-
P
in
sample
]
=
(
[
Calibrator
Lp
(
a
)
-
P
]
)
(
signal
produced
by
anti
-
apoB
antibody
signal
produced
by
calibrator
)
wherein:
Calibrator Lp(a)-P is the concentration of the calibrator lipoprotein in nmol/L, signal produced by anti-apoB antibody is the fluorescence signal produced by the third antibody,
signal produced by calibrator is the fluorescence signal of the signal producing calibrator lipoprotein, and
wherein (ii) a molar mass of the Lp(a)-P present in the sample is based on the molecular weight (MW) of apo(a) present in the Lp(a) subforms determined by the following formula:
MW
of
Unknown
apo
(
a
)
=
{
(
F
*
Unk
F
*
Cal
)
(
x
mol
KIV
2
mol
apo
(
a
)
)
(
total
mol
apo
(
a
)
)
mol
apo
(
a
)
in
Unk
from
Mab
}
(
g
KIV
2
mol
KIV
2
)
+
(
g
non
-
KIV
2
mol
apo
(
a
)
)
wherein:
MW of Unknown apo(a) is molecular weight of apo(a) present in the subforms,
F*Unk is the fluorescence signal produced by the second antibody,
F*Cal is the fluorescence signal of the signal producing calibrator lipoprotein,
x
mol
KIV
2
mol
apo
(
a
)
is the number of KIV2 domain repeats per mole of apo(a) in the calibrator lipoprotein,
total mol apo(a) is the molar concentration of apo(a) in the calibrator lipoprotein,
mol apo(a) in Unk from Mab is the molar concentration of apo(a) from the Lp(a)-P subforms in the sample,
g
KIV
2
mol
KIV
2
is molecular weight of a KIV2 domain, and
g
non
-
KIV
2
mol
apo
(
a
)
is molecular weight or Lp(a) not including KIV2 domain.
2. The method of claim 1 , wherein the method further comprises washing unbound material from the solid phase support after the solid phase support has been contacted with the biological sample.
3. The method according to claim 1 , wherein the method further comprises assigning the Lp(a)-P subforms to low, mid, or high molar mass category, wherein the Lp(a)-P subforms having apo(a) with a molecular weight less than about 600 kD are assigned to the low molar mass category, the Lp(a)-P subforms having apo(a) with a molecular weight of between about 600 kD and 700 kD are assigned to the mid molar mass category, and the Lp(a)-P subforms having apo(a) with a molecular weight of greater than about 700 kD are assigned to the high molar mass category.
4. A method of assessing cardiovascular risk in a subject, comprising:
(a) performing the method of claim 1 in order to determine a particle number and molar mass of Lp(a)-P subforms in a subject's biological sample; and
(b) assessing the cardiovascular risk of the subject based on the particle number and the molar mass of the Lp(a)-P subforms, wherein assessing comprises assigning the subject to a risk category for predicting cardiovascular disease based on the Lp(a)-P particle number and molar mass.
5. The method of claim 4 , further comprising selecting a therapeutic regimen, or modifying an existing therapeutic regimen, for the subject based on the assigned risk category.
6. The method of claim 5 , wherein the therapeutic regimen comprises administering a drug and/or supplement.
7. The method of claim 6 , wherein the drug is selected from the group consisting of niacin, fenofibrate, estrogen, and raloxifene.
8. The method of claim 6 , wherein the drug is selected from the group consisting of niacin, ezetimibe, a statin, or a combination thereof.
9. The method of claim 5 , wherein the therapeutic regimen involves giving recommendations on making or maintaining lifestyle choices.
10. The method of claim 9 , wherein the lifestyle choices involve changes in diet, changes in exercise, reducing or eliminating smoking, or a combination thereof.