U.S. patent application number 09/008059 was filed with the patent office on 2002-11-28 for immunoassay for detection of very low density lipoprotein and antibodies useful therefor.
Invention is credited to KUNDU, SAMAR K..
Application Number | 20020177240 09/008059 |
Document ID | / |
Family ID | 21729614 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177240 |
Kind Code |
A1 |
KUNDU, SAMAR K. |
November 28, 2002 |
IMMUNOASSAY FOR DETECTION OF VERY LOW DENSITY LIPOPROTEIN AND
ANTIBODIES USEFUL THEREFOR
Abstract
The present invention provides a method for directly measuring
apolipoprotein B-100 (apoB) or cholesterol associated with very low
density lipoprotein (VLDL) in a fluid sample. In one embodiment the
method involves the specific capture of intact VLDL particles from
a fluid sample with a specific VLDL binding agent. The quantity of
VLDL-apoB present in the sample is then measured by detecting the
amount of VLDL-apoB bound to the binding agent-VLDL complexes
formed in the reaction. In an alternative embodiment of the method,
intact VLDL particles from a fluid sample are also captured with a
specific VLDL binding agent and thereafter the cholesterol
associated with the bound VLDL is determined. The cholesterol
contained in the binding-agent-VLDL complexes can be detected by
reacting the complexes with labeled cholesterol specific binding
agents and measuring the amount of label bound therto, or by
releasing the cholesterol in the complexes and measuring the amount
of cholesterol released. VLDL specific binding reagents are also
provided.
Inventors: |
KUNDU, SAMAR K.;
(LIBERTYVILLE, IL) |
Correspondence
Address: |
ABBOTT LABORATORIES
DEPT. 377 - AP6D-2
100 ABBOTT PARK ROAD
ABBOTT PARK
IL
60064-6050
US
|
Family ID: |
21729614 |
Appl. No.: |
09/008059 |
Filed: |
January 16, 1998 |
Current U.S.
Class: |
436/518 |
Current CPC
Class: |
Y10T 436/104165
20150115; G01N 2800/52 20130101; G01N 33/92 20130101; G01N 2800/044
20130101; C07K 16/18 20130101 |
Class at
Publication: |
436/518 |
International
Class: |
G01N 033/543 |
Claims
What is claimed is:
1. A method for determining the amount of apoB associated with VLDL
in a sample comprising: (a) mixing a sample and a VLDL-specific
binding agent for a time and under conditions to form
binding-agent-VLDL complexes; and (b) determining the amount of
apoB associated with VLDL bound to said binding-agent-VLDL
complexes.
2. The method of claim 1 wherein said VLDL-specific binding agent
is coupled to a solid support.
3. The method of claim 2 further comprising the step of separating
the solid support from the sample before determining the amount of
apoB bound to said binding-agent-VLDL complexes.
4. The method of claim 2 wherein the solid support is selected from
the group consisting of nitrocellulose, latex, nylon,
polystyrene.
5. The method of claim 2 wherein the solid support is selected from
the group consisting of beads, particles, magnetic particles, and
glass fiber.
6. The method of claim 1 further comprising the step of separating
said binding-agent-VLDL complexes prior to step (b).
7. The method of claim 6 wherein said VLDL-specific binding agent
is conjugated to a first charged substance and said separation
comprises: (a) contacting said binding-agent-VLDL complexes with an
insoluble solid phase material which is oppositely charged with
respect to said first charged substance, such that said solid phase
material attracts and attaches to said first charged substance; and
(b) separating said solid phase material and said sample.
8. The method of claim 7 wherein said charged substances are
anionic and cationic monomers or polymers.
9. The method of claim 1 wherein the VLDL-specific binding agent is
an antibody or fragment thereof that binds to substantially all
VLDL, to LDL at less than about 10% of VLDL binding, to IDL at less
than about 10% of VLDL binding, and to HDL at less than about 10%
of VLDL binding.
10. The method of claim 9 wherein said antibody is a monoclonal
antibody.
11. The method of claim 10 wherein said monoclonal antibody is
selected from the group consisting of 18-571-312, 18-140-196,
18-459-172, and 18-358-211.
12. The method of claim 10 wherein said monoclonal antibody is
18-358-211.
13. A method for determining the amount of apoB associated with
VLDL in a sample comprising the steps of: (a) contacting said
sample with an indicator reagent wherein said indicator reagent is
a monoclonal antibody or fragment thereof that specifically binds
to said apoB associated with VLDL and with a solid support coated
with VLDL for a time and under conditions to permit binding of said
indicator reagent with said VLDL in said sample and with said bound
VLDL; and (b) determining said amount of apoB associated with VLDL
in said test sample by detecting the reduction in binding of said
indicator reagent to said solid support as compared to the signal
generated from a negative sample to indicate the presence of VLDL
in said test sample.
14. The method of claim 13 wherein said indicator reagent is Mab
18-358-211.
15. A method for determining the amount of cholesterol associated
with VLDL in a sample comprising: a. mixing a sample and a
VLDL-specific binding agent for a time and under conditions to form
binding-agent-VLDL complexes; and b. determining the amount of
cholesterol bound to said binding-agent-VLDL complexes.
16. The method of claim 15 wherein said VLDL-specific binding agent
is coupled to a solid support.
17. The method of claim 15 further comprising the step of
separating said binding-agent-VLDL complexes prior to step (b).
18. The method of claim 15 wherein the VLDL-specific binding agent
is a monoclonal antibody, polyclonal antibody or fragment thereof
that binds to substantially all VLDL, to LDL at less than about 10%
of VLDL binding, to IDL at less than about 10% of VLDL binding, and
to HDL at less than about 10% of VLDL binding.
19. The method of claim 18 wherein said antibody is a monoclonal
antibody.
20. The method of claim 19 wherein said monoclonal antibody is
selected from the group consisting of 18-571-312, 18-140-196,
18-459-172, and 18-358-211.
21. The method of claim 16 further comprising the step of
separating the solid support from the sample before determining the
amount of cholesterol bound to said binding-agent-VLDL
complexes.
22. The method of claim 16 wherein the solid support is selected
from the group consisting of nitrocellulose, latex, nylon and
polystyrene.
23. The method of claim 16 wherein the solid support is selected
from the group consisting of beads, particles, magnetic particles,
and glass fiber.
24. The method of claim 15 wherein said determination comprises
releasing said cholesterol bound to said binding agent-VLDL
complexes and measuring the amount of cholesterol released.
25. The method of claim 15 wherein said determination comprises
mixing said binding-agent-VLDL complexes with a cholesterol
specific binding agent coupled to a detectable label for a time and
under conditions suitable to form binding-agent-VLDL-cholesterol
specific binding agent complexes and determining the amount of
label bound to said binding-agent-VLDL-cholesterol specific binding
agent complex.
26. The method of claim 17 wherein said VLDL-specific binding agent
is conjugated to a first charged substance; and said separation
comprises: (a) contacting said binding-agent-VLDL complexes with an
insoluble solid phase material which is oppositely charged with
respect to said first charged substance, such that said solid phase
material attracts and attaches to said first charged substance; and
(b) separating said solid phase material and said sample.
27. The method of claim 26 wherein said charged substances are
anionic and cationic monomers or polymers.
28. The method of claim 17 wherein said determination comprises
releasing said cholesterol bound to said binding-agent-VLDL
complexes and measuring the amount of cholesterol released.
29. The method of claim 17 wherein said determination comprises
mixing said binding-agent-VLDL complexes with a cholesterol
specific binding agent coupled to a detectable label such that a
second complex is formed and determining the amount of label bound
to said second complex.
30. An antibody or fragment thereof specific for VLDL wherein said
antibody binds to substantially all VLDL, to LDL at less than about
10% of VLDL binding, to IDL at less than about 10% of VLDL binding,
and to HDL at less than about 10% of VLDL binding.
31. The antibody of claim 30 selected from the group consisting of
18-571-312, 18-140-196, 18-459-172, and 18-358-211.
32. The antibody of claim 31 which is produced by a hybridoma cell
line having ATCC Accession No. HB-12392.
33. A hybridoma cell line that produces a monoclonal antibody which
binds to substantially all VLDL, to LDL at less than about 10% of
VLDL binding, to IDL at less than about 10% of VLDL binding, and to
HDL at less than about 10% of VLDL binding.
34. The hybridoma cell line of claim 33 wherein said monoclonal
antibody is selected from the group consisting of 18-571-312,
18-140-196, 18-459-172, and 18-358-211.
35. The hybridoma cell line of claim 33 having ATCC Accession No.
HB-12392.
36. A monoclonal antibody specific for VLDL prepared by the method
comprising the steps of: (a) immunizing a mouse or a rat with Apo
CIII; (b) making a suspension of the mouse or rat spleen cells; (c)
fusing the spleen cells with mouse or rat myeloma cells in the
presence of a fusion promoter; (d) culturing the fused cells; (e)
determining the presence of anti-VLDL antibody in the culture
media; (f) cloning a hybridoma producing antibody that binds to
substantially all VLDL, to LDL at less than about 10% of VLDL
binding, to IDL at less than about 10% of VLDL binding, and to HDL
at less than about 10% of LDL binding; and (g) obtaining the
antibody from said hybridoma.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to diagnostic methods for
detection and quantification of lipoproteins and cholesterol
associated with lipoproteins. More particularly, the invention
relates to assay methods for direct measurement of apolipoprotein B
(apoB) associated very low density lipoproteins (VLDL) and
cholesterol associated with VLDL using specific monoclonal
antibodies.
BACKGROUND OF THE INVENTION
[0002] Very low density lipoprotein (VLDL) constitutes one of the
major plasma lipoproteins. VLDL particles are synthesized in the
liver and are involved in triglyceride metabolism and transport of
these lipids from the liver. The end products of VLDL catabolism
are low density lipoproteins (LDL), another major class of
lipoprotein particles in plasma.
[0003] It has been suggested that disturbances in the metabolism of
apoB containing lipoproteins such as VLDL and LDL correlate with
incidences of atherosclerosis (Hurt-Camejo et al. (1997)
Arteriosclerosis, Thrombosis, and Vascular Biology 17(6):
1011-1017). Furthermore, an increase in VLDL levels has been
associated with hypertriglycerimedia, hyperlipidemia or familial
combined hyperlipidemia (Betteridge (1989) Diabet Med 6: 195-218;
Schaefer et al. (1985) New Engl J. Med 312: 1300-1310; Shaefer et
al. (1993) Curr Opin Lipidol 4: 288-298). Hyperglyceridemia also
has been shown to correlate with an increased incidence of coronary
heart disease (Gianturco et al. (1991) Curr Opin Lipidol 2:
324-328; Manninen et al. (1992) Circulation 85: 37-45; Grundy et
al. (1992) Arch Intern Med 152: 28-34). Many patients with
hyperglycerimedia have very low levels of high density lipoprotein
(HDL), another major lipoprotein in plasma. It is recommended to
treat patients with coronary heart disease who concurrently have
hypertriglyceridemia and low levels of HDL with drugs and
pharmacologic reagents, even when these patients have acceptable
levels of total and LDL cholesterol (Larsen et al. (1993) Curr Opin
Lipidol 4: 34-40); Stein and Myers (1995) Clin Chem 41:
1421-1426).
[0004] Two methods are presently used for the quantitation of VLDL,
both of which involve the measurement of VLDL-cholesterol. The
first method uses the factor triglyceride/5 as VLDL-cholesterol
concentration (Friedewald et al. (1972) Clin Chem 18: 499-502). In
this method, it is assumed that all plasma triglycerides are
associated with VLDL and chylomicrons and that other VLDL remnants
are not present. Chylomicrons are microscopic lipid particles that
appear in the blood transiently after a fat-containing meal, are
rich in triglycerides and usually have no significant effect on the
total-cholesterol concentration. Although these assumptions are not
strictly true, the factor triglyceride/5 usually provides good
measure of VLDL-cholesterol when the subject is fasting and the
triglyceride concentrations do not exceed 400 mg/dL.
[0005] The second method for quantitating VLDL uses
ultracentrifugation. In this method, an aliquot of plasma is used
to measure the total cholesterol concentration in the sample. A
second aliquot of plasma is centrifuged (105,000.times.g) at a
plasma density concentration of 1.006 g/mL for 18 hours at
4.degree. C. After centrifugation, the upper layer containing VLDL
is quantitatively removed and the cholesterol concentration in the
isolated VLDL is measured. Alternatively, an aliquot of the
remaining bottom layer, which does not contain VLDL, is used to
measure the cholesterol concentration ([d>1.006g/mL chol]). The
cholesterol concentration of VLDL ([VLDL-chol]) is then calculated
using the following equation:
[VLDL-chol]=[Total-chol]-[d>1.006 g/mL chol]
[0006] Both methodologies suffer from a variety of problems. For
example, the use of the factor triglyceride/5 is unacceptable in
cases where a subject is not fasting, or where triglyceride
concentrations exceed 400 mg/dL. Moreover, this method should not
be used for Type III hyperlipoproteinemic patients that contain
floating beta-VLDL (Belcher et al. (1991) Methods for Clinical
Laboratory Measurement of Lipid and Lipoprotein Risk Factors, Eds.
Rifai and Wamick, MCC Press, Washington, D.C., pp. 75-86). Although
several studies have been conducted to determine better ways to
measure VLDL-cholesterol concentrations (McNamara et al. (1990)
Clin Chem 36: 36-42; Warnick et al. (1990) Clin Chem 36: 15-19;
Delong et al. (1986) JAMA 256: 2372-2377), no significant
improvement has yet been made.
[0007] The problem with the ultracentrifugation method of
VLDL-cholesterol quantitation is that it is both time consuming and
expensive to perform. Furthermore, since the method requires
specialized equipment, facilities and laboratory skills, it is not
suitable for routine analysis of patient samples. To complicate
these matters, no alternative methodologies for measuring VLDL,
such as assays which measure apoB associated with VLDL, are
currently available, either for analysis of patient samples or for
research purposes. Thus, there is a need for rapid, easily
performed, accurate and cost effective methods for quantitating
VLDL.
SUMMARY OF THE INVENTION
[0008] An objective of this invention is to generate monoclonal
antibodies that are specific for VLDL. Another object of this
invention is to develop a method of directly measuring
apolipoprotein B-100 (apoB) and cholesterol associated with VLDL
from plasma easily, cheaply, quickly and accurately without the
need of highly trained technicians or expensive equipment such as
ultracentrifuges. Yet another object of this invention is to
directly measure VLDL-cholesterol without the analytical
variability generally associated with the present method of
quantitative removal of VLDL layer in the ultracentrifugation
method even with highly trained technicians.
[0009] In one embodiment, the present invention provides a method
for determining the amount of apoB associated with VLDL in a sample
comprising the steps of: (a) mixing a sample and a VLDL-specific
binding agent for a time and under conditions to form
binding-agent-VLDL complexes; and (b) determining the amount of
apoB associated with VLDL bound to the binding-agent-VLDL
complexes. In a preferred embodiment, the VLDL-specific binding
agent is coupled to a solid support. In a more preferred
embodiment, the solid support is separated from the sample before
determining the amount of apoB bound to the binding-agent-VLDL
complexes. Preferred solid supports include nitrocellulose, latex,
nylon, polystyrene beads, particles, magnetic particles, and glass
fiber. In these embodiments, the VLDL-specific binding agent is an
antibody or fragment thereof that binds to substantially all VLDL,
to LDL at less than about 10% of VLDL binding, to IDL at less than
about 10% of VLDL binding, and to HDL at less than about 10% of
VLDL binding. Preferably, the antibody is a monoclonal antibody.
More preferably, the monoclonal antibody is selected from the group
consisting of 18-571-312, 18-140-196, 18-459-172, and 18-358-211. A
most preferred monoclonal antibody is 18-358-211.
[0010] In yet another embodiment, the method further comprises the
step of separating the binding-agent-VLDL complexes prior to
determining the amount of apoB associated with VLDL. In a preferred
method of this embodiment, the VLDL-specific binding agent is
conjugated to a first charged substance and the separation step
comprises contacting the binding-agent-VLDL complexes with an
insoluble solid phase material which is oppositely charged with
respect to the first charged substance, such that the solid phase
material attracts and attaches to the first charged substance and
separating the solid phase material and the sample. Preferably, the
charged substances are anionic and cationic monomers or
polymers.
[0011] In another embodiment, the invention provides a method for
determining the amount of apoB associated with VLDL in a sample
comprising the steps of: contacting the sample with an indicator
reagent wherein the indicator reagent is a monoclonal antibody or
fragment thereof that specifically binds to apoB associated with
VLDL and with a solid support coated with VLDL for a time and under
conditions to permit binding of the indicator reagent with the VLDL
in the sample and with the bound VLDL and determining the amount of
apoB associated with VLDL in the test sample by detecting the
reduction in binding of the indicator reagent to the solid support
as compared to the signal generated from a negative sample to
indicate the presence of VLDL in the test sample. In this
embodiment, the indicator reagent is preferably the monoclonal
antibody 18-358-211.
[0012] In another embodiment, the invention provides a method for
determining the amount of cholesterol associated with VLDL in a
sample and comprises the steps of mixing a sample and a
VLDL-specific binding agent for a time and under conditions to form
binding-agent-VLDL complexes; and determining the amount of
cholesterol bound to the binding-agent-VLDL complexes. In a
preferred embodiment, the VLDL-specific binding agent is coupled to
a solid support. In a more preferred embodiment, the solid support
is separated from the sample before determining the amount of
cholesterol bound to the binding-agent-VLDL complexes. Preferred
solid supports include nitrocellulose, latex, nylon, polystyrene,
beads, particles, magnetic particles, and glass fiber. In these
embodiments, the VLDL-specific binding agent is an antibody or
fragment thereof that binds to substantially all VLDL, to LDL at
less than about 10% of VLDL binding, to IDL at less than about 10%
of VLDL binding, and to HDL at less than about 10% of VLDL binding.
Preferably, the antibody is a monoclonal antibody. More preferably,
the monoclonal antibody is selected from the group consisting of
18-571-312, 18-140-196, 18-459-172, and 18-358-211.
[0013] In one alternative embodiment, determining the amount of
cholesterol comprises releasing the cholesterol bound to the
binding agent-VLDL complexes and measuring the amount of
cholesterol released. In a second alternative embodiment,
determining the amount of cholesterol comprises mixing the
binding-agent-VLDL complexes with a cholesterol specific binding
agent coupled to a detectable label for a time and under conditions
suitable to form binding-agent-VLDL-cholesterol specific binding
agent complexes and determining the amount of label bound to the
binding-agent-VLDL-cholesterol specific binding agent complex.
[0014] In yet another embodiment, the method further comprises the
step of separating the binding-agent-VLDL complexes prior to
determining the amount of cholesterol associated with VLDL. In a
preferred embodiment, the VLDL-specific binding agent is conjugated
to a first charged substance and the separation step comprises
contacting the binding-agent-VLDL complexes with an insoluble solid
phase material which is oppositely charged with respect to the
first charged substance, such that the solid phase material
attracts and attaches to the first charged substance and separating
the solid phase material and the sample. Preferably, the charged
substances are anionic and cationic monomers or polymers. One
alternative embodiment of this method involves releasing the
cholesterol bound to the binding-agent-VLDL complexes and measuring
the amount of cholesterol released. A second alternative embodiment
invovles mixing the binding-agent-VLDL complexes with a cholesterol
specific binding agent coupled to a detectable label such that a
second complex is formed and determining the amount of label bound
to the second complex.
[0015] The present invention also provides an antibody or fragment
thereof specific for VLDL wherein the antibody binds to
substantially all VLDL, to LDL at less than about 10% of VLDL
binding, to IDL at less than about 10% of VLDL binding, and to HDL
at less than about 10% of VLDL binding. Preferably, the antibody is
selected from the group consisting of 18-571-312, 18-140-196,
18-459-172, and 18-358-211. A most preferred antibody is produced
by a hybridoma cell line having ATCC Accession No. HB-12392.
[0016] The present invention also provides a hybridoma cell line
that produces a monoclonal antibody which binds to substantially
all VLDL, to LDL at less than about 10% of VLDL binding, to IDL at
less than about 10% of VLDL binding, and to HDL at less than about
10% of VLDL binding. Preferably, the hybridoma cell line produces a
monoclonal antibody selected from the group consisting of
18-571-312, 18-140-196, 18-459-172, and 18-358-211. A most
preferred hybridoma cell has ATCC Accession No. HB-12392.
[0017] The present invention yet further provides a monoclonal
antibody specific for VLDL prepared by the method comprising the
steps of: (a) immunizing a mouse or a rat with Apo CIII; (b) making
a suspension of the mouse or rat spleen cells; (c) fusing the
spleen cells with mouse or rat myeloma cells in the presence of a
fusion promoter; (d) culturing the fused cells; (e) determining the
presence of anti-VLDL antibody in the culture media; (f) cloning a
hybridoma producing antibody that binds to substantially all VLDL,
to LDL at less than about 10% of VLDL binding, to IDL at less than
about 10% of VLDL binding, and to HDL at less than about 10% of LDL
binding; and (g) obtaining the antibody from the hybridoma.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1: Typical antibody titer plots of the monoclonal
antibody 18-358-211 obtained by incubating microtiter plates with
LDL, VLDL, HDL, and IDL bound to the plates in separate wells and
measuring the antibody bound to the lipoproteins by an ELISA.
[0019] FIG. 2: Antibody titer plots of four monoclonal antibodies
obtained by incubating antibodies with VLDL bound to the microtiter
plates and measuring the binding of antibodies to VLDL by an
ELISA.
[0020] FIG. 3: Typical competitive binding curves of the monoclonal
antibody 18-358-211 obtained by pre-incubating the antibody with a
lipoprotein, adding the mixture to the microtiter plate with VLDL
bound to the plate reaction wells and measuring the antibody bound
to the VLDL by an ELISA.
[0021] FIG. 4: Typical titration of LDL, VLDL, HDL, and IDL with
the monoclonal antibody 18-358-211 obtained by incubating the
lipoproteins with the antibody bound to the microtiter plates and
measuring the binding of the lipoproteins to the antibody by an
ELISA.
[0022] FIG. 5: Titration of VLDL with four monoclonal antibodies
obtained by incubating VLDL with monoclonal antibodies bound to the
microtiter plates and measuring the binding of VLDL to the
antibodies by an ELISA.
[0023] FIG. 6: A typical cholesterol standard curve for a specific
VLDL-cholesterol immunocapture assay of this invention.
[0024] FIG. 7: A correlation curve for VLDL-cholesterol
measurements by the immunocapture assay using the monoclonal
antibody 18-358-211-Sepharose and the ultracentrifuge method.
[0025] FIG. 8: A is typical binding curves of the HRPO-labelled
anti-HDL polyclonal antibody with HDL. B is a typical binding curve
of the HRPO-labelled anti-apoB monoclonal antibody with LDL, VLDL,
IDL and HDL. Both are as described in Example 2.
[0026] FIG. 9: A typical calibration curve plot of VLDL-cholesterol
concentration versus absorbance using the immunoassay method of
Example 10.
[0027] FIG. 10: A correlation curve for VLDL-cholesterol
measurements by the immunoassay method using the monoclonal
antibody 18-358-211 and ultracentrifuge method.
DETAILED DESCRIPTION OF THE INVENTION
[0028] I. Definitions
[0029] Unless otherwise stated, the following terms shall have the
following meanings:
[0030] The term "fluid sample" or "test sample", as used herein,
includes biological samples which can be tested by the methods of
the present invention and include human and animal body fluids such
as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph
fluids, and various external secretions of the respiratory,
intestinal and genitorurinary tracts, tears, saliva, milk, white
blood cells, and the like, and biological fluids such as cell
culture supernatants. Any substance which can be adapted for
testing with the reagents described herein and assay formats of the
present invention are contemplated to be within the scope of the
present invention.
[0031] The term "analyte", as used herein, is the substance to be
detected which may be present in the test sample. The analyte can
be any substance for which there exists a naturally occurring
specific binding member (such as, an antibody), or for which a
specific binding member can be prepared. Thus, an analyte is a
substance that can bind to one or more specific binding members.
Analytes include but are not limited to antigenic substances,
haptens, antibodies, and combinations thereof. The term
"anti-analyte", as used herein, refers to an analyte specific
binding member.
[0032] A "specific binding member" or "specific binding agent", as
used herein, refers to one member or partner of a specific binding
pair. A "specific binding pair" refers to two different molecules
wherein one of the molecules through chemical or physical means
specifically binds to the second molecule. A typical example of
specific binding members or agents which constitute a specific
binding pair are an antigen and an antibody. Other specific binding
pairs can include biotin and avidin, carbohydrates and lectins,
cofactors and enzymes, enzyme inhibitors and enzymes, effector and
receptor molecules, and the like. Furthermore, specific binding
pairs can include members that are analogs of the original specific
binding members, for example, an analyte-analog. Immunoreactive
specific binding members include antigens, antigen fragments,
antibodies, antibody fragments, both monoclonal and polyclonal, and
complexes thereof.
[0033] The term "ancillary specific binding member", as used
herein, refers to a specific binding member which binds to an
analyte specific binding member and includes for example, an
antibody to an antibody.
[0034] The term "hapten" as used herein, refers to a partial
antigen or non-protein binding member which is capable of binding
to an antibody, but which is not capable of eliciting antibody
formation unless coupled to a carrier protein.
[0035] A "capture reagent" as used herein, refers to an unlabeled
specific binding member which is specific either for the analyte as
in a sandwich assay, for the indicator reagent or analyte as in a
competitive assay, or for an ancillary specific binding member, as
in an indirect assay. The capture reagent can be directly or
indirectly bound to a solid phase material before the performance
of the assay or during the performance of the assay, thereby
enabling the separation of immobilized complexes from the test
sample.
[0036] An "indicator reagent" as used herein comprises a specific
binding member conjugated to a label. Indicator reagents include
labeled specific binding members which directly bind to analytes of
interest and labeled ancillary specific binding members. "Solid
phases" ("solid supports") are known to those in the art and
include the walls of wells of a reaction tray, test tubes,
polystyrene beads, magnetic beads, nitrocellulose strips,
membranes, microparticles such as latex particles, sheep (or other
animal) red blood cells, and Duracytes.RTM. (red blood cells
"fixed" by pyruvic aldehyde and formaldehyde, available from Abbott
Laboratories, Abbott Park, Ill.) and others. The "solid phase" is
not critical and can be selected by one skilled in the art. Thus,
latex particles, microparticles, magnetic or non-magnetic beads,
membranes, plastic tubes, walls of microtiter wells, glass or
silicon chips, sheep (or other suitable animal's) red blood cells
and Duracytes.RTM. are all suitable examples. Suitable methods for
immobilizing peptides on solid phases include ionic, hydrophobic,
covalent interactions and the like. A "solid phase", as used
herein, refers to any material which is insoluble, or can be made
insoluble by a subsequent reaction. The solid phase can be chosen
for its intrinsic ability to attract and immobilize the capture
reagent. Alternatively, the solid phase can retain an additional
receptor which has the ability to attract and immobilize the
capture reagent. The additional receptor can include a charged
substance that is oppositely charged with respect to the capture
reagent itself or to a charged substance conjugated to the capture
reagent. As yet another alternative, the receptor molecule can be
any specific binding member which is immobilized upon (attached to)
the solid phase and which has the ability to immobilize the capture
reagent through a specific binding reaction. The receptor molecule
enables the indirect binding of the capture reagent to a solid
phase material before the performance of the assay or during the
performance of the assay. The solid phase thus can be a plastic,
derivatized plastic, magnetic or non-magnetic metal, glass or
silicon surface of a test tube, microtiter well, sheet, bead,
microparticle, chip, sheep (or other suitable animal's) red blood
cells, Duracytes.RTM. and other configurations known to those of
ordinary skill in the art.
[0037] It is contemplated and within the scope of the present
invention that the solid phase also can comprise any suitable
porous material with sufficient porosity to allow access by
detection antibodies and a suitable surface affinity to bind
antigens. Microporous structure generally are preferred, but
materials with gel structure in the hydrated state may be used as
well. Such useful solid supports include but are not limited to
nitrocellulose and nylon. It is contemplated that such porous solid
supports described herein preferably are in the form of sheets of
thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm. The
pore size may vary within wide limits, and preferably is from about
0.025 to 15 microns, especially from about 0.15 to 15 microns. The
surface of such supports may be activated by chemical processes
which cause covalent linkage of the antigen or antibody to the
support. The irreversible binding of the antigen or antibody is
obtained, however, in general, by adsorption on the porous material
by poorly understood hydrophobic forces. Other suitable solid
supports are known in the art.
[0038] The term "label", as used herein, refers to any substance
which can be attached to specific binding agents, such as
antibodies, antigens, cholesterol binding agents, Lp(a) specific
binding agents and analogs thereof, and which is capable of
producing a signal that is detectable by visual or instrumental
means. Various suitable labels for use in the present invention can
include chromogens, catalysts, fluorescent compounds,
chemiluminescent compounds, radioactive elements, colloidal
metallic (such as gold), non-metallic (such as selenium) and dye
particles (such as the particles disclosed in U.S. Pat. Nos.
4,313,734, 4,954,452, and 4,373,932), enzymes, enzyme substrates,
and organic polymer latex particles (as disclosed in co-owned U.S.
Pat. No. 5,252,459, issued Oct. 12, 1993), liposomes or other
vesicles containing such signal producing substances, and the like.
A large number of enzymes suitable for use as labels are disclosed
in U.S. Pat. No. 4,275,149. Such enzymes include phosphatases and
peroxidases, such as alkaline phosphatase and horseradish
peroxidase which are used in conjunction with enzyme substrates,
such as nitro blue tetrazolium, 3,5', 5,5'-tetranitrobenzidine,
4-methoxy-1-naphthol, 4-chloro-1-naphthol,
5-bromo-4-chloro-3-indolyl phosphate, chemiluminescent enzyme
substrates such as the dioxetanes described in U.S. Pat. Nos.
4,857,652 (issued Aug. 15, 1989), 4,931,223 (issued Jun. 5,1990),
4,931,569 (issued Jun. 5,1990), 4,962,192 (issued Oct. 9, 1990),
and 4,978,614 (issued Dec. 18, 1990), and derivatives and analogs
thereof. Fluorescent compounds such as fluorescein,
phycobiliprotein, rhodamine and the like, including their
derivatives and analogs are suitable for use as labels.
[0039] The linking of labels, i.e. labeling of peptides and
proteins is well known to those of ordinary skill in the art. For
example, monoclonal antibodies produced by a hybridoma can be
labeled by metabolic incorporation of radioisotope-containing amino
acids provided as a component in the culture medium. (See, for
example, Galfre et al., (1981) Meth. Enzymol., 73: 3-46). The
techniques of protein conjugation or coupling through activated
functional groups are particularly applicable. (See, Avrameas et
al., (1978) Scand. J. Immunol., 8(7): 7-23. Rodwell et al. (1984)
Biotech., 3: 889-894 and U.S. Pat. No. 4,493,795).
[0040] Cholesterol binding agents bind specifically to cholesterol
and include digitonin, tomatine, filipin, amphotericin B and
specific binding proteins such as polyclonal and monoclonal
antibodies and other synthetic and recombinant proteins that
specifically bind cholesterol, cholesterol esters and/or the
cholesterol associated with lipoprotein particles. A number of
cholesterol binding agents are known in the literature. These
include saponins such as digitonin (Berezin et al. (1980) Vopr Med
Khim 26: 843-846; Tsybul's kaya et al. (1986) Bioorg Khim 12:
1391-1395), tomatine (Schultz and Sanders (1957) Z Physiol Chem.
308: 122-126; Eskelson et al. (1967) Clin Chem 13: 468-474),
filipin (Boemig et al. (1974) Acta Histochem 50: 110-115; Behoke et
al. (1984) Eur J Cell Biol 35: 200-205), amphotericin B (Braitburg
et al. (1984) J Infect Dis 149: 986-997), Triterpene Glycoside
Halotoxin A1 and related compounds (Ivanov et al. (1986) Vopr Med
Khim 32: 132-134). Both monoclonal and polyclonal antibodies to
cholesterol are also known (J Immunol (1964) 92: 515; Nature (1965)
407: Proc Natl Acad Sci USA (1988) 85: 1902).
[0041] Digitonin, tomatine, amphotericin B and anti-cholesterol
antibodies can be used in the quantitation of cholesterol and its
esters in lipoprotein particles. Digitonin and tomatine can be
chemically modified and then conjugated to horseradish peroxidase
(HRPO) and alkaline phosphatase (AP). Amphotericin B and
anti-cholesterol antibodies can be coupled directly to HRPO and AP.
These four HRPO and AP conjugates bind to cholesterol and its
esters associated with VLDL. The binding affinity of the enzyme
conjugates to VLDL follows the order:
digitonin>tomatine>anti-cholesterol
antibodies>amphotericin B. Because digitonin conjugates and
tomatine conjugates bound more effectively to the cholesterol
components of VLDL, these conjugates are preferred in the present
invention.
[0042] II. The Invention
[0043] The present invention provides a method(s) for determining
the amount of apoB associated with VLDL in a fluid sample. A
VLDL-specific binding agent and a sample are mixed and incubated
for a time and under conditions suitable to form binding agent-VLDL
complexes. Thereafter, the amount of apoB associated with the VLDL
present in the sample is determined from the amount of VLDL present
in the binding-agent-VLDL complexes. The present invention also
provides methods for determining the amount of cholesterol
associated with VLDL in a fluid sample. In this case, a
VLDL-specific binding agent and a sample are mixed and incubated as
described above; thereafter the amount of cholesterol associated
with the VLDL in the complex is measured. The present invention
also provides reagents, such as, for example, VLDL-specific binding
agents, for use in the methods described herein.
[0044] a. Reagents
[0045] VLDL-specific binding agents of the present invention
include VLDL-specific binding proteins, such as monoclonal (Mab)
and polyclonal antibodies (Pab) and other VLDL specific synthetic
or recombinant proteins that specifically bind VLDL particles.
Preferably, the VLDL-specific binding agent is selective for only
VLDL, but some recognition of or binding to other lipoproteins can
be tolerated. For example, an antibody selected for its ability to
bind only to VLDL particles present in a sample can minimally
capture other lipoproteins (i.e bind other lipoproteins by up to
10% of VLDL binding and still be utilized in the invention. In
addition, preferably the VLDL-specific monoclonal antibody should
not cross-react with non-lipoprotein materials present in a sample.
In a more preferred embodiment, a VLDL-specific binding agent
selectively binds to VLDL, but not to other lipoproteins. For
example, a more preferred VLDL specific binding agent binds only to
VLDL and not to other lipoproteins, such as HDL, LDL, IDL, and
Lp(a). A most preferred VLDL specific binding agent is a monoclonal
antibody.
[0046] The term antibody is also meant to include both intact
molecules as well as fragments thereof, such as, for example, Fab
and F(ab').sup.2 which are capable of binding antigen. Fab and
F(ab').sup.2 fragments lack the Fc fragment of intact antibody and
may have less non-specific binding than an intact antibody (Wahl,
et ai., J. Nucl Med. 24: 316-325, 1983). Such fragments also may be
used for the detection and quantitation of VLDL cholesterol
particles according to the methods disclosed herein in the same
manner as intact antibodies. Such fragments are well known in the
art and are typically produced by enzymatic degradation of an
antibody, such as with pepsin, papain, or trypsin. Alternatively,
antibodies and antibody fragments can be prepared using recombinant
antibody methods such as those described in U.S. patent
applications Ser. Nos. 513,957, 693,249, 789,619, 776,391, 799,770,
799,772, and 809,083, wherein antibodies or antibody fragments are
produced from the RNA of an antibody producing B-cell from an
immunized animal, such as a rat or mouse, using known recombinant
techniques.
[0047] VLDL-specific binding agents according to the present
invention also include bacteriophage described in U.S. Pat. No.
4,797,363. Bacteriophage tail or head segments are capable of
selectively binding antigens. By mutation and selection processes,
bacteriophage having the necessary binding characteristics to
selectively bind lipoprotein cholesterol particles can be
obtained.
[0048] VLDL-specific binding agents according to the present
invention also include nucleic acid sequences, such as DNA and RNA,
which selectively bind to lipoprotein cholesterol particles. A
library of nucleic acid sequences are tested for the desired
binding characteristics and the sequences that are specific for
lipoprotein cholesterol particles are isolated and replicated.
Weintraub et al., WO 92/05285, and Gold et al., WO 91/19813 both
disclose methods for the preparation of DNA and RNA sequence which
are antigenic specific.
[0049] The VLDL-specific binding agent can be attached directly or
indirectly to a solid support, for example, by absorption,
adsorption, covalent coupling directly to the support or indirectly
through another binding agent (such as an anti-antibody), or the
like utilizing methods known in the art. The type of attachment or
binding will typically be dependent upon the material composition
of the solid support and the type of VLDL-specific binding agent
used in the assay. For example, nitrocellulose, polystyrene and
similar materials possess chemical properties that permit
absorption or adsorption of proteins to a solid phase composed of
this material Other materials, such as, latex, nylon, and the like
contain groups that permit covalent coupling of the VLDL-specific
binding agent to the solid support. Chemical groups, such as,
amines and carboxylic acids are coupled through the activation of
the carboxylic acid group with, for example, carbodiimide
compounds, to form an amide linkage. Other linking methods are
well-known in the art particularly for coupling proteins to solid
phases and one skilled-in-the-art can easily conceive of a variety
of methods for covalently coupling the specific binding agent to
the solid support. The solid support can take the form of a variety
of materials, for example, the solid support may be in the form of
a bead particle, a magnetic particle, a strip or a layered
device.
[0050] b. Methods for Determining VLDL in a Fluid Sample
[0051] The present invention utilizes a VLDL specific binding agent
to form a binding complex with VLDL particles in a sample. In one
embodiment, the method is performed by combining all components of
the test mixture simultaneously, i.e. a VLDL-specific binding
agent, a test sample, and any indicator reagent(s) for detecting
VLDL and then determining the amount of apoB or cholesterol
associated with VLDL. In a second preferred embodiment, a test
sample is combined with a VLDL-specific binding agent and then
separated from the binding agent-VLDL complexes formed before
measuring the amount of apoB or cholesterol associated with VLDL in
the complexes. More preferably, the VLDL particles are captured by
a VLDL-specific binding agent directly or indirectly bound to a
solid support. This methodology simplifies the separation of the
resulting binding agent-VLDL complexes from the sample. Thus, in a
preferred embodiment, the specific-VLDL particles of interest are
separated from other lipoprotein particles (i.e. HDL, Lp(a), IDL
and LDL cholesterol particles) in the sample before the
determination of apoB or cholesterol associated with VLDL is
made.
[0052] Separation of the binding-agent-VLDL complexes from the
sample or more specifically from the other lipoprotein particles in
the sample can be accomplished in a variety of ways. When the
binding agent is coupled to a solid support, the solid support can
be removed from the sample or the sample can be removed from the
solid support. For example, when the solid support is a microtiter
plate or another type of reaction well device, such as the devices
described in U.S. Pat. Nos. 5,075,077 and 4,883,763, and U.S.
patent application Ser. No. 523,629, the sample can be removed from
the wells and the plate washed of any residual sample. When the
solid support is a particle, such as a latex or magnetic particle,
the solid support can be separated from the sample by filtration
through a fiber matrix, such as the devices described in U.S. Pat.
Nos. 4,552,839 and 5,006,309, U.S. patent applications Ser. Nos.
554,975, 611,235 and 425,651, and Fiore et al. (1988) Clin. Chem.
34: 1726-1723 or by attraction to a magnet followed by removal of
the particles or the sample. Alternatively, the binding-agent-VLDL
complexes can be separated or removed by filtration such as by the
Ion Capture Methodology described in EP patents 0326100 and
0406473, both of which enjoy common ownership. These applications
describe the use of ion capture separation, in which specific
binding members used in an assay are chemically attached to a first
charged substance and a porous matrix having bound thereto a second
charged substance that binds to the first charged substance. A
specific binding pair is formed and separated from the reaction
mixture by an electrostatic interaction between the first and
second charged substances. The specific binding member is
preferably covalently coupled to the first charged substance.
Examples of charged substances include anionic and cationic
monomers or polymers, such as polymeric acids, e.g. polyglutamic
acid, polyaspartic acid, polyacrylic acid and polyamino acids;
proteins and derivative proteins, such as albumin; anionic
saccharides, such as heparin or alginic acid; polycations, such as
GafQuat.TM. L-200 and Celquat.TM. H-100. The art is replete with
examples of solid supports, as well as techniques in the separation
of samples from solid supports.
[0053] Alternatively, the methods of the present invention may be
performed without the need for a separation step, as described in
PCT Publication No. WO94/20636, published Sep. 15, 1994. PCT
Publication No. WO94/20636 teaches genetically engineered proteins,
such as hybrid enzymes and their preparation and use in
quantitative and qualitative assays. In the method systems
described, a hybrid enzyme is provided which comprises a starting
enzyme and a foreign amino acid moiety that either replaces or is
inserted into an amino acid sequence of the starting enzyme at a
region close to the enzyme's active site. The foreign moiety may be
either a first member of a specific binding pair or a linking
moiety to which a ligand may be coupled or conjugated. In either
case, the resulting hybrid enzyme exhibits the enzymatic activity
of the starting enzyme. Furthermore, the foreign moiety of the
hybrid enzyme can still bind to its corresponding specific binding
pair member or to an anti-ligand and as a consequence of such
binding, modulate or modify the activity of the hybrid enzyme.
Thus, in an assay system comprising a hybrid enzyme, the enzymatic
activity will change depending upon the presence or the amount of
analyte in the test sample.
[0054] The hybrid enzyme provides a basis for assays to detect, (1)
the presence or the amount of an antibody directly or (2) the
presence or the amount of an antigen indirectly by competition for
binding to a binding molecule. One assay system which utilizes a
hybrid enzyme comprises the steps of (1) contacting a test sample
containing an analyte of interest, a hybrid enzyme capable of
binding to the analyte and a binding molecule of the analyte to
form a reaction mixture; (2) contacting the reaction mixture with a
substrate for the starting enzyme; and (3) monitoring the change,
if any, in enzymatic activity of the hybrid enzyme. As an example,
in the case of a VLDL-apoB competitive assay, the monoclonal
antibodies of the present invention may be used as a binding
molecule of the analyte (VLDL). Other assay formats, such as a
direct assays are also envisioned.
[0055] The amount of apoB or cholesterol associated with VLDL in a
fluid sample can be determined by a variety of assay formats. A
preferred assay format, for example, is a sandwich assay. This
method comprises contacting a test sample with a solid phase
(hereinafter represented by the symbol ".vertline.-") to which at
least one capture reagent (i.e. anti-analyte) is bound, to form a
mixture. The mixture of test sample and capture reagent bound to a
solid phase is incubated for a time and under conditions sufficient
to allow .vertline.-capture reagent/analyte complexes to form.
These complexes then are contacted with an indicator reagent
comprising a second anti-analyte previously conjugated to a label.
This second mixture is incubated for a time and under conditions
sufficient for .vertline.-capture reagent/analyte/indicator reagent
complexes to form. The presence of the .vertline.-capture
reagent/analyte/indicator reagent complexes is determined by
detecting the measurable signal generated. In such an assay, the
capture reagent bound to the solid support may be, for example, a
first antibody which binds to an antigen in the test sample, and
the indicator reagent may be a second antibody which also binds to
the antigen but at a site different from the first antibody. It is
also within the scope of the present invention to use one antibody
as a capture agent and a fragment of an antibody as an indicator
reagent. In addition, sandwich-type assays may be configured in a
reverse orientation to that described above, i.e. with an antigen
serving as the capture reagent to test for the presence of antibody
in a test sample. In this case, the indicator reagent is a second
labeled antibody or fragment thereof which also binds to the
complex of antigen/antibody bound to a solid support.
[0056] Detection of complexes formed in sandwich and other assays
may be performed indirectly. In an indirect sandwich assay format,
complexes of .vertline.-capture reagent/analyte/second capture
reagent are formed, none of which are labeled. Instead, an
ancillary specific binding member which binds to the second capture
reagent acts as the indicator reagent. For example, when the second
capture reagent is a mouse antibody to the analyte of interest, the
complex of capture reagent/analyte/mouse antibody may be detected
using an ancillary antibody which is labeled, such as labeled goat
anti-mouse antibody. Furthermore, the use of biotin and antibiotin,
biotin and avidin, biotin and streptavidin, and the like, may be
used to enhance the generated signal in the assay systems described
herein.
[0057] For purposes of illustration, the following sandwich formats
may be utilized: in a first format, VLDL particles present in a
plasma sample are specifically captured by a VLDL specific
monoclonal antibody immobilized on a solid support. After removing
the other lipoprotein particles, the apoB associated with the VLDL
bound to the solid support is quantitated using an apoB specific
monoclonal antibody or polyclonal antibody (which are labeled) as
an indicator reagent. Since apoB is common to LDL, VLDL, IDL and
Lp(a), any monoclonal or polyclonal antibody that cross reacts to
the apoB of one or more of these lipoproteins is suitable as an
indicator reagent (provided such antibody is not specific to the
apoB of the specific lipoprotein, with the exception, of course, of
VLDL). Such antibodies are well known to those of ordinary skill in
the art (see, for example, WO 93/18067, published Sep. 16, 1993).
Preferably, in these formats, the indicator reagent is labeled with
an enzyme.
[0058] In addition to the foregoing sandwich assay formats,
competitive assays are also contemplated by the invention. In one
format, labeled VLDL may compete with the VLDL to be determined in
a fluid sample for binding to a VLDL specific monoclonal antibody
which has been immobilized on a solid support. In a second format,
VLDL in the sample competes with VLDL attached to the solid support
for binding by a labeled VLDL specific antibody. It is fully
expected that other known assay formats may be advantageously
adopted by the skilled artisan and these are within the scope of
the invention, to be utilized with the unique antibodies herein set
forth and described.
[0059] Another alternative is based on an immunochromatographic
assay format (such as described in U.S. Pat. No. 4,954,452 and U.S.
Pat. No. 5,229,073, for example) in which the lipoprotein particles
in the test sample bind to a labeled VLDL binding agent. The
resulting complexes then travel along a test strip by capillary
action. The labeled Lp(a) complexes are then captured by a high
affinity VLDL specific antibody immobilized on the test strip,
followed by detection and measurement of the captured labeled VLDL
complexes. Typically, the test strip is comprised of a porous or
bibulous membrane and the result is determined by a visual readout
of a detectable signal. Other test strip assay formats are also
within the scope of the invention.
[0060] The use of scanning probe microscopy (SPM) for immunoassays
also is a technology to which the monoclonal antibodies of the
present invention are easily adaptable. In scanning probe
microscopy, in particular in atomic force microscopy, in the
capture phase, for example, at least one of the monoclonal
antibodies of the invention is adhered to a solid phase and a
scanning probe microscope is utilized to detect antigen/antibody
complexes which may be present on the surface of the solid phase.
The use of scanning tunneling microscopy eliminates the need for
labels which normally must be utilized in many immunoassay systems
to detect antigen/antibody complexes. Such a system is described in
Publication No. WO 92/15709, published Sep. 17, 1992.
[0061] c. Methods for Determining VLDL-cholesterol
[0062] A VLDL specific binding agent can be used in an immunoassay
method for the quantitation of VLDL-cholesterol in a fluid sample.
This involves the specific capture of the VLDL particles in the
sample by the VLDL-specific antibody immobilized on the solid
support followed by quantitation of cholesterol in the captured
VLDL particles by a cholesterol-binding agent which is coupled
directly or indirectly to a label. The VLDL-cholesterol bound
cholesterol binding agent is then quantitated by detection and
measurement of the label. A variety of methods for quantitating
cholesterol are available and are well known to those of ordinary
skill in the art (see for example, WO 93/18067, published Sep. 16,
1993).
[0063] One embodiment of the method is illustrated by the following
sandwich assay example. The method involves incubating the sample
with a solid phase having an VLDL-specific binding agent, such as
the monoclonal antibody 18-358-211 immobilized on a solid phase and
preferably, blocking the remaining non-specific binding sites of
the solid phase such as with bovine serum albumin or alkali-treated
casein. VLDL particles are captured by the antibody on the solid
phase. Digitonin or tomatine enzyme conjugates are then incubated
with the solid phase. The conjugate binds to the cholesterol
associated with the VLDL particles on the solid phase. The quantity
or presence of enzyme bound to the solid phase or the quantity of
unbound conjugate remaining after incubation with the solid phase
is determined by incubation of enzyme substrate with the solid
phase or the solution containing unbound conjugate. The presence of
cholesterol associated with the captured VLDL particles is then
determined from the presence of enzyme associated with the solid
phase or a reduction of enzyme activity in the solution containing
unbound conjugate as compared with the original conjugate solution
added to the solid phase. The quantity of cholesterol associated
with the captured VLDL particles is proportional to the quantity of
enzyme associated with the solid phase or inversely proportional to
the quantity of unbound conjugate.
[0064] In an alternative embodiment, after capture of a VLDL
particle by a VLDL specific binding agent, the amount of
cholesterol in or on a VLDL particle can be determined directly by
a variety of methods. Such methods may be: chemical by using the
Liebermann-Burchard method or modifications of their method;
enzymatic by using a cholesterol-specific enzyme such as
cholesterol oxidase; through the formation of a
cholesterol-specific binding complex; or through the release of the
cholesterol from VLDL followed by detecting the amount of
cholesterol released using any of the above methods. One
skilled-in-the-art may conceive of yet other methods of detection
applicable to this method.
[0065] For purposes of illustration, a VLDL-cholesterol measurement
can be made as follows: VLDL particles present in a plasma sample
are specifically captured by an VLDL-specific monoclonal antibody
immobilized on a solid support. After separating the solid support
from the other unbound plasma lipoproteins, the cholesterol content
of the bound VLDL particles is estimated by releasing the
cholesterol and its esters with a detergent solution. A standard
cholesterol assay reagent comprising cholesterol ester hydrolase,
cholesterol oxidase and horseradish peroxidase is added. The
liberated hydrogen peroxide is then quantitated using a Tinder dye
reagent comprising of 4-aminoantipyrine and
3,5-dichloro-hydroxybenzenesulfonic acid similar to that described
in the art (see Sidel et al. (1983) Clin Chem 29: 1075-1079). The
cholesterol concentration in a given sample is quantitated on the
basis of the color generation.
[0066] The measurement of VLDL-cholesterol can also be accomplished
indirectly by removing all the other lipoproteins from the sample.
The selective binding agents of a group of selected lipoproteins
can be used to remove these lipoproteins from a sample, leaving
behind substantially only one lipoprotein in the sample.
Measurement of the cholesterol in the sample after this group of
lipoproteins have been removed gives an indication of the amount of
cholesterol present in the remaining lipoprotein. For example,
selectively removing HDL, LDL, IDL and Lp(a) will essentially leave
behind VLDL in the sample. Measurement of the cholesterol in the
remainder of the sample will give an estimation of the
VLDL-cholesterol present in the sample. The cholesterol levels
associated with the other lipoproteins could be measured by simply
changing the group of selected lipoproteins removed from the
sample. Moreover, lipoprotein specific binding agents, such as
antibodies, that are not selective for only one lipoprotein, such
as an antibody that binds to both VLDL and LDL, but not Lp(a), can
be used to remove the antibody cross-reacting lipoproteins (VLDL
and LDL) in the measurement of cholesterol associated with a
non-cross reacting lipoprotein (Lp(a)) using this indirect
method.
[0067] This indirect approach can also improve the efficacy of
lipoprotein specific binding agents, used in the direct measurement
of a specific lipoprotein, that are not sufficiently selective for
the lipoprotein of interest. By removing an antibody cross-reacting
specific lipoprotein from the sample prior to specifically
measuring the lipoprotein cholesterol of interest, the effect of
such cross-reactivity is eliminated.
[0068] Moreover, the sequential removal and measurement of specific
lipoprotein cholesterol levels from the same aliquot of sample
permits the use of less selective lipoprotein binding agents in the
measurement of lipoprotein cholesterol levels later in the
sequence. For example, an antibody that binds to both VLDL and LDL
could be used to selectively capture VLDL if the LDL present in the
sample had previously been removed.
[0069] The following examples are illustrative of the invention and
are in no way to be interpreted as limiting the scope of the
invention as defined in the claims. It will be appreciated that one
skilled-in-the-art can conceive of many other devices and methods
for use of which the present inventive concepts can be applied.
Throughout the entire specification, it is intended that citations
to the literature, including patents and patent applications, are
expressly incorporated by reference.
GENERAL METHODOLOGIES
[0070] 1. Development of Monoclonal Antibodies against VLDL:
[0071] Apolipoprotein CIII (Apo CIII) was used as immunogen to
develop VLDL specific monoclonal antibodies. Apo CIII is primarily
associated with VLDL as a major lipoprotein. It is also present in
the HDL fraction as a minor component. Apo CIII contains 79 amino
acid residues with a molecular weight of 9,000 (Brewer et al.
(1974) J. Biol Chem 249: 4975). The objective of using Apo CIII as
a target immunogen was to develop monoclonal antibodies that would
interact with a preferred conformation dictated by VLDL and not by
HDL.
[0072] a. Immunization:
[0073] Six female 4-6 week old RBF/dn mice (Charles River,
Wilmington, Mass.) were immunized with Apo CIII (Calbiochem, La
Jolla, Calif.). The dose level was 25 .mu.g in 0.1 mL of a 1:1
ratio of the Apo CIII solution in Freund's Complete Adjuvant (Difco
Laboratories, Detroit, Mich.). The adjuvant emulsion route of
injection was equally distributed interperitoneally and
subcutaneous. The mice were given subsequent immunizations of Apo
CIII on weeks three and five, 25 .mu.g in 0.1 mL of a 1:1 ratio of
Freund's incomplete Adjuvant. The adjuvant emulsion route of
injection was equally distributed interperitoneally and
subcutaneous. Three days prior to the fusion, mice were given an
immunization of 25-50 .mu.g Apo CIII in 0.9% saline by IV tail vein
injection.
[0074] b. Sera Evaluation:
[0075] Ten days following the third immunization, sera samples were
taken by orbital vein puncture and analyzed for APO CIII specific
antibody titer by enzyme immunoassay (EIA). Microtiter wells were
coated with 100 microliters (.mu.L) of 1 .mu.g/mL Apo CIII in
phosphate buffered saline (PBS) or 100 .mu.l PBS and incubated at
room temperature overnight. The following day the plates were
blocked for 30 minutes with 200 .mu.L per well of 3% bovine serum
albumin (BSA) in PBS. After washing the plate, 50 .mu.L of sera
sample was added per well, at log 2 serial dilutions starting at a
1:100 dilution, and incubated 1 hour. The plates were washed and 50
.mu.L of diluted goat anti-mouse IgG+M-HRPO (Kirkegaard and Perry
Laboratories, Gaithersburg, Md.), was added to the plate for a 30
minute incubation period. The plate was washed a final time and the
color developed using o-phenyienediame.2HCl (OPD) (Abbott
Laboratories, Abbott Park, Ill.). The relative intensity of optical
density readings identified mouse numbers 1 and 5 to have the
highest Apo CIII titer with minimal BSA background. Thus, these
mice were selected for fusion ten weeks following the first
immunization.
[0076] c. Fusion:
[0077] On the day of fusion, the two mice were euthanized by
cervical dislocation and a splenectomy was performed. Splenocytes
were flushed out and washed in Iscoves's Modified Dulbecco's Medium
(IMDM) (GIBCO, Grand Island, N.Y.) and centrifuged at 1000 rpm for
5 minutes. The splenocytes were combined with SP2/0 myeloma cells
at a 1:1 ratio, washed in IMDM, and centrifuged. The supernatant
was removed and 1 mL of 50% polyethylene glycol (PEG) (American
Type Culture Collection, Rockville, Md.) was added to the pellet
for one minute as the pellet was gently dispersed by tapping and
swirling. Thirty milliliters of IMDM were added to the mixture and
centrifuged as previously described. The supernatant was decanted
and the pellet resuspended in IMDM with Hypoxanthine Aminopterin
Thymidine (HAT) (GIBCO, Gaithersburg, Md.), 15% Fetal Bovine Serum
(FBS) (Hyclone Laboratories, Logan, Utah), Origen Hybridoma Cloning
Factor (Igen, Rockville, Md.), and Salmonella typhimurium mitogen
(STM) (1% v/v) (RIBI Immunochem Research, Inc., Hamilton, Mont.).
The fusion cells were plated into 96-well tissue culture plates at
3.times.10.sup.5 cells per well. The fusion cells were given media
changes by aseptically aspirating half the tissue culture
supernatant and feeding with IMDM with 1% v/v HT (hypoxanthine and
thymidine) Supplement (GIBCO, Gaithersburg, Md.), and 10% v/v FBS
at days five and seven. The fusion protocol was referenced from
Galfre, G. and Milstein, C. (1981), Preparation of Monoclonal
Antibodies: Strategies and Procedures, Meth Enzymol 73: 1-46.
[0078] d. Fusion Screening:
[0079] The primary screening of the fusion occurred on day ten with
confluent cultures. An EIA was performed in a similar manner to the
assay used for testing sera samples. Microtiter wells were coated
with 100 .mu.L of 1 .mu.g/mL Apo CIII in PBS and incubated at room
temperature overnight After washing and blocking as previously
described, 50 .mu.L of culture supernatant was added and incubated
1 hour. The plates were washed and goat anti-mouse HRPO conjugate
was added to each well. The addition of conjugate was followed by
washing and color development with OPD. Since the relative
intensity of optical density readings identified hybrids 18-358,
18-571, 18-459 and 18-140 as having about three times that of
negative control, normal mouse serum (NMS) (Organon Teknika-Cappel,
Malvern, Pa.), these hybrids were expanded. Thereafter, they were
selected for cloning because the optical density readings indicated
specific binding to Apo CIII with minimal non-specific binding to
BSA.
[0080] e. Hybrid Cloning:
[0081] All four hybrids described above were cloned by limiting
dilutions starting at 1-100, 10-fold to 10.sup.6. The cloning media
used was IMDM with 10% v/v FBS and 1% v/v HT Supplement. A 200
.mu.gL cell suspension was added to each of the 96 wells in the
tissue culture plate.
[0082] f. Clone Selection:
[0083] The clone screening occurred on day ten with confluent
cultures. Clones were selected based on EIA reactivity specific to
Apo CIII with minimal nonspecific binding to a BSA background. The
EIA screening protocol used was as described above.
[0084] g. Isotypes:
[0085] The isotypes of the monoclonal antibodies secreted from the
cell lines identified as 18-358-211,18-571-312, 18-459-172 and
18-140-196 were determined on an Isotype AL-STAT kit (Sargstat
Medical Corp., Menlo Park, Calif.). Assays of each were performed
according to the vendor recommendations and indicated that
18-150-196 was IgG2b and the other three monoclonal antibodies were
IgG1.
[0086] A murine hybridoma which produces Mab 18-358-211 has been
deposited under the terms of the Budapest Treaty in the American
Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.
20852, USA, and has been accorded accession number ATCC HB-12392.
The deposit is provided for convenience only, and is not required
to practice the present invention in view of the teachings provided
herein.
[0087] h. Antibody Production:
[0088] Cell lines 18-358-211, 18-571-312, 18-459-172 and 18-140-196
were expanded in volume with IMDM with 5% v/v Fetal Clone Serum
(Hyclone Laboratories, Logan, Utah) in tissue culture flasks at a
cell density between 1.times.10.sup.4 cells/mL and 5.times.10.sup.5
cells/mL until they could be expanded into roller bottles. The
roller bottle cells were allowed to grow for maximum antibody
production, i.e. normally until viability fell below 5%.
[0089] i. Antibody Purification:
[0090] Cultures were removed from roller bottle incubators and
cells were allowed to settle for three days at 4-8.degree. C. Cell
supernatants were filtered through 0.45 .mu.m filters and
concentrated approximately 20.times. on an Amicon Concentrator
(Amicon Corp., Beverly, Mass.). Concentrated supernatants were
filtered through an additional 0.45 .mu.m filter. These materials
were then purified by Protein A-Sepharose column chromatography as
described by Ey et al., (1978), Immunochem 15: 429-436. The
purified and dialyzed antibody was tested for Apo CIII reactivity
by EIA as previously described.
[0091] 2. Evaluation of Monoclonal Antibodies: The following
methods were used to evaluate the antibodies.
[0092] a. Specificity of the Mabs in a Direct Binding ELISA using
Lipoprotein Coated Microtiter Plates:
[0093] Lipoprotein fractions (LDL, HDL, VLDL, and IDL) purified by
ultracentrifugation (see Example 2, infra) were coated on separate
wells of a Maxisorb Nunc Immuno Plate as follows: one hundred
microliters (100 .mu.L) of each lipoprotein fraction at a
lipoprotein-cholesterol concentration of about 1 .mu.g/mL in 20 mM
phosphate buffered saline at pH 7.0 (PBS) was dispensed into
separated wells of the microtiter plate. The plate was incubated at
37.degree. C. for one hour and then washed five times with PBS
containing 0.05% (v/v) Tween 20 (PBS-Tween 20). Non-specific
binding sites were blocked with 200 .mu.L of 10% (v/v) fetal bovine
serum (FBS) in PBS at 37.degree. C. for one hour and the plate then
washed five times with PBS-Tween 20. Each monoclonal antibody was
diluted in 3% (v/v) FBS in PBS to a final antibody concentration of
about 2 .mu.g/mL and the diluted monoclonal antibody solutions were
serially diluted on the plate. After incubation at 37.degree. C.
for one-half hour, the plate was washed five times with PBS-Tween
20. Thereafter one hundred microliters (100 .mu.l ) of horseradish
peroxidase (HRPO) labeled goat anti-mouse IgG (obtained from
Kirkegeard and Perry Laboratories, MD), diluted in 3% FBS in PBS to
a final concentration of about 1.25 .mu.g/mL, were added to each
reaction well and the plate was incubated at 37.degree. C. for one
half hour. The plate was then washed eight times with PBS-Tween 20.
One hundred microliters (100 .mu.l) of freshly prepared HRPO
substrate solution, containing one o-phenylenedianine (OPD) tablet
per five milliliters (5 mL) of citrate buffer at pH 6 (both
available from Abbott Laboratories, Abbott Park, Ill.) were added
to each well. The color reaction was stopped after five minutes by
adding 100 .mu.l of 1 N H.sub.2SO.sub.4 to the reaction wells. An
absorbance reading of each reaction well was then obtained with a
Bio-Tek microplate reader at 490 nm. Typical binding curves for
each lipoprotein tested with monoclonal antibody 18-358-211 are
shown in FIG. 1. A summary of test results are presented in Table 1
which shows the binding effeciencies of the lipoproteins relative
to LDL at an antibody concentration of about 0.1 .mu.g/mL. As Table
1 shows, none of the four monoclonal antibodies bound to LDL and
IDL. Furthermore, antibody 18-358-211 showed no cross-reactivity
with HDL. The other three monoclonal antibodies showed very weak
cross reactivity with HDL (3-8%).
1TABLE 1 Binding of Monoclonal Antibodies to Lipoproteins on a
Solid Phase % Cross-reactivity Mab* VLDL LDL IDL HDL 18-571-312 100
0 0 5 18-140-196 100 0 0 8 18-459-172 100 0 0 3 18-358-211 100 0 0
0 *At 0.1 .mu.g/mL antibody concentration
[0094] b. Specificity of the Mabs to VLDL in Competitive Assays
using Lipoprotein Coated Microtiter Plates:
[0095] The specificities of the monoclonal antibodies were
determined by competitive binding of the monoclonal antibodies to
the other lipoproteins in microplate wells coated with VLDL. The
VLDL-coated plates were prepared as described previously (see
section 2a above). Each monoclonal antibody was diluted with 3%
(v/v) FBS in PBS to a concentration that was two times the
monoclonal antibody concentration at 50% VLDL-binding as determined
from the binding curves generated in Section 2a above. The binding
curves of the four monoclonal antibodies curves are shown in FIG.
2. Purified lipoprotein samples were diluted in PBS starting at 50
.mu.g/mL cholesterol concentration. Fifty microliters (50 .mu.L) of
each lipoprotein solution were then serially diluted with PBS in
reaction wells blocked by 10% (v/v) FBS in PBS. To each of these
wells were added 50 .mu.L of the diluted monoclonal antibody
solutions. The monoclonal antibody-lipoprotein mixtures were
incubated at room temperature for one-half hour on a rotator at 100
rpm. The contents from each well were then transferred to
VLDL-coated reaction wells and the plates were incubated at
37.degree. C. for one-half hour. The amount of monoclonal antibody
bound to the VLDL-coated reaction wells was measured according to
the method described in Section 2a above. Typical competitive
binding curves are shown in FIG. 3. A summary of the test results
are presented in Table 2. The cross-reactivities were determine at
66.67% inhibition of binding by a competing lipoprotein using the
following equation: 1 Cross - reactivity ( % ) = Amount needed by
VLDL Amount needed by competitor .times. 100
[0096] The results seen in Table 2 indicate extensive binding of
monoclonal antibody 18-571-312 to HDL in contrast to the binding
curves obtained in Section 2a above. In Section 2a, this monoclonal
antibody shows no binding to HDL, whereas in the inhibition assay,
both VLDL and HDL show equal affinity for this monoclonal antibody.
This result indicates that the affinities of some monoclonal
antibodies toward lipoproteins differ depending on whether the
reaction is performed with a solid phase (FIG. 2) or in a fluid
phase (FIG. 3).
2TABLE 2 Competitive Binding of Monoclonal Antibodies by ELISA %
Cross-reactivity Mab* VLDL LDL IDL HDL 18-571-312 100 0 0 100
18-140-196 100 0 0 0 18-459-172 100 0 8 4 18-358-211 100 0 0 0 *At
66.67% inhibition of binding
[0097] c. Specificity of the Mabs in a Direct Binding ELISA using
Antibody Coated Microtiter Plates:
[0098] Monoclonal antibodies were coated on wells of microtiter
plates as follows: Antibodies were diluted in PBS to a
concentration of 10 .mu.g/mL. One hundred microliters (100 .mu.l)
of each antibody solution were dispensed into separate reaction
wells and incubated at room temperature on a rotator at 100 rpm for
two hours. The plates were then washed five times with PBS-Tween 20
and blocked with 200 .mu.L of 10% FBS in PBS by incubation at
37.degree. C. for one hour. The plates were then washed five times
with PBS-Tween 20.
[0099] Each monoclonal plate was then serially diluted with VLDL,
LDL, IDL, and HDL in PBS, starting with a lipoprotein-cholesterol
concentration of 5 mg/dL cholesterol, so that each well contained a
total of 100 .mu.l of solution. After incubation at 37.degree. C.
for one-half hour, the plates were washed five times with PBS-Tween
20. One hundred microliters (100 .mu.L) of 0.5 .mu.g/mL
anti-ApoB-HRPO or 1 .mu.g/mL anti-HDL-HRPO conjugate (prepared
according to Example 3) in 3% FBS in PBS were added to the
respective wells. LDL-, VLDL- and IDL- containing wells received
anti-ApoB conjugate and HDL-containing wells received anti-HDL
conjugate. After incubation at 37.degree. C. for one-half hour,
HRPO substrate was added and the absorbance measured as described
in Section 2a above. FIG. 4 shows typical binding curves of
lipoproteins for the monoclonal antibody 8-358-211 (coated on a
solid phase) and FIG. 5 shows a comparison of the binding of VLDL
to the four monoclonal antibodies. A summary of test results are
presented in Table 3 which shows the binding efficiencies of the
lipoproteins relative to VLDL at an antibody concentration of 3.13
.mu.g/mL.
3TABLE 3 Binding of Lipoproteins to Mabs on a Solid Phase %
Cross-reactivity Mab* VLDL LDL IDL HDL 18-571-312 100 8 5 5
18-140-196 100 35 0 7 18-459-172 0 100 0 3 18-358-211 100 5 7 5
*3.13 .mu.g/mL lipoprotein-cholesterol concentration
[0100] As seen in Table 3, monoclonal antibodies 18-358-211 and
18-571-312 showed minimum cross-reactivity (less than 10%) with
LDL, IDL and HDL. In contrast, Mab 18-459-172 bound to LDL only and
Mab 18-140-196 showed high reactivity (35%) with LDL.
[0101] The selection of a preferred monoclonal antibody that could
be used in the VLDL capture assay of the present invention was
based on the consistency of the specificity results in the direct
titration assay (as exemplified in Section 2a and Table 1),
inhibition assay (as exemplified in Section 2b and Table 2) and
also by the direct binding of lipoproteins to monoclonal antibodies
immobilized on a solid phase (as exemplified in Section 2c and
Table 3). Monoclonal antibody 18-358-211 was the only Mab which
performed consistently in all three experiments described above and
therefore was selected in developing the VLDL-cholesterol
assays.
[0102] 3. Immobilization of Monoclonal Antibody to Solid Phase:
[0103] An assay system was designed to selectively capture VLDL
particles on an antibody coated solid phase and assay for
cholesterol in the bound VLDL. Since cholesterol or other lipids
associated with lipoproteins are very hydrophobic, it is desirable
to use solid phases which are hydrophillic in a solid phase assay
for cholesterol. Moreover, the solid phase should have a high
binding capacity and be non-porous to avoid preferential inclusion
of lipoproteins in a porous solid phase. Also, in the case where
antibody is to be immobilized on a solid phase, the activity and
the orientation of the immobilized antibody must be substantially
preserved. CNBr-activated Sepharose 4B (Pharmacia LKB) was used to
demonstrate the feasibility of an VLDL-specific cholesterol assay.
However, other hydrophilic solid phases, such as carbolink
hydrazide agarose beads (Pierce Chemicals, Rockford, Ill.),
Sulfolink coupling agarose beads (Pierce Chemicals), Trisacryl
(IBF), HEMA-epoxy Bio Gel, HEMA vinylsulfone Bio Gel (Altech
Associates, Deerfield, Ill.), glycosylated silica gel or control
porous glass, hydrophilic latex beads, and other like cellulosic
materials can also be used.
[0104] 4. Evaluation of Antibody Immobilized Solid Phases:
[0105] The antibody immobilized solid phases were evaluated in
terms of their binding efficiencies by incubating the solid phases
with two plasma samples and then determining the amount of VLDL
bound by measuring the amount of cholesterol in the bound solid
phases.
[0106] The protocol for a preferred lipoprotein capture assay is
described in Example 8. The assay was performed using reagents
prepared as described in Example 4. A typical cholesterol standard
curve is shown in FIG. 6.
[0107] The efficiency of VLDL capture on a Sepharose 4B matrix
having monoclonal antibody 18-358-211 bound thereto is shown in
Table 4.
4TABLE 4 Efficiency of VLDL Capture on Mab 18-358-211-Sepharose
Matrix Amount Mab VLDL-Chol Plasma Amount of % VLDL Matrix* (.mu.L)
(mg/dL) Sample No. Plasma (.mu.L) Captured 25 60 9 10 98 25 60 9 20
96 50 60 9 10 100 50 60 9 20 98 25 50 11 10 100 25 50 11 20 100 50
50 11 10 100 50 50 11 20 98 *Antibody-Matrix Concentration = 4.28
.mu.g/mL
[0108] As Table 4 shows, under all conditions tested, at least 96%
of VLDL particles were captured from plasma sample nos. 9 and 11,
even using a 25 .mu.L matrix volume. In developing the
VLDL-cholesterol immunocapture assay, a 50 .mu.L matrix volume and
10 .mu.L plasma volume were used. As also seen in Table 4, the
binding capacity of a 50 .mu.L matrix volume is at least two-fold
of 60 mg/dL VLDL-cholesterol concentration if a 10 .mu.L plasma
volume is used, i.e. using this methodology, a VLDL-cholesterol
concentration up to 120 mg/dL can be completely captured.
[0109] 5. VLDL-Immunocapture Assay:
[0110] The protocol for a VLDL-immunocapture assay is described in
Example 9. The VLDL-cholesterol concentrations were correlated with
a reference ultracentrifugation method described in Example 7. One
hundred subjects whose lipid profiles are shown in Table 5 were
used in this study. Eighty-seven normal subjects, nine patients
with coronary heart disease who were under lipid lowering drug
treatment (indicated by asterisk *) and four diabetic patients
(indicated by double asterisks **) participated in this study.
[0111] The VLDL-cholesterol concentrations determined by the
ultracentrifuge method and the immunocapture assay using monoclonal
antibody 18-358-211-Sepharose are presented in Table 6. The
correlation between the ultracentrifuge method and the
immunocapture assay is shown in FIG. 7. The correlation coefficient
(r) was 0.95 with an intercept of -0.964 and a slope of 1.02. The
results demonstrated that monoclonal antibody 18-358-211 is capable
of capturing all VLDL particles of heterogeneous sizes (Musliner et
al. (1986) Arteriosclerosis 6: 79-87).
[0112] 6. Cholesterol Binding Agents
[0113] a. VLDL-Cholesterol Standards:
[0114] A plasma sample with a known VLDL-cholesterol concentration
as determined by the ultracentrifuge method was used to generate a
VLDL-cholesterol standard curve. Standards for VLDL-cholesterol
concentrations within the range of about 20 mg/dL and 30 mg/dL were
prepared by diluting the plasma sample with 1% alkali-treated
casein in 20 mM phosphate buffered saline (PBS) at pH 7.4.
[0115] b. Preparation of Digitonin-Peroxidase Conjugates:
[0116] Digitonin (2.5 mg/mL in water) (water soluble containing 50%
digitonin and sodium deoxycholate commercially available from SIGMA
Chemical Company, St. Louis, Mo.) was oxidized with sodium
meta-periodate by adding a solution of periodate (1.68% w/v in
water) to the digitonin solution to a final concentration of 0.02 M
periodate (Tschesche and Wulff (1963) Tetrahedron 19: 621-634). The
mixture was stirred at 4.degree. C. for one hour and then dialyzed
against 20 mM phosphate buffered saline (PBS), pH 8.0, at 4.degree.
C. overnight. A solution of 0.25 M ethyienediamine in 20 mM PBS, pH
8.0, was added to the oxidized digitonin solution to a final
concentration of 0.05 M ethylenediamine and incubated at 4.degree.
C. The mixture was then reduced by two additions of 100 .mu.L of a
sodium borohydride solution per 30 mg of digitonin, after 30
minutes and after 60 minutes. After incubating at 4.degree. C. for
two hours, the mixture was dialyzed against 0.01 M carbonate
buffer, pH 9.5, at 4.degree. C. overnight.
[0117] Five milligrams (5 mg) of horseradish peroxidase (HRPO, 155
Ku/mg, commercially available from Amano International Enzyme Co.,
Troy, Va.) was dissolved in water to a final concentration of 4
mg/mL HRPO. The HRPO was oxidized by adding a freshly prepared
solution of 0.2 M sodium meta-periodate (50 .mu.L/milligram of
HRPO) and incubating the mixture in the dark at room temperature
for 20 minutes. The mixture was then dialyzed against 2 liters of 1
mM acetate buffer, pH 4.5, at 4.degree. C. for 4 hours.
[0118] The ethylenediamine derivatized digitonin solution and the
oxidized HRPO solution were mixed in digitonin:HRPO weight ratios
of 1:5. To the reaction was added 0.2 M carbonate buffer, pH 9.5
(50 .mu.L buffer/mg digitonin), and the pH was adjusted to 9.5 as
necessary. The reaction mixture was stirred in the dark at room
temperature for two hours and 100 .mu.L of sodium borohydride
solution (4 mg/mL in water) was added to the reaction. After
incubating for two hours at 4.degree. C., the reaction was dialyzed
against 20 mM PBS, pH 7.4, at 4.degree. C. overnight To each
mixture was added fatty-acid free bovine serum albumin (to a final
concentration of 5 mg/mL). The solution was then sterile filtered
through a 0.22 .mu.m filter (Coaster Labs) and stored at
-20.degree. C.
[0119] c. Anti-APO CIII Coated Plates: VLDL specific Mab 18-358-211
was diluted in 20 mM PBS, pH 7.4, to a final concentration of 15
.mu.g/mL. One-hundred microliters of the solution was added to each
well of Maxisorb Nunc Immuno plates (Nalge-Nunc International,
Naperville, Ill.) and incubated at room temperature with gentle
shaking for two hours. The plates were washed five times with
PBS-Tween and then blocked with 200 .mu.L of 5% BSA in 20 mM PBS by
incubation at 37.degree. C. for one hour. The plates were stored at
4.degree. C. with plastic sealers. Before use, the plates were
washed five times with PBS-Tween (if HRPO conjugates were to be
used in the assay) or TRIS-Tween (for assays utilizing AP
conjugates).
[0120] d. Generation of VLDL-Cholesterol Standard Curves:
[0121] VLDL-cholesterol standards (100 .mu.L/well, in duplicate)
were incubated in the monoclonal antibody coated plates (Section 6c
above) at 37.degree. C. for one hour. After washing the plates five
times with PBS-Tween, 100 .mu.L of digitonin-HRPO conjugate at 0.5
.mu.g/mL in 1% casein in PBS was added to each well and incubated
at 37.degree. C. for one hour. The plates were washed with
PBS-Tween eight times. OPD (100 .mu.L of standard solution prepared
from one OPD tablet/10 mL citrate buffer, pH 6; both commercially
available from Abbott Laboratories, Ill.) was added to the wells.
After incubation for 10 minutes, the color reaction was stopped
with 100 .mu.L of 1 N H.sub.2SO.sub.4. The plates were read at 490
nm on a microplate reader (Bio-Tek Instruments, Wingoski, Vt.).
Standard curves (absorbance vs VLDL-cholesterol concentration) were
generated from the results. A typical calibration curve plot of
VLDL-cholesterol concentration by the sandwich assay is shown in
FIG. 9.
[0122] e. Evaluation of VLDL-Cholesterol in Plasma Samples:
[0123] Plasma samples in ethylenediaminetetraacetic acid (EDTA)
were collected from normal individuals and patients and frozen at
-20.degree. C. until use. Thawed samples were not used after two
days storage at 4.degree. C. The samples were diluted 125-fold in
1% casein in PBS and assayed for VLDL-cholesterol according to the
procedure of Section 6d above with the modification that the
diluted samples were used in place of the standards. Along with the
samples, standards were also assayed in duplicate as described in
Section 6d. For each microtiter plate, a standard curve was
generated and the values of the samples were determined using a
point-to-point fitted computer program. Seventy five subjects (Nos.
1-75 in Table 5) were used in this study. The VLDL-cholesterol
concentrations determined by the ultracentrifuge method and the
assay of the present invention using monoclonal antibody
18-358-211-Sepharose and digitonin-HRPO are presented in Table 6.
The correlation between the ultracentrifuge method and the sandwich
assay is shown in FIG. 10. The correlation coefficient (r) was
0.923 with an intercept of -0.251 and a slope of 1.00. The results
demonstrate that Mab 18-358-211 is capable of capturing VLDL
particles of heterogeneous sizes as demonstrated in the
immunocapture assay of this invention.
EXAMPLE 1
Preparation Of Lipoprotein Fractions
[0124] Blood samples from normal fasting subjects were collected
into ethylenediamine-tetraacetic acid (EDTA) and the red blood
cells were removed by centrifugation. The plasma samples were then
analyzed for Lp(a) using a TERUMO ELISA kit (Terumo Medical Corp.,
Elkton, Md.). Plasma samples containing less than 1 mg/dL Lp(a)
cholesterol were selected for the purification of VLDL, IDL, LDL
and HDL. Lipoprotein subtractions were prepared in a Beckman
Ultracentrifuge with a SW 40 Ti rotor by successive
ultracentrifugation at 4.degree. C. (Havel et al. (1955) J Clin
Invest 4: 1345-1355). VLDL was collected at a density of about
1.006 g/mL; IDL was collected at a density range of about
1.006-1.019 g/mL; LDL was collected at a density range of about
1.019-1.050 g/mL; and HDL was collected at a density range of about
1.080-1.255 g/mL. All fractions were isolated by the tube-slicing
technique (Beckman Instruments, SPINCO Division, Palo Alto,
Calif.). The lipoprotein fractions were dialyzed exhaustively
against 0.15 M sodium chloride containing 0.1% EDTA and 0.1% sodium
azide, pH 7.4 at 4.degree. C. The IDL, LDL and HDL fractions were
sterile filtered through 0.2 .mu.m membrane filters (Nalgene) and
VLDL through a 0.45 .mu.m membrane filter (Nalgene) and stored at
4.degree. C. The purity of each lipoprotein fraction was evaluated
by electrophoresis under non-denaturing polyaccrylamide gradient
gel electrophoresis (Lefevre et al. (1987) J Lipid Res 28:
1495-1509). Gradient slab gels of 2-16% and 4-30% and
electrophoresis apparatus GE-24 (Pharmacia LKB) were used in the
analysis. The lipoprotein fractions containing no
cross-contamination were used in the studies.
EXAMPLE 2
Preparation Of Peroxidase Conjugates Of Anti-ApoB And Anti-Hdl
Antibodies
[0125] Mab 1-1182-137, developed at Abbott Laboratories using
intact LDL particles as an immunogen, was used. This monoclonal
antibody shows equal affinity with LDL, VLDL, and IDL in direct
binding on ELISA (as described in Section 2a) and also in an
inhibition assay as described in Section 2c. A polyclonal antibody
against HDL in goat obtained from MEDIX Corporation was also
used.
[0126] Horseradish peroxidase (1 mg=155 Ku, Amano International)
was dissolved in water (250 .mu.L) and oxidized with freshly
prepared 0.2 M sodium meta-periodate (50 .mu.l) at room temperature
in the dark for 20 minutes. The oxidized peroxidase was then
dialyzed against 2 liters of 1 mM acetate buffer (pH 4.5) at
4.degree. C. for four hours.
[0127] Monoclonal antibody against apoB (Mab 1-1182-137, 1.9
mg/mL), which was dialyzed against 0.01 M carbonate buffer (pH 9.5)
at 4.degree. C., was treated with 20 .mu.L of 0.2 M carbonate
buffer (pH 9.5). The antibody and the dialyzed peroxidase were then
mixed at room temperature in the dark for two hours. To this
mixture 24 .mu.L of freshly prepared sodium borohydride (Aldrich, 4
mg/mL in water) was added and then incubated at 4.degree. C. in the
dark for two hours. The peroxidase-antibody conjugate was then
dialyzed against two liters of 20 mM phosphate buffered saline (pH
7.4) at 4.degree. C. and stored at -20.degree. C. in small
aliquots.
[0128] Similar procedure was adopted to prepare the peroxidase
conjugate of anti-HDL polyclonal antibody.
[0129] The binding curves of anti-apoB and anti-HDL-peroxidase
conjugates with lipoproteins are shown in FIGS. 8A and 8B,
respectively. A Maxisorb Nunc Immuno plate was coated with 100
.mu.l of different lipoproteins by incubation at 37.degree. C. for
30 minutes. After blocking the non-specific sites with 200 .mu.l of
10% FBS in PBS at 37.degree. C. for one hour, and washing five
times with PBS-Tween 20, 100 .mu.L of peroxidase conjugates (2.5
.mu.g/mL diluted in 3% FBS in PBS) was added to the first row of
wells and were serially diluted in horizontal wells. The plates
were incubated at 37.degree. C. for 30 minutes, washed eight times
with PBS-Tween 20. One hundred microliters of OPD substrate
solution was added to each well. After incubation at room
temperature for five minutes, the color reaction was stopped with
100 .mu.l of 1 N H.sub.2SO.sub.4. The plate was then read at 490 nm
on a microplate reader.
EXAMPLE 3
Covalent Attachment Of Monoclonal Antibody To Cnbr-Activated
Sepharose 4b
[0130] One gram of CNBr-activated Sepharose 4B (Pharmacia LKB,
Piscataway, N.J.) was suspended in about 15 mL of 1 mM HCI. The gel
was then transferred to a coarse-porosity sintered-glass funnel and
washed with 25 mL of 0.1 M carbonate buffer in 0.5 M sodium
chloride, pH 8.3 (coupling buffer). A gentle vacuum was applied to
remove the buffer. The moist gel cake was then transferred to a
glass tube with a screw-capped stopper. Monoclonal antibody
18-258-211 (15 mg, concentration 2.6 mg/mL), which was dialyzed
against the coupling buffer at 4.degree. C., was then added to the
gel. The mixture was then mixed gently end-over-end using an
infiltration wheel at 4.degree. C. for 20 hours. The supernatant
was checked by measuring the absorbance at 280 nm for the unbound
antibody. For all monoclonal antibodies used, more than 95% were
bound to the gel. The gel was then transferred to a coarse-porosity
sintered-glass funnel, washed with 50 mL of the coupling buffer and
then with 25 mL of 0.1 M TRIS-HCI buffer, pH 8.0 (blocking buffer).
The gel was then transferred to a glass tube, and mixed with 10 mL
of the blocking buffer at room temperature for two hours. The
antibody-immobilized gel was then washed with three cycles of
alternating pH. Each cycle consisted of a wash with acetate buffer
(0.1 M, pH 4) containing sodium chloride (0.5 M) followed by a wash
with TRIS buffer (0.1 M, pH 8) containing sodium chloride (0.5 M).
The final wash was done with 100 mL of TRIS-HCI buffer (0.05 M pH
7.4) containing sodium chloride (0.15 M) and sodium azide (0.01%)
(storage buffer). The gel was stored as a 25% suspension (14 mL) in
the storage buffer at 4.degree. C. Assuming 100% of the monoclonal
antibody bound to the gel, 200 .mu.L of gel suspension contains 214
.mu.g of the monoclonal antibody.
EXAMPLE 4
Preparation Of Cholesterol Assay Reagents
[0131] a. Reagents:
[0132] Two separate reagents were prepared and were mixed together
at the time of the assay. The first reagent formula was comprised
of 1.62 g of 3,5-dichloro-2-hydroxy-benzenesulfonic acid sodium
salt (Aldrich, Milwaukee, Wis.) and 0.428 g of horseradish
peroxidase (specific activity 82.3 EZ/mg, Amano International)
dissolved in 20.4 mL 0.05 M of
3-(N-morpholino)-2-hydroxypropanesulfonic acid sodium salt (MOPSO,
SIGMA) at pH 7. The second reagent formaia was comprised of 0.0113
g MgCI.sub.2. 6H.sub.2O, 0.0276 g anhydrous CaCI.sub.2, 0.51 g
lactose, 0.51 g dextran (M.W. 17,900, Pharmachem, Terrytown, N.Y.),
1.02 g bovine serum albumin (fatty acid free, SIGMA), 0.30 g
glycerol, 0.187 g 4-aminoantipyrine (ALDRICH, Milwaukee, Wis.)),
0.1486 g cholesterol ester hydrolase (specific activity 8.4 EZ/mg,
Amano International), 0.086 g cholesterol oxidase (specific
activity 6.2 EZ/mg, Boehringer-Mannheim) dissolved in 10.2 mL of
0.25 M MOPSO buffer (pH 7). The first and the second reagents were
stored separately in small aliquots at -20.degree. C. in dark.
[0133] Both reagents have been found to be stable for at least 16
months in terms of their assay performances, in terms of
correlation and slope with known cholesterol standards. Eight and
one half microliters of each of the reagents were used in the
assay. The peroxidase activity of 8.5 .mu.L of first reagent was
14.7 EZ and the enzyme activities of cholesterol ester hydrolase
and cholesterol oxidase of the second reagent were 1.04 EZ and
0.445 EZ, respectively.
[0134] b. Reaction Buffer:
[0135] The reaction buffer (ICMT) which is also the extraction
buffer contained the following materials: 0.05 M MOPSO (pH 7)
(Sigma), 1% IgePal CO-530 (GAF), 0.2% Triton-X-100.RTM. (Bio-Rad)
and 0.3% cholic acid (Sigma). The buffer was sterile filtered and
was stored at room temperature. The buffer is stable for at least
one year.
EXAMPLE 5
Preparation Of A VLDL-Cholesterol Standard Curve
[0136] Two hundred microliters of monoclonal antibody-immobilized
Sepharose 4B gel suspension from Example 3 was transferred to an
appropriate number of Eppendorf microcentrifuge tubes. Each tube
was completely filled with 5% BSA in PBS and mixed on a TOMY micro
tube mixer (Peninsula Laboratories) at room temperature for one
hour in order to block the non-specific binding sites of the
plastic tube. Tubes were then centrifuged on a table-top centrifuge
for about one minute and the supernatant carefully aspirated. One
hundred microliters of a plasma sample diluted in PBS was added to
a tube of gel suspension. Gel suspensions were mixed on a TOMY
mixer at room temperature for one hour. Gel suspensions were then
washed twice with about 1 mL of PBS by mixing for one minute,
centrifuging for one minute and aspirating the supernatant from
each. ICMT solution (prepared as in Example 4(b)) was added to each
tube to a final volume of 750 .mu.L. Cholesterol assay reagents
(8.5 .mu.L of each of #1 and #2 from Example 4 (a)) were added to
each tube. The suspensions were mixed on a TOMY mixer for about
eight minutes, centrifuged for one minute and the absorbances of
the supernatant solutions were read on a DU 7400 Spectrophotometer
at 515 nm. The concentrations of the gel-bound cholesterol were
determined from a cholesterol standard curve. The standard curve
(shown in FIG. 6) was prepared with purified VLDL samples having
concentrations of 1.56, 3.12, 6.25, 12.5 and 25 mg/dL following the
assay protocol described above.
EXAMPLE 6
VLDL-Cholesterol Immunocapture Assay
[0137] Two hundred microliters of monoclonal antibody-immobilized
Sepharose 4B gel suspension from Example 3 (about 214 .mu.g of
antibody) was transferred to an appropriate number of Eppendorf
tubes which were previously treated with 5% BSA in PBS to block all
non-specific binding sites. One hundred microtiters of individual
plasma samples (containing acid-citrate-dextrose or EDTA
anticoagulant), diluted ten-fold in PBS, were added to each 200
.mu.L of gel suspension. The gel suspensions were mixed on a TOMY
mixer at room temperature for one and a half hours. The gel
suspensions were then washed with about 1 mL of PBS by mixing for
one minute, centrifuging for one minute and aspirating the
supernatants. ICMT solutions (described in Example 4(b)) were added
to each tube to a final volume of 750 .mu.L. Cholesterol assay
reagent (8.5 .mu.l of each of #1 and #2 from Example 4(a)) were
added to each tube. The suspensions were mixed on a TOMY mixer for
about eight minutes, centrifuged for one minute and the absorbances
of the supetnatant solutions were read on a DU 7400
Spectrophotometer at 515 nm. The concentrations of VLDL-cholesterol
in the plasma samples were determined by multiplying the
concentration obtained from the standard curve shown in FIG. 6 by
10. The results are shown in Table 6 and FIG. 7.
EXAMPLE 7
Ouantitation Of VLDL-Cholesterol By Ultracentrifugation
[0138] Plasma samples (2 mL each) were transferred to ultraclear
tubes (Beckman, 11.times.34 mm) and then overlayered with 0.3 mL of
d 1.006 g/mL KBr solution. The samples were centrifuged on a TLS 55
swinging bucket rotor at 40,000 rpm at 4.degree. C. for 20 hours
using a T-1 00 Beckman ultracentrifuge. The upper VLDL layers were
carefully pipetted out without disturbing the bottom layer.
Phosphate buffered saline, pH 7.4 was added to the centrifuged tube
to bring the volume to the original mark (2.3 mL). Adequate
recovery was verified by comparing the sum of cholesterol in each
of the fractions to the total cholesterol of the sample. The
cholesterol concentrations of the upper VLDL and the lower
d>1.006 g/mL (infranet cholesterol) were determined with VISION
cholesterol assays (Abbott Laboratories, Abbott Park, Ill.).
VLDL-cholesterol concentrations were calculated as the difference
between total plasma cholesterol and infranet cholesterol. The
results are shown in Table 5 and were used in the correlation
studies in Table 6.
EXAMPLE 8
VLDL-Cholesterol Sandwich Assay
[0139] The monoclonal antibody 18-358-211 was diluted in 20 mM PBS,
at pH 7.4, to a concentration of 15 .mu.g/mL. One hundred
microliters of the antibody solution was added to the wells of
Maxisorb Nunc Immuno plates and the plates were incubated at room
temperature on a rotator at 100 rpm for two hours. The plates were
washed five times with PBS-Tween solutions and then blocked with
200 .mu.l of 5% w/v BSA in 20 mM PBS, at pH 7.4, by incubation at
37.degree. C. for one hour. The plates can be stored at 4.degree.
C. with plastic sealers at least for ten days prior to use.
[0140] Plasma samples (Nos. 1-75 in Table 5) were diluted 125-fold
with 1% w/v alkali-treated casein in 20 mM PBS at pH 7.4. One
hundred microliters of the diluted samples were added to each well
of the antibody plates and the plates were incubated at 37.degree.
C. for one hour. After washing the plates five times with
PBS-Tween, 100 .mu.l of HRPO-digitonin conjugate (0.5 .mu.g/mL in
1% w/v alkali-treated casein in 20 mM PBS at pH 7.4) were added to
each well. The plates were incubated at 37.degree. C. for one hour
and then washed ten times with PBS-Tween. One-hundred microliters
of a freshly prepared solution of o-phenylenediamine in citrate
buffer (substrate commercially available from Abbott Laboratories)
were added to each well and after five minutes, the reaction was
quenched with 100 .mu.L of 1 N H.sub.2SO.sub.4. The absorbance of
each well was measured on a Bio-Tek microplate reader at 490 nm.
The VLDL-cholesterol concentration was then determined from a
standard curve of absorbance versus VLDL-cholesterol concentration
(shown in FIG. 9). The results of the sandwich assay and the
correlation curve between the ultracentrifuge method and sandwich
assay for VLDL-cholesterol are shown in Table 6 and FIG. 10. The
correlation between the two methods were fairly good with a
correlation coefficient(r)=0.923; intercept=-0.251 and slope=1.00.
The correlation between the two methods could have been even better
if the VLDL-cholesterol concentrations could have been more direct.
Moreover, calculation by using two assays (total cholesterol and
infranet cholesterol in the ultra centrifuged fraction) obviously
can lead to an error, particularly with plasma samples with low
VLDL cholesterol concentrations.
EXAMPLE 9
VLDL-Cholesterol Calibration Curve For Immunocapture Assay
[0141] VLDL-cholesterol standards were prepared from plasma samples
as described in Section 6. Calibrators having VLDL-cholesterol
concentrations of 0, 1.56, 3.125, 6.25, 12.5 and 25 mg/dL were
assayed by the method described in Example 9. The concentrations
were multiplied by 125 to generate the standard curve because the
plasma samples were diluted 125-fold prior to performing the assay.
A plot of VLDL-cholesterol concentration versus absorbance was
prepared from the resulting data. FIG. 8 is illustrative of such a
plot. The VLDL-cholesterol concentration in unknown samples can be
determined from the calibration curve. Generally the calibrators
and the plasma samples were assayed on the same plate to minimize
the effect of variations in the reagents, materials and conditions.
The number and concentration of calibrators can be readily altered
depending on the desired accuracy of the results.
[0142] The embodiments described and the alternative embodiments
presented are intended as examples rather than as limitations.
Thus, the description of the invention is not intended to limit the
invention to the particular embodiments disclosed, but it is
intended to encompass all equivalents and subject matter within the
spirit and scope of the invention as described above and as set
forth in the following claims.
5TABLE 5 LIPID PROFILES OF PLASMA SAMPLES Sample Total-C HDL-C
Trig.sup.1 FE-LDL.sup.2 FE-LDL.sup.3 No. mg/dL mg/dL mg/dL mg/mL
mg/mL 1 155 68 107 107 107 2 220 44 201 136 144 3 197 55 44 133 128
4* 230 47 109 150 166 5 177 38 104 118 119 6 182 70 37 104 104 7
217 41 127 150 156 8 135 43 77 77 78 9* 227 32 365 122 137 10 282
43 180 203 196 11 275 50 114 202 174 12 199 65 249 84 102 13 231 50
73 166 163 14 179 49 64 117 117 15 136 35 92 83 85 16 228 59 110
147 138 17 142 51 51 81 81 18 170 64 45 97 97 19 205 65 126 115 125
20 127 45 58 71 75 21 172 64 70 94 99 22 207 67 50 130 134 23 211
40 99 150 147 24 178 56 70 87 90 25 156 63 51 83 75 26 225 45 93
161 158 27 n/a r/a n/a n/a n/a 28 166 51 64 102 104 29 142 51 44 82
82 30 162 67 162 62 67 31 161 70 94 72 70 32 187 45 215 99 106 33
147 49 67 85 81 34 144 38 78 90 87 35 185 78 36 100 101 36 177 39
133 111 115 37 148 29 168 86 89 38 182 58 84 108 93 39 203 36 57
156 150 40 119 45 64 62 64 41 138 48 90 71 76 42 156 26 125 105 109
43 190 46 102 123 108 44 258 36 368 149 148 45* 209 41 146 139 124
46 160 60 41 92 97 47 181 47 60 123 123 48 222 38 106 162 158 49
246 57 146 159 161 50 184 48 88 119 119 51 280 45 209 193 180 52
229 53 115 153 148 53 164 45 124 94 95 54 208 42 161 134 135 55 245
50 91 177 162 56 185 54 54 120 124 57 180 66 62 102 99 58* 164 42
85 105 105 59* 244 55 132 163 140 60 167 33 154 103 120 61 175 53
102 102 104 62 151 55 169 62 75 63 180 36 205 103 107 64 230 54 122
151 142 65 181 42 92 121 116 66 184 50 132 108 110 67 206 37 247
120 129 68 139 46 59 71 75 69 167 46 73 106 105 70 215 63 78 136
134 71 148 71 53 67 73 72 156 42 64 102 91 73 162 53 95 90 95 74
216 34 142 154 152 75 154 50 69 90 86 76 n/a n/a n/a n/a n/a 77 136
38 65 85 72 78 160 49 61 99 89 79 165 72 98 73 75 80 147 46 62 89
77 81 160 49 86 94 72 82 173 29 98 124 113 83 161 38 96 104 82 84
176 90 45 77 83 85 161 48 84 96 95 86 n/a n/a n/a n/a n/a 87 198 80
54 107 110 88 207 75 79 115 120 89 115 46 67 56 58 90 n/a n/a n/a
n/a n/a 91 n/a n/a n/a n/a n/a 92 180 55 49 115 110 93* 238 40 214
155 163 94* 289 36 299 193 202 95* 228 70 91 140 144 96* 309 39 68
256 266 97** 200 34 341 98 129 98** 135 38 53 86 89 99** 237 53 316
121 138 100** 200 41 234 112 106 .sup.1Trig = triglyceride
concentration .sup.2FE = Friedewald Equation: [LDL-Chol] =
[Total-Chol} - [HDL-Chol] - [Trig/5] .sup.3UC = Ultracentrifuge
.beta.-quantitation: [LDL-Chol] = [d .1.006 g/mL Infranate-Chol] -
[HDL-Chol] *Patients with coronary heart disease who are on lipid
lowering drugs **Diabetic patients
[0143]
6TABLE 6 CORRELATION BETWEEN VLDL-CHOLESTEROL ASSAYS UC-VLDL.sup.1
IC-VLDL.sup.2 IA-VLDL.sup.3 Sample No. mg/dL mg/dL mg/dL 1 14 9 11
2 20 17 20 3 14 12 9 4 17 13 20 5 17 15 18 6 8 10 10 7 20 17 18 8
14 11 12 9 60 68 73 10 46 55 51 11 51 49 56 12 32 21 21 13 14 10 14
14 13 12 12 15 12 10 9 16 22 21 22 17 10 8 8 18 9 8 5 19 15 12 18
20 7 12 8 21 14 14 21 22 10 11 11 23 15 12 15 24 14 13 9 25 10 8 6
26 14 11 14 27 8 6 4 28 11 10 9 29 12 15 12 30 28 29 30 31 26 20 22
32 37 35 44 33 18 13 12.4 34 21 18 12.4 35 11 16 12 36 23.5 23.9 28
37 31 23 30 38 33 25 24 39 19 19 17 40 14 12.4 14 41 16 17 18 42 22
21.5 18 43 40 21 36 44 77 80 76 45 42 31 31 46 10 13 15 47 16 14.4
18 48 21 20 17 49 29 33 27.4 50 20.5 20.7 20 51 57 61 49 52 31 28
28 53 27 23 29 54 18 28 30 55 30 20 19 56 13 16.8 21 57 16 15.6
16.8 58 18 16 18 59 40 41 34 60 18 23 23.7 61 20 19.2 19.5 62 27
32.8 47 63 39 49 49 64 39 44 31 65 22.5 24.5 22 66 31 31.6 30 67 41
50 49 68 13 13 14 69 21 19.3 18 70 24 23 17 71 10 14 10 72 15 19 14
73 19 18 21 74 33 33.7 31.2 75 22 28 32 76 10 9 77 13 10 78 12 14
79 14 10 80 12 10 81 16 11 82 20 18 83 19 19 84 9 13 85 10 8 86 7
12 87 12 15 88 14 18 89 11 9 90 15 9 91 8 4 92 15 14 93 37 43 94 51
50 95 11 11 96 14 17 97 37 37 98 8 5 99 35 31 100 52 53
.sup.1Measured by Ultracentrifugation according to Example 7.
.sup.2Measured by immunocapture according to Example 6.
.sup.3Measured by sandwich immunoassay according to Example 8.
* * * * *