U.S. patent number 4,677,057 [Application Number 06/710,038] was granted by the patent office on 1987-06-30 for diagnostic assay for the presence of apolipoproteins associated with plasma high density lipoproteins.
This patent grant is currently assigned to Scripps Clinic and Research Foundation. Invention is credited to Linda K. Curtiss, Thomas S. Edgington.
United States Patent |
4,677,057 |
Curtiss , et al. |
June 30, 1987 |
**Please see images for:
( Certificate of Correction ) ** |
Diagnostic assay for the presence of apolipoproteins associated
with plasma high density lipoproteins
Abstract
Monoclonal receptors that immunologically bind to human
apolipoprotein A molecules, particularly apo-A-I and apo-A-II, are
described as are their methods of use and articles of manufacture
containing them.
Inventors: |
Curtiss; Linda K. (San Diego,
CA), Edgington; Thomas S. (La Jolla, CA) |
Assignee: |
Scripps Clinic and Research
Foundation (La Jolla, CA)
|
Family
ID: |
24852362 |
Appl.
No.: |
06/710,038 |
Filed: |
March 11, 1985 |
Current U.S.
Class: |
436/518; 435/7.5;
435/7.9; 435/7.92; 435/948; 435/975; 436/533; 436/540; 436/548;
436/808; 436/809; 436/811; 530/359; 530/388.25; 530/808; 530/809;
530/864 |
Current CPC
Class: |
C07K
16/18 (20130101); G01N 33/92 (20130101); G01N
2800/044 (20130101); Y10S 435/975 (20130101); Y10S
436/811 (20130101); Y10S 530/864 (20130101); Y10S
435/948 (20130101); Y10S 436/809 (20130101); Y10S
530/808 (20130101); Y10S 530/809 (20130101); Y10S
436/808 (20130101) |
Current International
Class: |
C07K
16/18 (20060101); G01N 33/92 (20060101); G01N
033/53 (); G01N 033/543 (); C12N 015/00 (); C12N
005/00 () |
Field of
Search: |
;260/112B,112R
;435/948,172.2,240,211,7 ;935/89,93,95,103,106,108,110
;436/548,518,533,538,539,540,544,545,808,809,811
;530/359,387,388,808,809 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4399217 |
August 1983 |
Halmquist et al. |
|
Other References
Kohler and Milstein, Nature, vol. 256, pp. 495-497 (1975). .
El Morshidy, Dissertation Abstracts International B, vol. 45, No.
3, p. 821, 1984. .
Steinberg et al., Clin. Chem., vol. 29, No. 3, pp. 415-426, 1983.
.
Ventrex Laboratories Inc. Catologue, R1A for Apolipoprotein A-1,
1984..
|
Primary Examiner: Marantz; Sidney
Assistant Examiner: DeSantis; Patricia L.
Attorney, Agent or Firm: Dressler, Goldsmith, Shore, Sutker
& Milnamow Ltd.
Claims
What is claimed is:
1. A method for assaying for the presence of human apolipoprotein A
in a sample to be assayed comprising the steps of:
(a) providing a monoclonal receptor that is secreted by a hybridoma
having an ATCC accession number selected from the group consisting
of HB 8741, HB 8743, HB 8744 and HB 8745 and whose antibody
combining site immunologically binds to human apolipoprotein A, but
does not immunologically bind to human apolipoproteins B, C, D and
E;
(b) admixing a known amount of said receptor with an aliquot of a
sample to be assayed for the presence of an human apolipoprotein to
form an admixture;
(c) maintaining said admixture for a predetermined time period
sufficient for said receptor to immunologically bind to human
apolipoprotein A present in said sample and form an immunoreactant;
and
(d) determining the amount of said receptor bound in said
immunoreactant and thereby the presence of said human
apolipoprotein A.
2. The method according to claim 1 wherein said receptor is an
antibody that immunologically binds to apolipoprotein A-I, and is
screted by a hybridoma having an ATCC accession number selected
from the group consisting of HB 8741, HB 8744 and HB 8745.
3. The method according to claim 1 wherein said receptor is an
antibody that immunologically binds to apolipoprotein A-II, and is
secreted by a hybridoma having the ATCC accession number HB
8743.
4. The method according to claim 1 including the additional steps
of: (i) providing an apolipoprotein A bound by said receptor that
is affixed to a solid matrix as a solid support antigen;
(ii) admixing the maintained admixture of step (c) present as a
liquid admixture with said solid support to form a solid-liquid
admixture;
(iii) maintaining said solid/liquid admixture for a predetermined
time period sufficient for said receptor of said liquid admixture
to immunologically bind to said antigen and form an
immunoreactant;
(iv) separating said solid and liquid phases; and
(v) determining the amount of said receptor bound in said
immunoreactant.
5. The method according to claim 4 wherein said receptor includes a
linked indicating means that signals the formation of said
immunoreactant and by which the amount of said receptor bound in
said immunoreactant is determined.
6. The method according to claim 1 wherein said sample aliquot is
affixed to a solid matrix as a solid support antigen prior to
forming the admixture of step (b), the admixture formed is a
solid/liquid admixture, and including the further step of
separating said solid/liquid admixture prior to determining the
amount of bound receptor.
7. The method according to claim 6 wherein the amount of said bound
receptor is determined by the steps of:
(i) admixing a known amount of an indicating means-containing
reagent that reacts with the bound receptor of the immunoreactant
to form a second solid/liquid phase admixture, said reagent being
free from reaction with said solid support antigen, and said
indicating means signalling the presence of said bound
receptor;
(ii) maintaining said second admixture for a predetermined time
period sufficient for said admixed reagent to react with said bound
receptor and form a bound reaction product;
(iii) separating the solid and liquid phases; and
(iv) determining the amount of said bound reaction product
present.
8. The method according to claim 7 wherein said indicating
means-containing reagent is a .sup.125 I-linked antibody that
immunologically binds to said receptor.
9. The method according to claim 1 wherein said admixture of step
(b) is a liquid admixture and includes a known amount of a
radiolabeled apolipoprotein A-containing antigen, and said
determination of bound receptors is carried out by the further
steps of:
(i) admixing an excess of an antibody that precipitates said
receptors but does not precipitate human apolipoprotein or said
admixed radiolabeled antigen, to form a second liquid
admixture;
(ii) maintaining said second liquid admixture for a predetermined
period of time sufficient for said admixed antibody to precipitate
said receptors, and form a precipitate and a supernatant;
(iii) separating said precipitate from said supernatant; and
(iv) measuring the radioactivity present in said precipitate.
10. The method according to claim 1 wherein said receptor is
affixed to a solid matrix of latex particles as a solid support
prior to said admixture of step (b), said admixture formed is a
dispersion of said receptor-affixed latex particles in an aqueous
medium, the formation of said immunoreactant causes said latex
particles to agglutinate, and the amount of said receptors bound in
said immunoreactant is determined by the time required for said
latex agglutination to occur.
11. The method according to claim 1 wherein said receptor is
produced by hybridoma ATCC HB 8741.
12. The method according to claim 1 wherein said receptor is
produced by hybridoma ATCC HB 8743.
13. The method according to claim 1 wherein said receptor is
produced by hybridoma ATCC HB 8744.
14. The method according to claim 1 wherein said receptor is
produced by hybridoma ATCC HB 8745.
15. A diagnostic assay system comprising at least one package that
contains an effective amount of a monoclonal receptor that is
secreted by a hybridoma having an ATCC accession number selected
from the group consisting of HB 8741, HB 8743, HB 8744 and HB 8745
and immunologically bind to human apoliproprotein A, but does not
immunologically bind to human apolipoproteins B, C, D, and E.
16. The diagnostic system according to claim 15 further including a
second package that includes, affixed to a solid matrix as a solid
support antigen, a known amount of an apolipoprotein A that is
immunologically bound by said receptor.
17. The diagnostic system according to claim 16 wherein said solid
support is a well of a microtiter plate.
18. The diagnostic system according to claim 15 wherein said
receptor is affixed to a solid matrix as a solid support.
19. The diagnostic system according to claim 18 wherein said solid
matrix is a latex particle.
20. A hybridoma having the ATCC accession number HB 8741.
21. A hybridoma having the ATCC accession number HB 8743.
22. A hybridoma having the ATCC accession number HB 8744.
23. A hybridoma having the ATCC accession number HB 8745.
24. A method for assaying the amount of human high density
lipoprotein in a sample to be assayed comprising the steps of:
(a) providing a mixture of monoclonal receptors containing
effective amounts of receptors secreted by the hybridoma having the
ATCC accession number HB 8743 that immunoreact with apolipoprotein
A-II and receptors secreted by any two of the hybridomas having the
ATCC accession numbers HB 8741, HB 8744 and HB 8745 that
immunoreact with apolipoprotein A-I;
(b) admixing a known amount of said mixture with a sample to be
assayed for the presence of human high density lipoprotein to form
an admixture;
(c) maintaining said admixture for a predetermined period of time
sufficient for said receptors to immunoreact with said
apolipoproteins A-I and A-II to form immunocomplexes; and
(d) determining the amount of said receptors bound in said
immunocomplexes and thereby the amount of said high density
lipoprotein in the sample.
25. The method according to claim 24 wherein said admixing of step
(b) is carried out in an aqueous liquid phase.
26. The method according to claim 25 wherein the amount of said
receptors bound is determined by the steps of:
(i) admixing an excess of a labeled antibody that immunoreacts with
the bound receptor molecules of the immunocomplexes formed in step
(c) and precipitates said immunocomplexes:
(ii) maintaining the admixture formed in step (c) for a
predetermined time period sufficient for said labeled antibody to
immunoreact and bind to said bound receptors and precipitate said
immunocomplexes containing said bound receptor; and
(iii) determining the amount of labeled antibody in the precipitate
so formed.
27. The method according to claim 24 wherein said admixture formed
in step (b) is a solid/liquid phase admixture.
28. A method for assaying for the presence of human apolipoprotein
A-II in a sample to be assayed comprising the steps of:
(a) admixing a sample to be assayed for the presence of human
apolipoprotein A-III with an effective amount of a monoclonal
receptor secreted by the hybridoma having the ATCC accession number
HB 8743 that immunoreacts with human apolipoprotein A-II to form an
admixture;
(b) maintaining said admixture for a predetermined period of time
sufficient for said receptor to bind to human apolipoprotein A-II
present in said sample and form an immunoreactant; and
(c) determining the presence of an immunoreactant formed in step
(b), and thereby the presence of human apolipoprotein A-II in said
sample.
29. A method for assaying for the presence of human apolipoprotein
A-I in a sample to be assayed comprising the steps of:
(a) admixing a sample to be assayed for the presence of human
apolipoprotein A-I with an effective amount of a mixture of
monoclonal receptors that immunoreact with human apolipoprotein A-I
secreted by two of the hybridomas having ATCC accession numbers
selected from the group consisting of HB 8741, HB 8744 and HB 8745
to to form an admixture;
(b) maintaining said admixture for a predetermined period of time
sufficient for said receptors to bind to human apolipoprotein A-I
in said sample and form an immunoreactant; and
(c) determining the presence of an immunoreactant formed in step
(b), and thereby the presence of human apolipoprotein A-I in said
sample.
30. A monoclonal receptor that is secreted by a hybridoma having
the ATCC accession number HB 8741 and immunologically binds to
human apolipoprotein A-I.
31. A monoclonal receptor that is secreted by a hybridoma having
the ATCC accession number HB 8744 and immunologically binds to
human apolipoprotein A-I.
32. A monoclonal receptor that is secreted by a hybridoma having
the ATCC accession number HB 8745 and immunologically binds to
human apolipoprotein A-I.
33. A monoclonal receptor that is secreted by a hybridoma having
the ATCC accession number HB 8743 and immunologically binds to
human apolipoprotein A-II.
Description
DESCRIPTION
1. Technical Field of the Invention
The present invention relates to epitope-specific reagents that
bind apolipoproteins, and particularly to monoclonal receptors that
form immunoreactants with apolipoprotein A thereby permitting a
determination of the immunochemical heterogencity of
lipoproteins.
Background of the Invention
A. Atherosclerosis and Lipoproteins
Atherosclerosis is the disease in which cholesterol and other
lipids, accumulating on the walls of arteries, form bulky plaques
that inhibit the flow of blood and may lead to the formation of a
clot, obstructing an artery and causing occlusive thrombotic or
embolic disease such as a heart attack or stroke. Up to 50 percent
of all deaths in the United States are caused by atherosclerosis
and its secondary complications.
Human atherosclerosis is defined as the accumulation of selected
lipids, including cholesterol, and cells in the walls of arteries
and with time produces occlusive lesions. Although the etiology of
atherosclerosis is multi-factorial, a large body of clinical,
pathologic, genetic and experimental evidence suggests that
abnormalities of lipoprotein metabolism can contribute to the
development of atherosclerosis. These lipids are carried in the
blood stream as lipid-protein complexes called lipoproteins.
Atherosclerosis, and particularly that form known as coronary
artery disease (CAD), is a major health problem. Atherosclerosis
and its related vascular diseases acounted for 983,000 deaths in
1983; and CAD alone accounts for more deaths annually than all
forms of cancer combined. In the United States, more than 1 million
heart attacks occur each year and more than five hundred thousand
people die as a result of this disease. In direct health care
costs, CAD costs the United States more than $60 billion a year.
This enormous toll has focused attention on ways to identify
particular populations at risk for CAD so that the disease can be
controlled with diet, behavioral modification (exercise), and
specific therapeutic agents.
Four major classes of cholesterol-associated plasma lipoprotein
particles have been defined, and have their origin in the intestine
or liver. These particles are involved in the transport of the
neutral lipids including cholesterol and triglycerides. All classes
of plasma lipoproteins have apolipoproteins associated with the
lipid-protein complex; and the apolipoproteins play requisite roles
in the function of these lipoproteins.
The first class is the chylomicrons. They are the largest of the
lipoproteins and are rich in triglycerides. The site of origin of
the chylomicrons is the intestine.
While apolipoproteins are a quantitatively minor proportion of the
mass of chylomicrons, apolipoproteins A-I, A-II and A-IV are
significantly associated with chylomicrons, and intestinal
synthesis of these A apolipoproteins has been found. Much of the
chylomicron complement of A apolipoproteins is lost, and C and E
apolipoproteins are acquired when chylomicrons are exposed to
plasma or HDL in vitro. Intestinal production of the A
apolipoproteins (apo-A) may be regulated by factors other than fat
absorption and chylomicron formation.
The next class of lipoproteins is the very low density
lipoproteins, VLDL. The VLDL particle is involved in triglyceride
metabolism and transport of these lipids from the liver. The
apolipoproteins, apo-B and apo-E are the major constituents of the
VLDL particle.
The third lipoprotein is called low density lipoprotein (LDL), and
is a specific product of the catabolism of VLDL. The predominant
apolipoprotein in the LDL particle is apolipoprotein B, or apo-B.
Analytical techiques have revealed that apo-B is also the specific
apolipoprotein associated with chylomicrons and VLDL.
The results of the now classic Framingham study (1971) showed a
clear correlation between risk for CAD and serum cholesterol
levels. This study also demonstrated that elevated levels of low
density lipoprotein (LDL) cholesterol are associated with increased
risk of CAD. Recently, a study conducted by the Lipid Research
Clinics Coronary Primary Prevention Trial (1984) has demonstrated
that plasma levels of cholesterol and LDL cholesterol can be
reduced by a combined regime of diet and drugs, and that this
reduction of plasma cholesterol results in reduction of the
incidence of CAD mortality.
The cholesterol of atherosclerosis plaques is derived in part, if
not mostly from low-density lipoprotein (LDL). LDL is a large
spherical particle whose oily core is composed of about 1500
molecules of cholesterol, each attached by an ester linkage to a
long chain fatty acid. This core of cholesterol is enclosed by a
layer of phospholipid and unesterified cholesterol molecules. The
phospholipids are arrayed so that the hydrophilic heads are on the
outside, allowing the LDL to be in hydrated suspension in the blood
or extracellular fluids.
The cholesterol delivered to, and liberated from LDL particles
taken up by cells, controls cell's cholesterol metabolism. An
accumulation of intracellular cholesterol modulates three
processes.
First, it reduces the cell's ability to make its own cholesterol by
turning off the synthesis of an enzyme, HMG CoA reductase, that
catalyzes a step in cholesterol's biosynthetic pathway. Suppression
of the enzyme leaves the cell dependent on external cholesterol
derived from the receptor-mediated uptake of LDL.
Second, the incoming LDL-derived cholesterol promotes the storage
of cholesterol in the cell by activating an enzyme denominated
lipoprotein acyltransferase. That enzyme esterifies fatty acids to
excess cholesterol molecules, making cholesteryl esters that are
deposited in storage droplets.
Third, and most significant, the accumulation of cholesterol within
the cell drives a feedback mechanism that makes the cell stop
synthesizing new LDL receptors. Cells thereby adjust their
complement of external receptors so that enough cholesterol is
brought into the cells to meet the cells' varying demands but not
enough to overload them. For example, fibroblasts that are actively
dividing, so that new membrane material is needed, maintain a
maximum complement of LDL receptors of about 40,000 per cell. In
cells that are not growing, the incoming cholesterol begins to
accumulate, the feedback system reduces receptor manufacture and
the complement of receptors is reduced as much as tenfold.
On the other hand, it has been shown that another circulating
lipoprotein, high density lipoprotein (HDL) is implicated in a
state of elevated cholesterol associated with lowered risk of
atherosclerosis. Apolipoprotein A is a ligand of the HDL particle.
The amount of HDL provides an inverse correlation with the
predicted incidence of atherosclerosis.
High density lipoprotein (HDL) contains two major apolipoproteins,
apo-A-I and apo-A-II. Apo-A-I is the major protein component of all
primate HDL. All HDL particles contain apo-A-I, and therefore
immuno quantification of HDL has usually involved the quantitation
of apo-A-I. HDL particles containing only apo-A-II have not been
described.
One function of apo-A-I is the activation of the plasma enzyme,
lecithin-cholesterol acyltransferase (LCAT). This enzyme is
required for the esterification of free cholesterol for transport
to the liver. In the absence of apo-A-I, cholesterol in the blood
is not esterified and thus cholesterol is not cleared from the
blood. The specific role in HDL metabolism served by apo-A-II has
not been defined.
Many studies have shown that elevated HDL levels correlate with a
reduced incidence of CAD. Some authors have speculated that HDL
removes cholesterol from peripheral sites, such as the arterial
wall, therefore attributing anti-atherogenic properties to HDL.
Higher concentrations of HDL cholesterol are correlated with a
lower incidence of and/or a decreased severity of cardiovascular
disease, while elevated levels of LDL cholesterol are associated
with an increased risk of CAD. For the proper management of
patients with hyperlipidemia (excess lipids in the blood) and those
patients at special risk for CAD, it is desirable to frequently
determine levels of LDL and HDL cholesterol.
To date, assays of HDL cholesterol have been cumbersome and
inaccurate in determining blood levels of HDL. It would therefore
be beneficial to provide an assay that is easy to use and
accurately determines HDL blood levels.
B. Lipoprotein Structure and Function
It is important to understand that cholesterol does not exist free
in plasma but is transported to tissue sites in the body by
lipoproteins. Cholesterol can be obtained from directed cellular
synthesis or by diet. However, cholesterol can be removed from the
host only by the liver, where it is converted to bile acids and
excreted.
Very low density lipoprotein (VLDL) carries cholesterol and
triglycerides to the liver for subsequent excretion, whereas, LDL
delivers cholesterol to extrahepatic tissues, including the
coronary arteries. Hence, the "bad" lipoprotein, LDL/apo-B, is
involved in the deposition of cholesterol in peripheral tissue.
Conversely, the "good" lipoprotein HDL/apo-A, removes cholesterol
from the tissues and returns cholesterol to the liver for
excretion.
Historically, many systems have been developed to isolate and to
characterize lipoproteins. These techniques are usually based upon
the physicochemical properties of the lipoprotein particles. The
two most frequently used techniques are ultracentrifugation and
electrophoresis.
Differential density gradient ultracentrifugation takes advantage
of the fact that the lipoproteins are lighter or less dense, than
other plasma proteins, and it is easy to separate the chylomicrons
(the lightest lipoproteins), VLDL, LDL and HDL from each other.
Electrophoretic techniques have been useful for the classification
of patients with hyperlipidemias. However, these techniques are not
easily carried out in an ordinary clinical laboratory.
One can also see that the simple quantitation of blood cholesterol
or triglycerides does not provide the physician with the specific
information about which lipoproteins are carrying these lipids and
their quantitation.
C. The Plasma Lipoproteins
Four major classes of plasma lipoproteins; i.e., chylomicrons,
VLDL, LRL and HDL, have been defined, and subclasses within these
undoubtedly exist. All lipoproteins have their origin in the
intestine or liver, or both, and appear to have a pseudomicellar
structure. Neutral lipids, and particularly, cholesterol esters and
triglycerides, are maintained in the lipoproteins in a soluble and
stable form through interactions with the apolipoproteins and
phospholipids, which are more polar.
Unesterified cholesterol is also present in these complexes. Its
polarity lies between that of the neutral lipids (cholesteryl
esters and triglycerides) and that of the more polar
apolipoproteins and phospholipids.
An outer surface consisting of apolipoproteins, unesterified
cholesterol, and phospholipids surrounds a water-insoluble core of
cholesteryl esters and triglycerides, protecting the apolar lipids
from the aqueous environment. This general structural concept has
been supported by low-angle x-ray scattering studies and by other
physical methods in which a variety of probes have been used to
explore the structure of the lipoproteins. An important function of
the plasma lipoproteins is thus the solubilization and transport of
the neutral plasma lipids.
D. The Apolipoproteins
Apolipoproteins are the lipid-free protein components of the plasma
lipoproteins obtained by treating intact lipoproteins with organic
solvents, detergents, or chaotropic agents. Not all proteins
captured with lipoproteins necessarily have a role in lipid
transport. A pertinent example is the recent recognition that the
serum amyloid A proteins, acute phase reactants, are transported in
plasma bound to HDL. These low molecular weight proteins may
comprise up to 30 percent of apo-HDL in inflammatory states, but it
is doubtful that they have specific lipid transport roles.
1. Apolipoproteins A-I and A-II
Two of the apolipoproteins of interest in the present invention are
apolipoprotein A-I (apo-A-I) and apolipoprotein A-II (apo-A-II).
These are discussed below.
Apo-A-I is the major protein component of all primate HDL. It
consists of a single chain of 243 to 245 residues; does not contain
cystine, cysteine, leucine, or carbohydrate; and exists in several
isoforms. Apo-A-I has an alpha helical content of about 55 percent
in the lipid-free state, which increases to about 75 percent upon
binding phospholipid. Repeating cycles of 11 helical residues have
been identified in this apolipoprotein. It has been suggested that
these units represent a single ancestral chain which, by gene
duplication, has generated a 22-residue repeat unit. These units
have close sequence homology and are believed to represent the
lipid-binding regions of the protein.
Apo-A-I is potent activator of LCAT, a plasma enzyme that catalyzes
the conversion of cholesterol and phosphatidylcholine to
cholesteryl ester and lysophosphatidylcholine, respectively.
Specific lipid-binding regions of apo-A-I have been found to
activate LCAT, and this activity has been associated with the
property of lipid binding. As already noted, liver and intestine
synthesize apo-A-I, but their relative contributions to the total
plasma content and the factors modulating apo-A-I production are
not well defined. Typically, more than about 90 percent of plasma
apo-A-I is associated with HDL, less than about 1 percent with VLDL
and LDL, and about 10 percent or less is associated with the
lipoprotein-free fraction of plasma.
Apo-A-II is also a major constituent of human HDL, accounting for
about one-third of the total protein and about 15 percent of HDL
mass. It exists as a dimer of two identical chains of 77 residues,
which are linked covalently at the cysteine of position 6 from the
amino-terminus by a disulfide bond, and its primary structure is
known. Both the monomeric and dimeric forms of apo-A-II are capable
of reassembling with phospholipid. The alpha helix content of
apo-A-II increases from about 40 to 65 percent on interaction with
egg lecithin, and specific lipid binding segments have been
identified and synthesized.
The specific role of apo-A-II in lipid transport has not been
identified, and it is a quantitatively minor HDL apolipoprotein in
most lower species. The bulk of plasma apo-A-II is found in HDL,
with less than about 5 percent in other density classes.
2. Clinical Importance of Apo-A Lipoproteins
Measurement of the major protein constituent of HDL, apo-A, is
clinically important. The results of a number of studies have
demonstrated that apo-A-I levels are decreased in subjects with
CAD. This observation stresses the protective role of plasma
apo-A-I in this patient group.
The results of several studies suggests that by measuring the
apo-A-I and apo-A-II levels accurately, it may be possible to
predict an individual's prognosis for atherosclerosis, specifically
for CAD.
BRIEF SUMMARY OF THE INVENTION
The present invention contemplates monoclonal receptors that
immunologically binding with apolipoprotein A, but are free from
immunoreaction with and binding to apolipoproteins B, C, D and E.
Particularly preferred monoclonal receptors are monoclonal
antibodies.
A method of preparing a monoclonal antibody that immunologically
binds with an apolipoprotein A constitutes another aspect of the
invention. In accordance with that method, a host animal such as a
mammal is immunized with an human apolipoprotein A such as HDL or
VLDL. Antibody-producing cells of the immunized host are collected
as by removing the host's spleen and preparing a suspension of
splenocytes. The antibody-producing cells so collected are fused
with cells of a myeloma cell line, preferably of the same animal
species as the immunized host, and typically in the presence of a
cell fusion promoter to form hybridoma cells. The hybridoma cells
are diluted and cultured in a medium that does not support growth
of unfused myeloma cells such as HT or HAT media. Such dilution and
culturing are typically carried out at an initial concentration of
about one hybridoma cell per cell growth well. The monocolonal
antibodies produced by the culture hybridomas are thereafter
assayed for the ability to immunologically bind with apolipoprotein
A. A hybridoma whose monoclonal antibodies immunochemically bind
with apolipoprotein A is selected and cloned, and is thereafter
recovered.
The particularly preferred monoclonal antibodies are produced by
hybridomas fused from myelomas denominated P3.times.63Ag8 (ATCC
TIB9), MPC-11 (ATCC CRL 167), S/P 2-O-Ag14 (ATCC CRL 1581), and
P3.times.63Ag8.653 (ATCC CRL 1580).
The above-described method of preparing monoclonal antibodies can
include culturing the hybridoma in vitro in a suitable medium and
recovering the antibody from the hybridoma supernatant, i.e., a
cell culture system. The above method can include injecting the
hybridoma into an animal host and recovering the antibodies from
ascites fluid of the host.
The present invention also includes the monoclonal antibodies
produced by any of the above-described methods, and the
above-denominated hybridomas.
A method for assaying the presence of an apolipoprotein A such as
HDL constitutes another aspect of the present invention. Here, a
monoclonal receptor such as a whole antibody of this invention is
provided, and a known amount is admixed with an aliquot of a sample
to be assayed for the presence of an apolipoprotein A to form an
admixture. The admixture is maintained for a period of time
sufficient for the receptor to immunologically bind with an
apolipoprotein A present in the sample and form an immunoreactant.
The amount of receptor bound in the sample is determined, thereby
determining the presence and quantity of the apolipoprotein A such
as HDL in the sample.
The methods of the present invention enable the practioner to assay
for total HDL present in the sample, as well as for independently
assaying for apo-A-I and apo-A-II. The methods also enable the
assay of subsets of apo-A-I that are immunologically bound by each
of the specific monoclonal receptors of the invention.
The invention further contemplates a diagnostic system such as a
kit that includes at least one package containing as an active
ingredient an effective amount of the monoclonal receptor
(epitope-specific reagent) of this invention which, when introduced
into a sample to be assayed (for example, serum), immunologically
binding with an apolipoprotein-A such as apo-A-I or apo-A-II, but
does not react with other classes of apolipoproteins including
apolipoproteins B, C, D and E or non-apolipoproteins, i.e., it is
specific.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a photograph of a polyacrylamide gel electrophoresis
(PAGE) separation showing apolipoprotein chain specificity of a
mouse monoclonal antibody of this invention for VLDL, LDL, HDL,
apo-A-I, and apo-A-II at concentrations of 30, 20, 20, 10, and 10
micrograms, respectively. The samples were electrophoresed in
7.5-20 percent polyacrylamide gradient slab gels containing 0.1
percent sodium dodecyl sulfate (SDS). The top left is a photograph
of a Coomassie Brilliant Blue R-250 protein-stained gel before
electrophoretic transfer of the apolipoproteins to nitrocellulose.
The abbreviations are: B, apo-B; HSA, human serum albumin; E,
apo-E; C, apo-C; A-I, apo-A-I; A-II(D), apo-A-II dimers; A-II(M),
apo-A-II monomers. The remaining photographs are 24-hour
autoradiographs of identical nitrocellulose paper transfers after
incubation with the individual hybridoma ascites fluids and
.sup.125 I-goat anti-mouse Ig [0.5 micro-Curies/milliliter (micro
Ci/ml)]. The antibody numbers above each autoradiograph refer to
the apolipoprotein specificity as determined by this procedure. The
monoclonal receptors denominated A-II-1, A-I-4, A-I-7, and A-I-9
were used at dilutions of 1:5000, 1:2000, 1:3000, and 1:2000,
respectively.
FIG. 2 is a graph of data illustrating maximum binding capacity of
mouse ascites fluids containing human apo-A-I- and
apo-A-II-specific monoclonal receptors (antibodies). The upper
portion of the figure shows binding of .sup.125 I-HDL. The lower
portion of the figure is a graph showing data of binding of
.sup.125 I-apo-A-I or .sup.125 I-apo-A-II. The fluid-phase RIAs
were incubated for 18 hours at 4 degrees C. and contained .sup.125
I-HDL, .sup.125 I-apo-A-I, or .sup.125 I-apo-A-II at final
concentrations of 66.7, 33.3, and 33.3 nanograms per milliliter
(ng/ml), respectively. The coefficient of variation for all data
points was less than 10 percent.
FIG. 3 is a photograph of a 24 hour autoradiograph after gel
electrophoresis for determining apolipoprotein composition of the
antibody bound and unbound portions of .sup.125 I-HDL. The .sup.125
I-HDL was contacted and maintained in contact (incubated) with
monoclonal antibodies A-I-7, A-II-1, and A-I-4 in antibody excess
for 18 hours at 4 degrees C. followed by precipitation of the
monoclonal antibody with optimal proportions of goat anti-mouse Ig
antiserum. The precipitates containing the bound fractions (B) and
the supernatants containing the unbound fractions (UB) were
recovered, dissolved in 1 percent sodium dodecyl sulfate (SDS), and
electrophoresed along with the starting .sup.125 I-HDL (S) on a
7.5-20 percent polyacrylamide gradient pore gel in the presence of
0.1 percent SDS. The notations are as follows: HSA represents human
serum albumin; (D) represents apo-A-II dimer and (M) represents
apo-A-II monomer.
FIG. 4 is a photograph of a 24-hour radioimmunoassay following
polyacrylamide gel electrophoresis of the five HDL subfractions
isolated by density gradient ultracentrigugation in KBr. A fraction
of plasma brought to a density of 1.063 grams per milliliter (g/ml)
with KBr was centrifuged for 48 hours at 10 degrees C. in a Beckman
60 Ti ultracentrifuge rotor at 54,000 rpm. The gradient was
fractionated from the top, and the 4.0-ml fractions were dialyzed
into 0.15 molar (M) NaCl containing 0.1 percent
ethylenediaminetetracetic acid (EDTA). The upper photograph shows
the protein-staining pattern of the fractions after electrophoresis
on a 7.5-20 percent acrylamide gradient gel in the presence of 0.1
percent SDS to visualize the apolipoproteins. The bottom photograph
shows the protein-staining pattern of the same density fractions
after electrophoresis on a 4-30 percent acrylamide gradient gel in
the absence of a denaturant for separation of intact lipoprotein
particles on the basis of size. The total cholesterol of HDL
fractions 1 through 5 was 498, 321, 231, 194, and 222 micrograms
per milligram (ug/mg) protein, respectively.
FIG. 5 is a bar graph representing the relative expression of
apo-A-I and apo-A-II epitopes in HDL subpopulations. The upper
portion shows HDL fractions 1 through 5 obtained by density
gradient ultracentrifugation. The lower portion shows HDL fractions
from the PBE 94 chromatofocusing column. Data shown were obtained
from logit-transformation analysis of the competitive RIAs. The
relative epitope expression in each density or chromatofocusing HDL
subfraction was obtained by assigning for each antibody a value of
1.0 to the fraction that required the least amount of protein
(ug/ml) for 50 percent inhibition of antibody binding. For each
antibody, other density or chromatofocusing fractions were then
expressed as a fraction of that value.
FIG. 6 is a photograph of an autoradiograph of representative HDL
chromatofocusing fractions following polyacrylamide gel
electrophoresis. A fraction of plasma having a density of
1.063-1.21 g/ml (20 mg protein in 2 ml) was dialyzed into
piperazine HCl, having a pH value of 5.8, chromatographed on a PBE
94 column, and eluted with Polybuffer 74. The Polybuffer was
removed from selected 4-ml column fractions (11, 18, 27, 32, 34,
and 37) by chromatography on Sephadex G-75 with 0.15 M NaCl, 1
millimolar (mM) EDTA, and 0.02 percent NaN.sub.3 (having a pH value
of 7.4) as eluant. The top photograph is the protein staining
pattern after electrophoresis on a 7.5-20 percent acrylamide
gradient gel in the presence of 0.1 percent SDS to identify the
particle size distribution of each fraction. The bottom photograph
is of the same HDL fractions after electrophoresis on a 4-30
percent polyacrylamide gradient pore gel to identify the particle
size distribution. Total cholesterol of HDL fractions 11 through 37
above was 294, 233, 183, 174, 190, 185, and 93 micrograms/mg
protein, respectively.
FIG. 7 is a photograph of an autoradiograph of a Western blot
analysis performed to delineate apo-A-I isoforms. Increasing
amounts of each radiolabeled apolipoprotein or lipoprotein were
paired with decreasing amounts of homologous non-radioiodinated
antigen so that a constant amount of total antigen was added to
each RIA to insure that radioiodination of the ligands did not
interfere with antibody binding. Varying proportions of labeled and
nonlabeled HDL (FIG. 7A) or soluble apolipoprotein (FIG. 7B) were
incubated with each monoclonal antibody for 18 hours at 4 degrees
C. Constant concentrations of 133.3 nanograms per milliliter
(ng/ml) HDL (.sup.125 I-HDL plus homologous HDL), and 33.3 ng/ml
apo-A-I or apo-A-II (.sup.125 I-apo-A-I plus homologous apo-A-I, or
.sup.125 I-apo-A-II plus homologous apo-A-II) were maintained.
In panel B, monoclonal antibody A-II-1 was incubated with apo-A-II,
and monoclonal antibodies A-I-4, A-I-7 and A-I-9 were incubated
with apo-A-I. Results were plotted as the mean counts per minute
(cpm) recovered in the precipitate after reaction with an optimal
proportion of goat anti-mouse Ig antiserum versus the percent of
.sup.125 I-antigen added. For the linear regression, correlation
coefficients were equal to or greater than 0.995 for all antigen
and antibody combinations shown. The linearity and concordance
indicated by the high correlation coefficients (r greater than or
equal to 0.995) identified that each antibody reacted with each
labeled and nonlabeled antigen pair with the same apparent
affinity.
In separate studies, .sup.125 I-HDL labeled by two different
methods, namely with either the Bolton-Hunter reagent or by the
lactoperoxidase procedure were found to be equivalent in their
reactivity for each antibody. In addition, a 1:16 dilution of a
polyvalent antisera obtained from a rabbit hyperimmunized with
human HDL precipitated 100 percent of 100 ug/ml of .sup.125 I-HDL.
Therefore, radioiodination of the antigens did not interfere with
the immunoreactivity or account for the inability of these
antibodies to bind 100 percent of the labeled antigen.
FIG. 8 is a graph showing radioiodination of .sup.125 I-HDL and
.sup.125 I-apolipoproteins. The antigens were subjected to mild
dissociating conditions that included heat and exposure to
detergents to insure that the epitopes recognized by these
antibodies were exposed and available for reaction with antibody.
Limiting amounts of monoclonal antibodies (Mab) A-I-4, A-I-7, A-I-9
and A-II-1 were added to .sup.125 I-HDL (final concentration 133
ug/ml) that had been incubated at either 4 degrees C. or 52 degrees
C. The isolated apolipoproteins, .sup.125 I-apo-A-I and .sup.125
I-apo-A-II were similarly heated before exposure to antibody.
Again, no significant increases in antibody binding were observed.
In fact, the binding of Mab A-I-7 to .sup.125 I-apo-A-I and
antibody Mab A-II-1 to .sup.125 I-apo-A-II was reduced by
heating.
To determine if higher temperatures during rather than before
antibody exposure would increase binding, reaction mixtures
containing .sup.125 I-HDL and antibody were incubated at 4 degrees
C., 24 degrees C., 37 degrees C., or 52 degrees C. for up to 18
hours. In no instance did incubation at 37 degrees C. or 52 degrees
C. increase binding above that observed at 4 degrees C. or 24
degrees C. and, as noted above, the binding of Mab A-I-7 and Mab
A-II-1 to .sup.125 I-HDL was reduced at 52 degrees C.
FIG. 9 is a photograph of an autoradiograph showing the
polyacrylamide gel electrophoresis of the individual .sup.125 I-HDL
ligands. Apolipoproteins and the molecular weights indicated in
FIG. 9 were obtained from other lanes of the same gels containing
molecular weight markers and unlabeled HDL from a pooled plasma
source after staining the gels for protein with Coomassie Brilliant
Blue R 250. The sex of the donor, and the specific activity and
acid precipitability of the .sup.125 I-HDL ligands, respectively
were: LK, female, 5.6 disintegrations per minute per picogram
(dpm/pg) and 99.2 percent; AD, female, 6.1 dpm/pg and 99.2 percent;
AL, female, 7.1 dpm/pg and 99.2 percent; JR, female, 6.2 dpm/pg and
99.3 percent; EW, male, 6.0 dpm/pg and 99.1 percent; PM, male, 5.8
dpm/ug and 99.0 percent; GM, male, 4.5 dpm/pg and 98.7 percent; and
CW, male 6.6 dpm/pg and 99.3 percent.
FIG. 10 is a graph showing the binding capacities of the apo-A
antibodies for .sup.125 I-HDL ligands obtained from eight unrelated
individuals. The percent of .sup.125 I-HDL that was maximally bound
from a pooled HDL source was consistently greatest with Mab A-I-7
(greater than 50 percent), less with antibody A-I-4 (40-50 percent)
and lowest with Mab A-I-9 (30-40 percent). This pattern of
reactivity was duplicated with the eight .sup.125 I-HDL ligands
(final concentration, 15 ng/ml) from the individual donors (FIG.
10A, B and C). Each line represents a different .sup.125 I-HDL
ligand. Data from female donors is indicated with solid lines,
males in hatched lines. No consistent sex differences were noted.
FIG. 10A, RIA with antibody A-I-4; FIG. 10B, RIA with antibody
A-I-7; FIG. 10C, RIA with antibody A-I-9; 10D, RIA with antibodies
A-I-4, A-I-7 and A-I-9 at a ratio of 1:16:8; FIG. 10E, RIA with
antibody AII-1; and FIG. 10F, RIA with antibodies A-I-4, A-I-9 and
A-II-1 at a ratio of 1:16:8.
DETAILED DESCRIPTION OF THE INVENTION
I. GENERAL DISCUSSION
The term "receptor" as used herein is meant to indicate a
biologically active molecule that immunologically binds to (or
with) an antigen. Such binding typicaly occurs with an affinity of
about 10.sup.5 liters per mole and is specific interaction of the
epitope of the antigen with the Fab portion of the receptor.
A receptor molecule of the present invention is an intact antibody
protein, substantially intact antibody or an idiotype-containing
polypeptide portion of an antibody (antibody combining site) in
subtantially pure form, such as in ascites fluid or serum of an
immunized animal. The terms "receptor" and "monoclonal receptor"
are used interchangeably herein in a generalized sense for a
molecular entity that contains the antibody combining site of a
monoclonal antibody of this invention. The terms "antibody",
"monoclonal antibody" and "Mab" are utilized interchangeably herein
for a whole antibody of this invention.
The term "ligand" as used herein is meant to indicate a molecule
that contains a structural part that is immunologically bound by a
specific receptor to form an immunoreactant. A ligand used in the
present invention is an apolipoprotein A-containing entity such as
a radioiodinated HDL antigen adhered to a solid matrix as described
in the radioimmunoassay described hereinafter.
Biological activity of a receptor molecule is evidenced by the
immunologic reaction of the receptor to its antigenic ligand upon
their admixture in an aqueous medium to form an immunoreactant, at
least at physiological pH values and ionic strengths. Preferably,
the receptors also bind to the antigenic ligand within a pH value
range of about 5 to about 9, and at ionic strengths such as that of
distilled water to that of about one molar sodium chloride.
Idiotype-containing polypeptide portions (antibody combining sites)
of antibodies are those portions of antibody molecules that contain
the idiotype and bind to the ligand, and include the Fab, Fab' and
F(ab').sub.2 portions of the antibodies. Fab and F(ab').sub.2
portions of antibodies are well known in the art, and are prepared
by the proteolytic reaction of papain and pepsin, respectively, on
substantially intact antibodies by methods that are well known. See
for example, U.S. Pat. No. 4,342,566 to Theofilopolous and Dixon.
Fab' antibody portions are also well known and are produced from
F(ab').sub.2 portions followed by reduction of the disulfide bonds
linking the two heavy chain portions as with mercaptoethanol, and
then alkylation of the resulting protein mercaptan with reagent
such as iodoacetamide. Intact antibodies are preferred, and will be
utilized as illustrative of the receptor molecules of this
invention.
A "monoclonal receptor" (Mab) is produced by clones of a single
cell called a hybridoma that produces (secretes) but one kind of
receptor molecule. "Polyclonal" antibodies (Pab) are antibodies
produced by clones derived from different cells that secrete
different antibodies that bind to a plurality of epitopes of the
immunogenic molecule. The preparation of Pab is discussed
hereinafter as part of the production of Mabs.
The hybridoma cell is fused from an antibody-producing cell and a
myeloma or other self-perpetuating cell line. Such receptors were
first described by Kohler and Milstein, Nature, 256, 495 (1975),
which description is incorporated herein by reference. Monoclonal
receptors are typically obtained from the supernatants of hybridoma
cell cultures, or, alternatively, from ascites fluid or other body
fluids obtained from non-human, warm blooded host animals into
which the hybridoma cells were introduced.
Antibodies are secreted by specialized cells called plasma cells
and to a quantitatively lesser degree by their precursor B cells
(bone marrow-derived lymphocytes). Each B cell or plasma cell
secretes one type of antibody having a single specificity, so
various antibodies of different specificites are each secreted by
different B cells and their derivative plasma cells. These B cells
may be cloned to provide a source of single antibodies. However,
these cells die in a few days in culture media and must be made
relatively "immortal" so that a supply of the desired antibodies
may be obtained. This is accomplished by removing the B cells and
plasma cells from the animal, typically from the spleen, fusing
them with a cancerous or myeloma cell to form a somatic cell hybrid
(hybridoma), and then cloning and propagating the hybridoma.
The antibody-producing cells that are employed may be obtained from
a non-human host animal immunized by injection of an immunogen, in
this instance a human apolipoprotein A, typically followed by one
or more booster injections with the same immunogen. The spleen is
isolated after a sufficient time period has elapsed for the host to
produce antibodies, this is typically about one month to about
three months after the first immunization.
Non-human, warm blooded animals usable in the present invention as
hosts may include poultry (such as a chicken or a pigeon), a member
of the ratitae bird group (such as an emu, ostrich, cassowary or
moa) or a mammal (such as a dog, cat, monkey, goat, pig, cow,
horse, rabbit, guinea pig, rat, hamster or mouse). Preferably, the
host animal is a mouse or rabbit.
It is preferred that a myeloma cell line be from the same species
as the antibody-producing cells. Therefore, fused hybrids such as
mouse-mouse hybrids [Shulman et al., Nature, 276, 269 (1978) or
rat-rat hybrids (Galfre et al., Nature, 277, 131 (1979)] are
typically utilized. However, some rat-mouse hybrids have also been
successfully used in forming hybridomas [Goding, "Production of
Monoclonal Antibodies by Cell Fusion", in Antibody as a Tool,
Marchalonis et al. eds., John Wiley & Sons Ltd., 273-289
(1982), hereinafter Marchalonis et al.]. Suitable myeloma lines for
use in the present invention include MPC-11 (ATCC CRL 167),
P3.times.63-Ag8.653 (ATCC CRL 1580), Sp 2/0-Ag14 (ATCC CRL 1581),
P3.times.63Ag8U.1 (ATCC CRL 1597), Y3-Ag12.3 (deposited at
Collection Nationale de Cultures de Microorganisms, Paris, France,
number I-078 and P3.times.63Ag8 (ATCC TIB9). Myeloma line
P3.times.63-Ag8.653 is preferred for use in the present
invention.
Monoclonal anti-apolipoprotein A receptors were formed as described
herein from murine (mouse) splenocytes fused with murine myeloma
cells. The polyclonal anti-apolipoprotein A antibodies described
were formed from rabbits. The hybridomas that produce the
monoclonal anti-apo-A-I and anti-apo-A-II receptors of this
invention were given the following designations for reference
purposes and were deposited with the American Type Culture
Collection (ATCC), Rockville, Maryland on Mar. 5, 1985 under the
following ATCC accession numbers.
______________________________________ ATCC Accession Hybridoma Mab
Number ______________________________________ HA61 H112F3.1A11
A-II-1 HB 8743 611 AV63C2.1F1 A-I-4 HB 8744 HA60 HA22GF.5F8 A-I-7
HB 8745 HA62 HA227A2.7D3 A-I-9 HB 8741
______________________________________
Receptors are typically utilized along with an indicator labeling
means or "indicating group" or a "label". The indicating group or
label is utilized in conjunction with the receptor as a means for
determining the extent of a reaction between the receptor and the
antigen.
The terms "indicator labeling means", "indicating group" or "label"
are used herein to include single atoms and molecules that are
linked to the receptor or used separately, and whether those atoms
or molecules are used alone or in conjunction with additional
reagents. Such indicating groups or labels are themselves
well-known in immunochemistry and constitute a part of this
invention only insofar as they are utilized with otherwise novel
receptors, methods and/or systems.
The indicator labelling means can be a fluorescent labelling agent
that chemically binds to antibodies or antigens without denaturing
them to form a fluorochrome (dye) that is a useful
immunofluorescent tracer. Suitable fluorescent labelling agents are
fluorochromes such as fluorescein isocyanate (FIC), flourescein
isothiocyanate (FITC), dimethylamino-naphthalene-S-sulphonyl
chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC),
lissamine, rhodamine B200 sulphonyl chloride (RB 200 SC) and the
like. A description of immunofluorescence analysis techniques is
found in Marchalonis et al., "Immunofluorescence Analysis",
189-231, supra, which is incorporated herein by reference.
The indicating group may also be an enzyme, such as horseradish
peroxidase (HRP) or glucose oxidase, or the like. Where the
principal indicating group is an enzyme such as HRP or glucose
oxidase, additional reagents are required to visualize the fact
that a receptor-ligand complex has formed. Such additional reagents
for HRP include hydrogen peroxide and an oxidation dye precursor
such as diaminobenzidine. An additional reagent useful with glucose
oxidase is 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid)
(ABTS).
An exemplary radiolabelling agent is a radioactive element that
produces gamma ray emissions. Elements which themselves emit gamma
rays, such as .sup.124 I, .sup.125 I, .sup.128 I, .sup.131 I,
.sup.132 I, and .sup.51 Cr represent one class of gamma ray
emission-producing radioactive element indicating groups.
Particularly preferred is .sup.125 I. Another class of useful
indicating groups are those elements such .sup.11 C, .sup.18 F,
.sup.15 O and .sup.13 N which themselves emit positrons. The
positrons so emitted produce gamma rays upon encounters with
electrons present in the animal's body. Also useful is a beta ray
emitter, such as .sup.111 indium.
A preferred radioactively labeled monoclonal receptor may be
prepared by culturing hybridoma cells in a medium containing
radioactive amino acids, as is well known, as well as by isolating
the monoclonal receptor and then labelling the monoclonal receptor
with one of the above radioactive elements as described in U.S.
Pat. No. 4.,381,292 to Bieber and Howard, incorporated herein by
reference.
Specific indicating means linked to reagents that react with the
receptors of this invention are discussed hereinafter.
Four previously identified monoclonal antibodies that bind to
apolipoproteins A of human plasma HDL were obtained from their
respective hybridomas and characterized. Each of these antibodies
was specific for the apolipoproteins of human HDL, based on binding
to delipidated and isolated apolipoproteins of HDL after transfer
to nitrocellulose and binding of the soluble apolipoproteins in
fluid phase.
The vast majority of the monoclonal antibodies obtained by
immunization of mice with native human HDL were specific for human
apo-A-I, suggesting greater immunogenicity of human apo-A-I for
BALB/c mice. This difference in immunogenicity between
apolipoproteins A-I and A-II was observed also when the isolated
apolipoproteins were used as immunogens. Thus, of the four
antibodies characterized in this study, three were specific for
three separate epitopes on apo-A-I. Only a single apo-A-II-specific
antibody was obtained and characterized.
Human plasma HDL of density 1.063-1.21 g/ml represents a
heterogenous mixture of HDL particles that differ with respect to
both lipid and protein composition. Using the hybridoma-produced
antibodies of this invention that define apo-A-I-specific and
apo-A-II-specific epitopes, immunochemical heterogeneity of HDL was
clearly evident.
Solid-phase immunoassays permitted analysis of antibody
specificity, but with fluid-phase assays it was possible to analyze
the heterogeneity of molecules with respect to expression of
individual epitopes. It was found that not all HDL particles
expressed the defined apo-A-I and apo-A-II epitopes that could be
bound by a given apo-A-I-specific antibody. The unbound HDL
contained apo-A-I and HDL, indicating heterogeneity of epitope
display by apo-A-I on different particles (FIG. 2).
The existence of at least two types of HDL; i.e., particles
containing apo-A-I and apo-A-II, and particles containing apo-A-I
but no apo-A-II, was verified with monoclonal antibody A-II-1.
Whereas this apo-A-II-specific antibody bound only a subset of
total HDL, it did bind all apo-A-II (FIG. 2). The unbound HDL was
devoid of detectable apo-A-II, appearing to contain only apo-A-I
(FIG. 3). Thus, all HDL particles possessing an apo-A-II chain
expressed this A-II epitope.
Heterogeneity of epitope expression by isolated apo-A-I was readily
evident. None of the apo-A-I-specific antibodies was able to bind
all apo-A-I molecules, either as HDL or soluble apo-A-I. The
inability of each of the anti-apo-A-I antibodies to identify its
complementary epitope on all A-I apolipoprotein chains was
examined. First, technical issues were excluded such as affinity,
quantity of available antibody, or radioiodination of the ligands.
Second, it was demonstrated that the antibodies did not selectively
bind different apo-A-I isoforms. Third, the use of dissociating
conditions (e.g., heat and nonionic detergents) designed to
mobilize and expose cryptic epitopes of the apolipoprotein on
either HDL or the isolated soluble state did not result in a
significant increase in the capacity of antibody to bind all
molecules.
Immunochemical heterogeneity of epitope expression by apo-A-I
organized on HDL was further supported by the demonstration that
the combination of three apo-A-I-specific antibodies could bind a
greater relative proportion of HDL than any single antibody (Table
I, hereinafter). Thus, the three apo-A-I epitopes recognized by the
anti-apo-A-I differed, and some particles existed that expressed
only one or another of these three epitopes.
Apo-A-I occurs predominantly, if not virtually entirely, associated
with lipid. The heterogeneity of epitope expression was determined
by lipoprotein-associated apo-A-I.
The possibility that these antibodies distinguished individual
epitopes of apo-A-I that were present or not on the basis of
allotypic (genetically determined individual differences) or sex
differences in apo-A-I was examined. For those studies, HDL was
isolated from single individual normo-lipidemic subjects. Compared
with pooled plasma, HDL isolated from the plasmas of four unrelated
normal donors of each sex had similar patterns of heterogeneous
epitope expression.
Because these antibodies did not appear to identify allotypic or
sex differences between individuals in their apo-A-I molecules, the
existence of multiple apo-A-I genes was considered since
differential gene regulation, or differential sites or rates of
metabolism might account for the observed heterogeneity of apo-A-I.
It appears that there is a single apo-A-I gene (Karathanasis et
al., Proc. Natl. Acad. Sci, USA, 80, 6147-6151 (1983). Alternative
splicing of the gene has not been described but has not been
examined.
Also, each of the epitopes is expressed by the different apo-A-I
charge isoforms. Thus, it was determined whether the apo-A-I
antibodies selectively distinguished apo-A-I on HDL that was
derived from different known major synthetic sources such as the
liver and the intestine. Thoracic duct lymph was used as an
enriched source of intestinal apo-A-I. The medium from human Hep G2
hepatoma cultures provided a source of pure hepatic apo-A-I.
Both the hepatic and intestinal apo-A-I contained molecules
expressing epitopes bound by antibodies A-I-7 and A-I-9; i.e.,
A-I-7 and A-I-9 epitopes. In addition, plasma VLDL fractions that
provide a source of both hepatic and intestinal (chylomicron
remnant) apo-A-I expressed only the epitope bound by antibody
A-I-9. Therefore, the results are not consistent with epitope
differences based on different synthetic sources.
The hypothesis that the three apo-A-I epitopes distinguish between
molecules differently organized on different HDL particles was in
part substantiated by separation of HDL on the basis of the
physical properties of density and charge. However, because density
and chromatofocusing fractions differed quantitatively but not
absolutely in the expression of individual apo-A-I epitopes, these
methods did not entirely resolve the responsible subsets of HDL.
Rather, they facilitate only enrichment or relative depletion of
particles expressing individual apo-A-I epitopes.
Physical fractionation of native HDL is unlikely to result in
complete segregation of specific apo-A-I epitopes expressed by
apo-A-I on HDL, since HDL particles appear not to exist that
exclusively contain only apo-A-I organized in a single
conformational format. However, immunochemical separation may
provide new information. Recent studies of immunopurified HDL have
shown that ultracentrifugation can alter HDL structure and suggest
that additional studies of the immunochemical properties of HDL
should be directed at the HDL particle as it exists in plasma
[McVicar et al., Proc. Natl. Acad. Sci. USA, 81, 356-1360
(1984)].
There is no reason to assume that conformational variation will be
identical for lipid-free and lipid-associated apo-A-I. For example,
protein-protein interactions resulting in the formation of soluble
oligomers of lipid-free apo-A-I have been observed in preliminary
studies to influence the degree of expression of epitope A-I-4;
whereas protein-lipid or lipoprotein interactions appear to have a
similar influence. Studies of the HDL density and chromatofocusing
subfractions demonstrate that apo-A-I is not organized the same on
different HDL particles.
The lighter, larger cholesterol-rich HDL (HDL.sub.2 -like) that are
enriched in apo-A-I relative to other apolipoproteins are rich in
apo-A-I that express predominantly the A-I-9 epitope. In contrast,
the more dense, smaller, cholesterol-poor HDL which contain
apo-A-II and other minor apolipoproteins are rich in apo-A-I that
express predominantly the A-I-7 epitope. Because these two types of
HDL particles may represent different metabolic states of HDL, the
different apo-A-I conformations on HDL may serve to direct HDL
particles to their proper enzymatic or cellular sites.
Some methods of quantitative analysis of plasma HDL have employed
immunologic assays for apolipoproteins A-I or A-II. The
immunochemical properties of these apolipoproteins as evident from
analysis with polyclonal antibodies have indicated the existence of
unusual and distinctive properties. The reactivity of
apo-A-II-specific antisera is for the most part comparable for
apo-A-II whether in free solution or associated with HDL [Mas et
al., Biochemistry, 14, 4127-4131 (1975)].
However, the HDL density class is composed of at least two types of
HDL particles; i.e., those possessing both apo-A-I and apo-A-II,
and those containing apo-A-I, but no apo-A-II. Because all HDL
particles appear to contain apo-A-I, immunologic analyses of
apo-A-I have been herein used in quantitating total plasma HDL. A
caveat is the difference in the ability of various antisera to
detect all apo-A-I in HDL or plasma. The reasons offered for this
discrepancy have centered around the hypothesis that some apo-A-I
epitopes on native HDL are sterically occult.
As noted before, hybridoma cell lines that secrete human
HDL-binding monoclonal antibodies were prepared to examine this
molecular aberration, to determine if the apparent immunochemical
heterogeneity of HDL and its apolipoproteins is valid, and to
obtain precise immunochemical reagents that permit quantitation of
all HDL particles in plasma as well as defined subsets of HDL.
Three mouse monoclonal antibodies (Mab's) specific for human
apolipoprotein (apo) A-I and one specific for human apo-A-II that
were prepared have been highly characterized and their binding of
high density lipoprotein (HDL) particles in solution was
determined. The apo-A-II-specific antibody bound 85 percent of
.sup.125 I-HDL and 100 percent of soluble .sup.125 I-apo-A-II.
However, none of the apo-A-I-specific antibodies bound more than 60
percent of either HDL or soluble apo-A-I.
These results suggested the existence of intrinsic immunochemical
heterogeneity of apo-A-I both as organized on HDL as well as in
free apo-A-I in solution. The validity of this observed
heterogeneity was supported by demonstrating that (i) increased
binding of HDL occurred when each of the apo-A-I antibodies was
combined with another to form an oligoclonal antibody mixture, and
(ii) approximately 100 percent binding of HDL occurred when any two
apo-A-I antibodies (antibodies denominated A-I-4 and A-I-7; i.e.,
Mab A-I-4, of hybridoma 611 AV63C2.1F1 (ATCC HB 8744) and Mab
A-1-7, of hybridoma HA60 HA22GF.5F8 (ATCC HB 8745) were combined
with the single apo-A-II antibody Mab A-II-1 produced by hybridoma
HA61 H112F3.1A11 (ATCC HB 8743).
To understand the basis for the heterogeneity of the expression of
apo-A-I epitopes on HDL, two hypotheses were examined. The first
hypothesis that these apo-A-I antibodies distinguished apo-A-I
molecules from different synthetic sources was not substantiated.
Two of the antibodies bound epitopes on apo-A-I molecules in both
thoracic duct lymph as an enriched source of intestinal HDL and the
culture supernatants of the hepatic cell line Hep G2 as a source of
hepatic HDL.
From the assays of this invention, it has been shown that the
monoclonal antibodies identified differences in the expression of
apo-A-I on HDL subpopulations that were distinguished on the basis
of size or net particle charge; i.e., organizational heterogeneity
appeared to provide the best available explanation for the
immunochemical heterogeneity of apo-A-I in HDL.
Relative differences in the expression of three distinct apo-A-I
epitopes were demonstrated in HDL subpopulations obtained by either
density gradient ultracentrifugation or chromatofocusing. In light
of these studies, it is concluded that there is intrinsic
heterogeneity in the expression of intramolecular loci representing
the apo-A-I epitopes identified by the monoclonal antibodies of
this invention. Such heterogeneity must be considered in analysis
of the biology and physiology of apo-A-I and lipoprotein particles
bearing this chain as well as any attempt to immunologically
quantitate or characterize HDL.
II. ASSAY METHODS
The monoclonal receptor molecules of the present invention are
particularly useful in methods for assaying the presence and amount
of an apolipoprotein A such as that of HDL in a sample to be
assayed such as blood, serum or plasma. As noted hereinafter, the
presence and amount of HDL and soluble apolipoproteins A may also
be assayed in other body fluids such as lymph, and in in vitro
materials such as hepatic cell cultures and the like.
Useful solid and liquid phase assay methods are discussed
hereinafter. However, the invention is not so limited. Further,
while the particularly described assay methods utilize a
radioactive element and determination of receptor bound in
apolipoprotein A/receptor-containing immunoreactants
(radioimmunoassay; RIA), the present invention is also not
specifically limited to such assays. Additional assay methods are
described hereinbelow with particular emphasis on solid phase
immunoassay methods.
Solid phase assay methods are comprised of an antigen or a receptor
of this invention affixed to a solid matrix as a solid support.
The antigen or receptor is typically affixed to the solid matrix by
adsorption from an aqueous medium, although several modes of
adsorption, as well as other modes of affixation, well known to
those skilled in the art may be used. Exemplary of such modes are
the reaction of the receptor or antigen with the reactive carboxyl
functionality produced by the reaction of cyanogen bromide with
glucose-containing matrices such as cross-linked dextrans or
cellulosics, glutaraldehyde linking as discussed hereinafter in
conjunction with latex particles, and the like.
Useful solid matrices are well known in the art. Such materials
include the cross-linked dextran available under the trademark
SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose;
beads of glass; polystyrene beads about 1 micron to about 5
millimeters in diameter available from Abbott Laboratories of North
Chicago, Ill.; polyvinyl chloride, polystyrene, cross-linked
polyacrylamide, nitrocellulose or nylon-based webs such as sheets,
strips or paddles; or tubes, plates or the wells of a microtiter
plate such as those made from polystyrene or polyvinylchloride.
Latex particles useful in agglutination-type assays are also useful
solid matrices. Such materials are supplied by the Japan Synthetic
Rubber Company of Tokyo, Japan, and are described as
carboxy-functional particles dispersed in an anionic soap. Typical
lots of such particles have an average diameter of 0.308 microns,
and may have an average carboxy-functional group distribution of
about 15 to about 30 square Angstroms per carboxy group.
Prior to use, the particles are reacted with a diamine such as
1,3-diamino-2-propanol to form a plurality of amide bonds with the
particle carboxy groups while maintaining free amine groups. The
free amines are thereafter reacted with a dialdehyde such as
glutaraldehyde and the receptor or antigen to form Schiff base
reaction products. The Schiff base reaction products are thereafter
reduced with a water-soluble reductant such as sodium borohydride
to provide a useful solid support.
Those skilled in the art will understand that there are numerous
methods for solid phase immunoassays that may be utilized herein.
Exemplary, useful solid phase assays include enzyme-linked
immunosorbant assays (ELISA) and fluorescence immune assays (FIA),
in addition to the specifically discussed RIA. However, any method
that results in a signal imparted by the reaction of apolipoprotein
A with a receptor of this invention is considered. Each of those
assay methods may employ single or double antibody techniques in
which an indicating means is utilized to signal the immunoreaction,
and thereby the binding of an apolipoprotein A that is to be
assayed with a receptor of this invention. Exemplary techniques may
be found explained in Maggio, Enzyme Immunoassay, CRC Press,
Cleveland, Ohio (1981); and in Goldman, Fluorescent Antibody
Methods, Academic Press, New York, N.Y. (1980).
Broadly, the presence of an apolipoprotein A such as that of human
HDL in a sample to be assayed includes the following steps.
(a) An effective amount of a monoclonal receptor of this invention
whose antibody combining site immunoreacts with and binds to human
apolipoprotein A, but is free from immunoreaction with and binding
to human apolipoproteins B, D, D, and E, or other known proteins or
ligands is provided. The receptor is also free from immunological
binding with any other protein or ligand found in plasma or serum
of normal individuals. This is typically accomplished by using an
aliquot of an appropriate hybridoma supernatant or ascites.
The effective amount of receptor will differ, inter alia, with the
particular receptor used, and with the particular assay method
utilized, as is well known. Also well known is the ease with which
the effective amount may be determined using standard laboratory
procedures by one skilled in preparing such assays.
(b) A known amount of the receptor is admixed with aliquot of a
sample to be analyzed for the presence of an apolipoprotein A such
as that of human HDL, to form an admixture. The admixture so formed
may be a liquid admixture as in the liquid phase RIA described
hereinafter, or that admixture may be a solid/liquid admixture as
where a solid support is utilized.
(c) In either event, the admixture so formed is maintained for a
predetermined period of time from minutes to hours, such as about
90 minutes to about 16-20 hours at a temperature of about 4 degrees
to about 45 degrees C. that is sufficient for the receptor to
immunoreact with and bind to apo-A present in the sample, and form
an immunoreactant.
(d) The amount of receptor bound in the immunoreactant is then
determined to thereby determine the amount of apo-A as in HDL
present in the sample. That amount may be zero, thereby indicating
that no apo-A is present in the sample, within the limits that may
be detected.
Individual receptors of this invention may be utilized or the
individual receptor molecules may be admixed for use. The
particular receptor or combination to use for assaying for the
presence of a particular apo-A-containing molecule may be
determined from the data of the RESULTS section (IV) that follows.
Thus, one may select a receptor that immunoreacts with and binds to
apolipoprotein A-I, or A-II, or both A-I and A-II.
For example, if it is desirable to analyze only apo-A-II molecules,
the receptor of choice (A-II-1) is that produced by the hybridoma
denominated HA61 H112F3.1A11 (ATCC HB 8743). If only apo A-I
subsets are desired, then each of the three different receptors
(A-I-4, A-I-7 or A-I-9) provide a reagent for each subset defined
by these receptors. Where the total HDL present in a sample is
desired, a mixture containing A-II-1 receptors plus receptors
produced by any two of the other three hybridomas of this
invention, i.e., receptors denominated A-1-4, A-1-7, or A-1-9 (from
hybridomas 611 AV63C2.1F1, ATCC HB 8744; HA60 HA22GF.5F8, ATCC HB
8745; or HA62 HA227A2.7D3, ATCC HB 8741; respectively).
In one embodiment of the above, general method, an apolipoprotein A
that is bound by the receptor used in the method such as human HDL
is provided affixed to a solid matrix as a solid support antigen.
The admixture of step (c), above, is present as a liquid admixture,
and is admixed with the solid support to form a solid/liquid phase
admixture. That solid/liquid phase admixture is maintained for a
predetermined time period such as about 16-18 hours at 4.degree. C.
that is sufficient for the receptor molecules in the liquid
admixture to immunoreact with and bind to the antigen and form an
immunoreactant. The solid and liquid phases are separated, and the
solid phase is usually rinsed to remove non-specifically bound
receptor molecules. The amount of receptor molecules bound
(specifically) in the immunoreactant is then determined.
Where the sample is free from apo-A molecules, the amount of
receptor in the solid phase immunoreactant is relatively high.
Conversely, where there is a relatively large amount of apo-A
molecules as where there is a large amount of human HDL present in
the sample, the amount of bound receptor is relatively lower.
Quantitative comparison of the result obtained with separately
obtained control results provides quantitation of the amount of
apo-A in the sample.
The determination of the amount of receptor bound may be by means
of an indicating means-containing reagent that reacts with the
bound receptor but does not react with the solid support antigen
such as .sup.125 I-labeled goat anti-mouse Ig, where the receptors
are mouse antibodies. The receptor may itself include a linked
indicating means such as a radioactive element or an enzyme that
signals the formation of an immunoreactant, or an added ligand
specific for another indicating receptor.
In another embodiment of the general method, the sample to be
assayed may be affixed to a solid matrix as a solid support antigen
prior to forming the admixture described in the general method in
step (b), above. It is understood that while several entities from
the sample may become affixed to the solid support, the useful
solid support antigen includes those entities such as HDL that
contain apolipoprotein A.
The sample may be affixed in several ways as are known, and
described previously. One exemplary method is by adsorption as is
discussed in connection with the solid phase RIA described
hereinafter.
When the sample is affixed to the solid support prior to formation
of the admixture of step (b), the admixture formed in that step is
a solid/liquid admixture in which the solid phase is the solid
support antigen and the liquid phase is the aqueous composition
that includes a receptor of this invention. The solid/liquid phase
admixture is maintained as already described, and is separated
prior to determining the amount of receptor that is bound in the
immunoreactant. The separated solid phase is typically rinsed prior
to that determination being made, as discussed before.
A convenient way to determine the amount of receptors bound in the
above-described method utilizes an indicating means-containing
reagent that reacts with the bound receptors to form a bound
reaction product, but does not bind to the solid support antigen.
The indicating means of the reagent signals the presence of the
bound receptor.
A known amount of a liquid composition including such a reagent is
admixed with the separated solid phase to form a second
solid/liquid admixture. That admixture is maintained for a
predetermined period of time sufficient for the reagent to react
with the bound receptor of the immunoreactant and form a bound
reaction product.
The solid and liquid phases are thereafter separated as described
before and the amount of bound reaction product is determined.
In the case of the specifically disclosed RIA, the reagent was goat
anti-mouse antibodies that immunoreact with and bind to the
mouse-derived receptor molecules. That reagent included linked
iodine-125 atoms (indicator) whose gamma radiation provided the
signal that bound receptor was present in the solid phase, and
consequently that an human apolipoprotein A was present in the
sample.
The indicating means may also be an enzyme or a fluorescent
molecule that is linked to the reagent for use in an enzyme-linked
immunosorbent assay (ELISA) or fluorescence immunoassay (FIA),
respectively.
For an ELISA, typically used enzymes linked to the reagent as a
signalling means include horseradish peroxidase, alkaline
phosphatase and the like. Each of those enzymes is used with a
color-forming reagent or reagents (substrate) such as hydrogen
peroxide and o-phenylenediamine; and p-nitrophenyl phosphate,
respectively.
Enzyme-linked antibody (conjugate) reagents of one animal raised to
the antibodies of another animal such as peroxidase-linked rabbit
anti-goat and goat anti-mouse antibodies, as well as
phosphatase-linked rabbit anti-goat, and rabbit anti-mouse
antibodies are commercially available from several suppliers such
as Sigma Chemical Company of St. Louis, Mo. Those indicating
means-containing reagents may be used where the receptor utilized
has an Fc portion of the "other animal", e.g., goat and mouse.
Similar assays may also be carried out using fluorochrome dyes
linked to an antibody as an indicating means-containing reagent to
signal the presence of receptors bound in an immunoreaction product
The fluorochrome dye is typically linked by means of an
isothiocyanate group to form the conjugate. Exemplary fluorochrome
dyes include fluorescein isothiocyanate (FITC), rhodamine B
isothiocyanate (RITC) and tetramethylrhodamine isothiocyanate
(TRITC). Conjugates such as FITC-linked rabbit anti-mouse, goat
anti-mouse, goat anti-rabbit and sheep anti-mouse antibodies are
commercially available from several sources such as Sigma Chemical
Company.
In addition to the RIA, ELISA and FIA techniques for determining
the presence of receptors of this invention bound to an antigen in
an immunoreactant, other well known techniques are also available.
In one technique, protein A of Staphylococcus aureus linked to a
signalling means such as .sup.125 I is utilized to determine the
presence of the receptors bound to the solid support.
In another technique, biotin linked to an antibody reagent is
utilized to signal the presence of the immunoreactant in
conjunction with avidin that is itself linked to a signalling means
such as horseradish peroxidase. Biotin-linked antibody conjugates
such as biotin-linked goat anti-rabbit, goat anti-mouse and rabbit
anti-goat IgG's are commercially available from Polysciences, Inc.
of Warrington, PA. Avidin-FITC, avidin-RITC, avidin-peroxidase and
avidin-alkaline phosphatase are also available commercially from
Polysciences, Inc. for use with the biotin-linked antibody
conjugates to provide the signal. Still other techniques are well
known to those skilled in this art.
In a still further embodiment of the before-described method, the
admixture formed in step (b) is a liquid admixture; i.e., the
sample to be assayed and the receptors are admixed in a liquid
composition that is typically aqueous. That admixture includes a
known amount of a radiolabeled apoliprotein A-containing
competitive antigen such as HDL, or free human apo-A-II or
apo-A-I.
Where such a liquid phase admixture is used, the amount of receptor
bound in the immunoreactant may be determined by admixing an excess
of an antibody that immunoreacts with, binds to and precipitates
the receptors with the liquid phase admixture, to form a second
liquid phase admixture. The precipitating antibody so used does not
immunoreact with, bind to or precipitate the apolipoprotein being
assayed for or the competitive antigen. An exemplary antibody is
the .sup.125 I-goat anti-mouse Ig used in a RIA described
hereinafter.
The second liquid phase admixture is maintained for a predetermined
period of time sufficent for the admixed antibody to immunoreact
with, bind to and precipitate the receptors of the immunoreactant,
and form a precipitate and a supernatant.
The precipitate and supernatant are separated; and the
radioactivity present in the precipitate is measured. That
measurement, when compared to control valves obtained with known
amounts of assayed apolipoprotein A, radioactive competitive
antigen, receptor and precipitating antibody, may be used to
provide the amount of receptor bound in the immunoreactant, and
thereby the amount of apolipoprotein A present in the sample
assayed.
A still further aspect of the invention contemplates the use of the
before-mentioned latex particles as a solid matrix of a solid
support. In an exemplary method, a receptor of this invention is
affixed to the latex particles, as described before, prior to the
admixture of step (b) of the previously described, general
method.
The sample to be assayed is admixed in an aqueous medium with those
particles to form a solid/liquid phase admixture that is a
dispersion of solid latex particles in an aqueous medium. The
admixture is maintained for a time period sufficient for an
immunoreactant to form, which formation causes the latex particles
to agglutinate.
The time required for the latex particles to agglutinate is
measured. That measurement provides a determination of the amount
of receptor bound in an immunoreactant, and thereby the presence
and relative amount of apolipoprotein A present in the sample by
comparison with values obtained with controls.
Similar agglutination methods may be performed with red blood cells
(hemagglutination) or with other agglutinatable particles or cells
following the above steps.
Still further assay methods within the before-described general
method may also be employed. Each of those methods differs from
those previously described by the manner in which the amount of
immunochemical binding is determined.
One group of such methods utilizes optical measurements for that
determination. In one exemplary procedure, a liquid admixture is
formed in before-described steps (b) and (c) and the turbidity of
the liquid admixture is measured and compared to control values. In
another embodiment, the change in light scattering after step (c)
is compared to control values.
A still further method utilizes the direct precipitation of the
immunoreactant formed. The amount of binding may also be determined
by noting changes in electrophoretic mobility of the liquid
admixture of step (c) under non-denaturing conditions.
Yet another method utilizes a receptor of this invention affixed to
a soid matrix such as SEPHAROSE beads as an affinity sorbant. Here,
the admixture formed in step (b) is a solid/liquid admixture that
physically separates the immunoreactant from the liquid portion of
the admixture. The liquid portion is thereafter subjected to
electrophoretic separation and compared to a similar separation
using another aliquot of the sample to determine whether an
apolipoprotein A was present in the sample.
It is to be noted that values obtained from appropriate controls
are stated as being utilized in several of the methods. It is to be
understood that such control values are obtained separately, and
may be so obtained before, during or after the recited steps.
III. DIAGNOSTIC SYSTEMS
The present invention also contemplates diagnostic systems,
preferably in kit form. Several embodiments of a diagnostic system
are contemplated. However, each diagnostic system comprises at
least one package that contains a known amount of a monoclonal
receptor of this invention that immunoreacts with and binds to
human apolipoprotein A, but is free from immunoreaction with and
binding to apolipoproteins B, C, D and E.
Exemplary packages include glass and plastic such as polyethylene
and polypropylene bottles or vials; plastic, plastic-metal foil,
and plastic-metal foil-paper envelopes, and the like. The receptor
may be packaged in an aqueous liquid form as in ascites or buffer,
but preferably, the receptor is supplied in dried form such as that
provided by lyophilization.
A known amount of the receptor is provided. That amount is at least
enough to carry out one assay. The provided receptor is typically
supplied in a form and amount that is designed to be diluted to a
prescribed volume with water, saline or a buffer such as
phosphate-buffered saline at pH 7.3-7.5.
In another embodiment, the system includes a second package that
includes a known amount of an apolipoprotein A with which the
receptor immunoreacts and binds to form an immunoreactant. The
apolipoprotein A is provided affixed to a solid matrix as a solid
support antigen.
Useful solid matrices are as already described. Preferably,
however, the solid matrix is the well of a microtiter plate. The
microtiter plate forms the package for the well, but may also be
separately enclosed in a paper envelope or plastic film to avoid
contamination of the wells.
In a further embodiment, the receptor is provided affixed to a
solid matrix as a solid support. Exemplary of such a solid support
are receptor-affixed latex particles that are dispersed in an
aqueous medium as previously described.
Additional packages may also be included in the system. Such
packages may contain (i) buffer salts in dry or liquid form, (ii)
substrates such as hydrogen peroxide and o-phenylenediamine, (iii)
an indicating means-containing reagent such as peroxidase-linked
goat anti-mouse antibodies in a liquid or dry form, and the
like.
It is also noted that the receptor that is required for a
diagnostic system of this invention may be any individual receptor
of this invention or may be a mixture that contains the
antibody-combining sites (idiotype polypeptide portions) of two or
more such receptors.
IV. RESULTS
A. Apoprotein Specificity
Each of the four monoclonal antibodies (designated A-I-4 antibody
from 611 AV63C2.1F1 hybridoma; A-I-9 antibody from HA62 HA227A2.7D3
hybridoma; A-I-7 antibody from HA60 HA22GF.5F8 hybridoma; and
A-II-1 from HA61 H112F3.1A11 hybridoma) was selected on the basis
of its capacity to bind intact HDL. Three were selected by
screening for antibodies that reacted with the immobilized
immunizing antigen using a solid-phase RIA. The fourth (A-II-1) was
selected on the basis of indirect precipitation of soluble .sup.125
I-HDL in a fluid-phase assay. In addition to the immunizing
antigen, the antibodies produced by each of the four hybridomas
bound to immobilized human HDL in a solid-phase RIA, suggesting
that each of these antibodies was specific for one of the
apolipoproteins of human HDL.
Antibody specificity was determined by Western blotting of the
electrophoretically separated apolipoproteins of human VLDL, LDL,
and HDL, as well as isolated apo-A-I and apo-A-II. Antibodies
A-I-4, A-I-7, and A-I-9 bound completely to apo-A-I of HDL and
isolated apo-A-I. Some of the antibodies identified trace amounts
of what appeared to be contaminating apo-A-I in both LDL and
isolated apo-A-II; i.e., proteins that were marginally visible in
the stained gel.
The one exception to this pattern of reactivity was antibody
A-II-1. This antibody bound to isolated human apo-A-II dimers and
apo-A-II monomers as well as the apo-A-II dimers and monomers of
human HDL (FIG. 1).
In addition, this antibody bound a trace VLDL protein of apparent
molecular weight of 52,000 daltons that was not readily observed in
the protein-stained gel. This protein, which appeared to be present
also in HDL, had a mobility that was intermediate between apo-E and
albumin, and may have been an apo-E-A-II dimer as described by
Weisgraber and Mahley, J. Biol. Chem., 253, 6281-6288 (1978).
Thus, three of the monoclonal antibodies were specific for apo-A-I,
and the fourth was specific for apo-A-II. The numerical antibody
designations shown in FIG. 1 reflect this apolipoprotein
specificity. In addition, each of the apo-A-I antibodies bound
multiple apo-A-I isoforms including A-I-1, A-I-2, and pro-A-I from
either HDL or isolated apo-A-I, after separation of those isoforms
in isoelectric focusing gels.
B. Lipoprotein Specificity
To characterize the reactivity of these antibodies for native HDL,
binding of the antibodies to .sup.125 I-HDL was studied in a
fluid-phase double-antibody RIA. Antibody binding was measured at a
final antigen concentration of 66.7 ng of .sup.125 I-HDL/ml.
Maximum binding of .sup.125 I-HDL by each of the four antibodies in
antibody excess varied from 18 to 56 percent for the
apo-A-I-specific antibodies and was 87 percent for the
apo-A-II-specific antibody (FIG. 2). It was notable that 100
percent binding of .sup.125 I-HDL was uniformly expressed by the
apolipoprotein chains as organized on all HDL particles.
As reported by Chung and Albers, J. Lipid Res. 23, 747-753 (1982),
HDL of density equal to 1.063 to 1.21 contains two types of
particles: (i) particles that contain apo-A-I and apo-A-II in an
approximate 2:1 molar ratio; and (ii) particles that contain
apo-A-I but no apo-A-II. Therefore, it was not surprising that the
apo-A-II antibody did not bind 100 percent of HDL. However, if all
HDL particles contained at least apo-A-I, other explanations must
exist for the inability of any one of the three apo-A-I antibodies
to bind all HDL. Because each of the antibodies bound all isoforms
of the isolated apolipoprotein after electrophoresis in SDS, the
ability of these antibodies to recognize the isolated
apolipoprotein in a fluid-phase RIA also was examined.
Antibody A-II-1 bound 100 percent of .sup.125 I-apo-A-II (FIG. 2).
Therefore, this protein chain appeared to be immunochemically
homogeneous in that all apo-A-II molecules expressed the epitope
defined by the A-II-1 antibody.
However, none of the apo-A-I-specific antibodies bound 100 percent
of soluble .sup.125 I-apo-A-I (FIG. 2). In antibody excess,
antibodies A-I-4, A-I-7, and A-I-9 bound 55, 60, and 13 percent of
.sup.125 I-apo-A-I, respectively.
To determine if there was a difference in the apolipoprotein
composition of .sup.125 I-HDL particles bound by each antibody, as
opposed to those particles that were not bound by antibody,
precipitates and supernatants formed in the presence of high
concentrations of monoclonal antibody (and a slight excess of
precipitating antibody to fully precipitate all monoclonal
antibody) were dissolved in SDS and electrophoresed on SDS-PAGE. A
representative autoradiograph of the bound (precipitate) and
unbound (supernatant) fractions of .sup.125 I-HDL after reaction
with antibodies A-I-7, A-II-1, and A-I-4 is shown in FIG. 3.
All apo-A-I-specific antibodies, including antibodies A-I-4 and
A-I-7, bound .sup.125 I-HDL particles that contained both apo-A-I
and apo-A-II, and the bound fractions were indistinguishable from
either the starting .sup.125 I-HDL or the unbound .sup.125 I-HDL;
i.e., the unbound .sup.125 I-HDL contained nonprecipitable apo-A-I.
In contrast, antibody A-II-1 appeared to bind most if not all of
the .sup.125 I-HDL that contained apo-A-II, because the unbound
supernatant fraction from this reaction mixture was free of
demonstrable apo-A-II dimers or monomers (FIG. 3). Thus, antibody
A-II-1 bound all HDL particles that contained apo-A-II, whereas
none of the A-I-specific antibodies were capable of binding all HDL
particles that contained only apo-A-I.
C. Incomplete Binding of Antigen
To explain the inability of the apo-A-I-specific antibodies to bind
to and facilitate total precipitation of either .sup.125 I-HDL,
soluble .sup.125 I-HDL or soluble .sup.125 I-apo-A-I, two general
possibilities were considered: (i) heterogeneity of apo-A-I with
respect to expression of epitopes; and (ii) nonoptimal conditions
of analysis of binding. In initial studies, the optimum time and
temperature was determined for the maximum binding of antibodies
A-I-4, A-I-7, A-I-9, and A-II-1 to .sup.125 I-HDL in fluid phase.
For each of these antibodies, maximal binding was observed within
18-20 hours at either 4 or 24 degrees C. The quantity of .sup.125
I-HDL that was bound by each antibody was maximal and independent
of the amount of antibody added under conditions of antibody
excess. In additional studies, it was shown that (a) antibody
binding was independent of the amount of antigen added; i.e.,
antibody affinity; (b) radioiodination of apo-A-I or HDL did not
interfere with antibody binding; (c) mild antigen dissociating
conditions such as heating and detergents did not expose additional
antigen epitopes; and (d) individual allytypic differences in
apo-A-I did not account for the incomplete binding of HDL.
Because none of the above manipulations led to complete binding of
HDL, the alternative possibility was considered that there may be
heterogeneity of apo-A-I. It was hypothesized that all apo-A-I
molecules in plasma were not absolutely identical; i.e., all
molecules of apo-A-I did not uniformly express the epitopes defined
by the three apo-A-I-specific antibodies. If each apo-A-I antibody
bound a different epitope on apo-A-I, and if all HDL particles
contained an apo-A-I expressing one or more of these epitopes, then
complete binding of all .sup.125 I-HDL particles would be observed
by combining the three apo-A-I-specific antibodies. When all
possible combinations of two or three apo-A-I-specific antibodies
were analyzed for binding, only incomplete binding of .sup.125
I-HDL was observed as shown in Table 1, below.
TABLE 1 ______________________________________ All .sup.125 I-HDL
Bound By One Apo-A-II- Specific And Three Apo-A-I-Specific
Antibodies .sup.125 I-HDL bound (% of maximum) Antibody Alone In
combination ______________________________________ A-I-4, A-I-7 44
.+-. 1; 61 .+-. 3 80 .+-. 3 A-I-4, A-I-9 44; 32 .+-. 2 63 .+-. 2
A-I-7, A-I-9 61; 32 76 .+-. 4 A-I-4, A-I-7, A-I-9 44, 61, 32 83
.+-. 3 A-II-1, A-I-4 67 .+-. 6, 44 92 .+-. 2 A-II-1, A-I-7 67; 61
93 .+-. 1 A-II-1, A-I-9 67; 32 87 .+-. 3 A-II-1, A-I-4, A I-7 67,
44, 61 100 .+-. 2 A-II-1, A-I-4, A I-9 67, 44, 32 98 .+-. 3 A-II-1,
A-I-7, A I-9 67, 61, 32 99 .+-. 1
______________________________________ .sup.125 IHDL was used at
66.7 ng/ml in the fluidphase RIA
In view of the before-discussed results, each of the
apo-A-I-specific antibodies must bind a different epitope because
as each additional antibody was added, additional apolipoprotein
A-I was bound, although all antibodies were present in excess. All
combinations of the A-I antibodies were present in excess. All
combinations of the A-I antibodies bound more HDL than any single
A-I antibody, and the oligoclonal mixture of the three apo-A-I
antibodies, and the oligoclonal mixture of the three apo-A-I
antibodies most closely approached complete binding of .sup.125
I-HDL. These results suggest that HDL particles may exist that
either do not contain apo-A-I or contain apo-A-I molecules that are
not recognized by any of these apo-A-I-specific antibodies.
Because complete binding of .sup.125 I-HDL could not be obtained
with any combination of the three apo-A-I-specific antibodies,
binding all .sup.125 I-HDL was examined by combining each of the
apo-A-I-specific antibodies individually with the apo-A-II-1
antibody (Table 2).
TABLE 2 ______________________________________ The Percent Of
.sup.125 I-HDL Bound By Each Antibody Was Independent Of The Amount
Of .sup.125 I-HDL Added .sup.125 I-HDL Added .sup.125 I-HDL Bound
(Percent of Maximum) (ng/ml) A-I-4 A-I-7 A-I-9
______________________________________ 10 31.8 .+-. 2.8 43.7 .+-.
3.1 30.2 .+-. 0.5 30 30.6 .+-. 4.1 42.0 .+-. 7.7 29.0 .+-. 2.3 100
33.3 .+-. 1.1 44.4 .+-. 0.6 29.2 .+-. 1.1 300 34.7 .+-. 3.2 44.4
.+-. 1.7 29.1 .+-. 1.2 ______________________________________
To insure that each of the apo-A-I-specific antibodies identified
all apo-A-I isoforms, HDL and isolated apo-A-I were separated by
isoelectric focusing in polyacrylamide gels and were Western
blotted to nitrocellulose for reaction with antibody. The left
panel of FIG. 1 is a photograph of a Coomassie Brilliant Blue R250
stained gel before electrophorectic transfer of the apolipoproteins
to nitrocellulose. The remaining three panels are 24 hour
autoradiographs of identical nitrocellulose paper transfers after
incubation with each of the individual antibodies and .sup.125
I-goat-anti mouse Ig (0.5 milliCi/ml). As shown, each apo-A-I
antibody bound multiple apo-A-I bands, suggesting that none of the
antibodies distinguished among the various isoforms. No combination
of one apo-A-I-specific antibody with antibody A-II-1 resulted in
100 percent binding of .sup.125 I-HDL.
However, the combination of any two apo-A-I-antibodies with the
single apo-A-II-specific antibody resulted in 100 percent binding
of .sup.125 I-HDL (Table 1). Those results confirm that all HDL
particles express at least one of the three apolipoprotein epitopes
defined by antibodies A-II-1, A-I-4, and A-I-7; A-II-1, A-I-4, and
A-I-9; or A-II-1, A-I-7, and A-I-9, and thus establish limits on
the degree of heterogeneity.
B. HDL and Apoprotein Affinity
Because complete binding of .sup.125 I-HDL could be achieved with
an oligoclonal mixture of monoclonal antibodies, the feasibility of
using these antibodies to accurately quantitate total plasma HDL
was further analyzed. The quantity of apo-A-I measured in HDL and
apo-HDL with polyclonal antisera has often been different,
suggesting that the affinities of antibodies might differ for
soluble apolipoproteins as compared with the same apolipoproteins
when they are organized on HDL. Competitive RIAs with .sup.125
I-HDL were used in which the ability of HDL and the isolated
apolipoprotein to compete for binding of .sup.125 I-HDL was
analyzed to identify differences in antibody affinities for HDL and
apo-HDL.
Slope analysis of the logit-transformed competitive curves
indicated that two of the antibodies, A-I-7 and A-II-1, had the
same affinity for the isolated apolipoprotein and for that
apolipoprotein when organized on HDL, whereas the other two
antibodies, A-I-4 and A-I-9 differed. For both antibodies A-I-4 and
A-I-9, the affinities were less for free apo-A-I than for apo-A-I
organized on HDL.
C. Expression of Apo-A-I and Apo-A-II Epitopes by Apoproteins of
Different Biosynthetic Origin
The apo-A-I and apo-A-II epitopes defined by the antibodies of this
invention were examined to determine if apolipoproteins from
different biosynthetic sites differed in epitope expression.
Included in this analysis were (a) conditioned culture medium from
the hepatic cell line Hep G2; (b) human lymph collected by thoracic
duct drainage; and (c) unfractionated whole plasma,
lipoprotein-depleted plasma, VLDL, and HDL from the same pooled
plasma source. Each of those samples was examined for epitope
expression by competitive inhibition immunoassay for each
monoclonal antibody using .sup.125 I-HDL as the ligand. Inhibition
was based on total protein added. The .sup.125 I-HDL used in each
immunoassay was obtained from a pooled plasma source.
Each apolipoprotein source was analyzed at three levels. First it
was determined whether the protein competitvely inhibited antibody
binding to .sup.125 I-HDL. Second, if inhibition was observed, the
affinity of the antibody for the competing protein was determined
by slope analysis and compared with the affinity for HDL. Third, if
similar affinities were observed, the quantitative expression of
the epitope by the competing protein (based on total protein added)
was compared with that expressed by either HDL or plasma. If the
affinities were not the same as determined by slope analysis, no
quantitative conclusions could be drawn.
The epitope defined by antibody A-I-4, which bound a subset of
apo-A-I present on 40-50 percent of .sup.125 I-HDL, was not
expressed by apo-A-I of VLDL or by hepatocyte-derived apo-A-I
present in culture medium from the Hep G2 cells. This epitope was
expressed in lipoprotein-deficient plasma (LPDP), but the affinity
for the epitope in LPDP was less than the epitope expressed by
apo-A-I organized on HDL. As demonstrated previously, the affinity
of this antibody for isolated Apo-A-I was less than its affinity
for HDL, indicating differences in the defined epitope. This
suggested that the majority of the apo-A-I in LPDP was not
associated with lipoprotein particles.
In contrast this epitope was expressed by apo-A-I in normal human
plasma (NHP) and in thoracic duct lymph (lymph) with an affinity
that was indistinguishable from that for plasma-derived HDL,
suggesting that the apo-A-I of NHP and lymph was associated with
lipid. On the basis of total protein added, more A-I-4 epitope was
detected in lymph than in NHP.
Antibody A-I-7, which identified a major apo-A-I epitope
denominated A-I-7, also did not bind apo-A-I of plasma VLDL. The
A-I-7 epitope expressed by molecules in the Hep G2 culture medium
and lymph interacted with a higher affinity compared with HDL,
whereas the A-I-7 epitope expressed by molecules in LPDP and NHP
interacted with its antibody with an affinity that was
indistinguishable from that of the same epitope expressed on
HDL.
As demonstrated above, the affinity of this antibody for isolated
apo-A-I and apo-A-I in HDL is the same. Thus, this antibody did not
appear to distinguish between free or lipid-associated apo-A-I. The
difference in affinity of the antibody for the A-I-7 epitope in
lymph and hepatocyte medium indicated a modification of apo-A-I in
these sources. On a quantitative basis, the amount of apo-A-I in
LPDP by reference to the A-I-7 epitope was 11 percent of the
apo-A-I present in normal human plasma.
The third apo-A-I epitope identified by antibody A-I-9 was
distinguished from epitope bound by antibodies A-I-4 and A-I-7 by
its expression by apo-A-I on VLDL, although it was not expressed by
molecules in LPDP. The A-I-9 epitope was expressed by apo-A-I from
Hep G2 cells in culture.
Compared with HDL, the A-I-9 epitope of apo-A-I of lymph had higher
affinity for antibody, that of VLDL had lower affinity, and the
same affinity was observed for Hep G2 culture medium and NHP. The
epitope bound by antibody A-I-9 was thus subject to fine
differences in structure on different apo-A-I.
The apo-A-II epitope identified by antibody A-II-1 was present in
all samples studied. Compared with HDL, the A-II-1 epitope was
expressed with the same affinity by molecules in Hep G2 culture
medium, LPDP, and NHP. This apo-A-II epitope interacted with
epitopes that appeared the same for isolated apo-A-II and apo-A-II
organized in HDL. The apo-A-II of LPDP represented 3.4 percent of
the apo-A-II present in NHP. Surprisingly, the binding affinity of
this antibody for apo-A-II in VLDL and lymph was slightly greater
than its affinity for HDL.
D. Expression of Apo-A-I and Apo-A-II Epitopes in HDL
Subfractions
Epitope expression by HDL subpopulations separated by density
gradient ultracentrifugation and chromatofocusing was examined to
determine if HDL subpopulations differing in apo-A-I and apo-A-II
epitopes could be distinguished on the basis of particle size or
composition. Five HDL density subfractions were isolated from a
single plasma source that was pooled from three donors. The
apoprotein composition of the subfractions was characterized by
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis), and the particle size distribution of particles
present in these HDL subfractions was characterized by PAGE (FIG.
4).
HDL density subfraction 1 (the lowest density HDL) was
distinguished by SDS-PAGE from fractions 2 through 5 by the
presence of apo-B, greater quantities of apo-E, and relatively
small amounts of apo-A-II (dimer and monomer) and apo-D. Because
these HDL subfractions were obtained from a single
ultracentrifugation to minimize apoprotein loss and potential
perturbation of the HDL particles, they also contained small
amounts of other plasma proteins (FIG. 4, top).
Electrophoresis of the HDL density subfractions on 4-30 percent
polyacrylamide gradient (PAGE) pore gels in the absence of SDS or
other dissociating agents demonstrated the presence of varying
proportions of HDL particles of at least two sizes. Predominantly
large HDL particles were present in density subfractions 1 and 2,
and small HDL particles predominated in subfractions 4 and 5 (FIG.
4, bottom). In addition, the HDL subfractions differed with respect
to their total cholesterol content. The light HDL fractions (1 and
2) contained the largest amount of free and esterified
cholesterol/mg of total protein.
When epitope expression by each HDL subfraction was analyzed by
competitive inhibition, complete inhibition of the binding of each
antibody could be achieved, confirming that each of the defined
epitopes was present in each HDL fraction. When the competitive
inhibition profiles were analyzed by logit transformation to
compare the qualitative epitope expression by each subfraction,
slope analysis indicated that the affinity of each epotope for its
antibody did not significantly differ one from another (p less than
or equal to 0.2). Thus, a relative assessment of the quantitative
expression of each epitope in each HDL subfraction was
feasible.
From the competitive inhibition regression line, the protein
required for 50 percent inhibition of antibody binding was
determined (Table 3). On a quantitative basis, epitopes A-I-4 and
A-I-9 were expressed at highest concentration by subfraction 2,
whereas epitope A-I-7 was most highly expressed by subfractions 4
and 5. Epitope A-II-1 was most abundant in subfraction 4 as set
forth in Table 3 below.
TABLE 3 ______________________________________ Quantitative
Expression Of Apolipoprotein A-I And A-II Epitopes And HDL
Subfractions Competitor concentration per antibody.sup.a A-I-4
A-I-7 A-I-9 A-II-1 ______________________________________ HDL
density fractions.sup.b 1 (light) 137 20.5 19.5 1.08 2 46 9.5 8.4
0.44 3 78 7.4 14.8 0.39 4 78 6.4 21.9 0.32 5 (heavy) 84 6.4 32.0
0.49 HDL chromatofocusing fractions.sup.c 11 (pH 5.0) 72 3.3 5.6
2.70 18 59 3.0 5.4 1.75 27 23 1.5 7.4 0.82 32 20 1.8 8.2 0.53 34 23
1.6 10.3 0.40 37 (pH 4.4) 109 6.0 27.0 1.38
______________________________________ .sup.a Concentration of
competing protein required to exhibit 50 percent inhibition of
antibody binding expressed in milligrams of total protein per
mililiter. .sup.b Fractions were obtained by density gradient
ultracentrifugation. Apoprotein compositions and the size
distributions are shown in Figure 4. Unfractionated homologous HDL
was used as the radiolabeled ligand and was used at a final
concentration of 66.7 ng/ml. Mean slopes and minimum correlation
coefficients of the logittransformed inhibition curves by all HDL
density subfractions with antibodies AI-4, AI-7, AI-9, and AII-1
were -3.01 .+-. 0.30, r greater than or equal to 0.995; -2.94 .+-.
0.13, r greater than or equal to 0.996; -2.27 .+-. 0.14, r greater
than or equal to 0.992; and -3.16 .+-. 0.15, r greater than or
equal to 0.997, respectively. Therefore, no differences were
observed in the affinity of each antibody for its epitope in each
HDL fraction. .sup.c Fractions were obtained by column
chromatography as described in the Materials and Methods section
(V). Unfractionated homologous HDL was used as the radioiodinated
ligand and was added at a final concentration of 66.7 ng/ml. Mean
slopes and minimum correlation coefficients of the logittransformed
inhibition curves by all HDL chromatofocusing fractions with
antibodies AI-4, AI-7, AI-9, and AII-1 were: -3.11 .+-. 0.24, r
greater than or equal to 0.995; 2.87 .+-. 0.21, r greater than or
equal t 0.994; -2.06 .+-. 0.17, r greater than or equal to 0.997;
and -3.36 .+-. 0.13, r greater than or equal to 0.998,
respectively.
A comparison of the quantitative expression of the four apoprotein
epitopes in the five HDL subfractions is illustrated in FIG. 5
(top). Relative epitope expression for each antibody was calculated
from Table 3 by assigning a value of 1.0 to the HDL subfraction
that contained, on a protein basis, the greatest quantity of the
epitope. All other HDL subfractions were then expressed
fractionally. As shown, the relative epitope expression varied for
each density subfraction with epitopes A-I-4 and A-I-9
predominating in the light HDL subfractions and epitopes A-I-7 and
A-II-1 predominating in the heavy HDL subfractions.
This same epitope analysis was performed on another set of HDL
subfractions separated on the basis of net charge by
chromatofocusing. A single plasma source pooled from three donors
was used to isolate a d=1.063-1.21 g/ml HDL fraction by
ultracentrifugation that was then chromatographed on a
Polybuffer-Exchange 94 column. The apoprotein composition of
representative subfractions as determined by SDS-PAGE, and the
particle size distribution of the same subfractions as determined
by PAGE are illustrated in FIG. 6. Fractions 11 and 18 were
distinguished from subfractions 32 and 34 by the smaller quantity
of apo-A-II (FIG. 6, top). PAGE demonstrated the presence of
varying proportions of at least two particle sizes. Subfractions 11
and 18 contained predominantly large cholesterol-rich HDL, whereas
subfractions 34 and 37 contained predominately small
cholesterol-poor HDL (FIG. 6, bottom).
When epitope analysis was performed, complete competitive
inhibition of the binding of each antibody was observed in excess
antigen. The affinity of each antibody for its complementary
epitope expressed by particles in each chromatofocusing subfraction
was equivalent (p equals 0.2). From the competitive inhibition
regression lines, the total protein required for 50 percent
inhibition was determined (Table 3). On a quantitative basis,
epitope A-I-9 was most abundant in subfractions 11 and 18, epitopes
A-I-4 and A-I-7 were most abundant in fractions 27, 32, and 34,
whereas epitope A-II-1 was present in fraction 34 (FIG. 6, bottom).
The most striking feature of the distribution of each of the
apoprotein epitopes was the predominance of epitope A-1-9 in HDL
particles eluted at pH 5.0 that appeared to contain only apo-A-I
(FIG. 6).
V. MATERIALS AND METHODS
A. Lipoproteins
During the course of these studies, lipoproteins were isolated from
nine different plasma pools, each made up of three or more
individual fasting donors. The isolated lipoproteins, including
LDL, density equal to 1.006 g/ml; LDL, density equal to 1.019 to
1.063 g/ml; and HDL density equal to 1.063 to 1.21 g/ml, were
dialyzed against lipoprotein buffer (LLB) containing 150 mM NaCl, 1
mM EDTA, 0.005 percent alpha-tocopherol, and 5 mM benzamidine, and
were stored under sterile conditions for no more than 21 days. In
selected studies to identify potential allelic differences, the HDL
(density equal to 1.063 to 1.21 g/ml) was isolated from plasmas
obtained from individual normolipidemic donors and treated in the
same manner.
HDL density subfractions were obtained from a single pooled plasma
source by isopycnic density gradient ultracentrifugation. After
removal of the lipoprotein of density less than or equal to 1.063
g/ml by a single 18-hour run at 200,000xg, the infranatant plasma
fraction (20 ml) was increased to a density of 1.21 g/ml and
centrifuged at 10 degrees C through 20 ml of 1.21 g/ml KBr for
about 4 to about 8 hours at 200,000xg. Five 4-ml fractions were
collected beginning at the top of the tube, and were dialyzed into
LLB for further analysis.
HDL chromatofocusing fractions were obtained from a separate pooled
plasma source essentially as described by Nestrock et al., Biochem.
Biophys. Act, 753, 65-73 (1983). The HDL (density equal to 1.063 to
1.21 g/ml) was isolated by ultracentrifugation and dialyzed into 25
mM piperazine hydrochloride, having a pH value of 5.8. Forty mg of
protein were applied to a 1.6.times.30 cm column of
Polybuffer-Exchanger 94 (Pharmacia Fine Chemicals, Piscataway,
N.J.; hereinafter Pharmacia) equilibrated with 25 mM piperazine
HCl, pH 5.8, and the HDL was eluted with Polybuffer 74 (Pharmacia)
diluted 1:15 with H.sub.2 O, having a pH value of 4.0. The effluent
was monitored for absorbance at 280 nanometers (nm) and for pH
value. Six HDL subpopulations corresponding to those described by
Nestrock et al., (supra), and eluting at pH maximal values of 5.0,
4.9, 4.8, 4.7, 4.5, and 4.4, respectively, were collected and
desalted by chromatography on Sephadex G-75 equilibrated with
LLB.
B. Isolation of Apoproteins A-I and A-II
Apoproteins A-I and A-II were isolated from
ether/ethanol-delipidated HDL by chromatography on DEAE-cellulose
in deionized 6 M urea as described below and by Blaton et al.,
Biochemistry; 16, 2157-2163 (1977). The isolated apolipoproteins
were stored in dilute solution in 0.1 percent sodium bicarbonate at
-20 degrees C.
C. Lipoprotein Characterization
Lipoproteins were analyzed for protein by a modification of the
method of Lowry [Lowry et al., J. Biol. Chem. 193, 265-275
(1951)]in the presence of SDS using a bovine albumin standard.
Lipoprotein concentrations were expressed as the mass of protein.
Total and free cholesterol were measured by the enzymatic
fluorometric method. Esterified cholesterol was taken as the
difference between total and free cholesterol. Results were
expressed as micrograms of cholesterol/mg of total protein.
The apolipoprotein composition of the lipoproteins was analyzed by
polyacrylamide slab gel electrophoresis in the presence of 0.1
percent SDS as described by Curtiss et al., J. Biol. Chem., 257,
15213-15221 (1982). The running gels contained a linear 7.5-20
percent acrylamide gradient. The apo-A-I isoforms were separated by
isoelectric focusing in a 6 percent polyacrylamide gel containing 8
M urea and 2 percent Ampholine (1 percent having a pH value between
4-6 and 1 percent having a pH value between 5-8) as described by
Weisgraber et al., J. Lipid Research, 21, 316-325 (1980).
Lipoproteins were delipidated by boiling for 3 minutes in 1 percent
SDS before electrophoresis, and the gels were stained after
electrophoresis with 0.1 percent Coomassie Brilliant Blue R-250 in
50 percent trichloracetic acid. Gels containing radioiodinated
lipoproteins were visualized by autoradiography.
Lipoprotein particle size distributions were determined by
lipoprotein polyacrylamide gradient pore gel electrophoresis using
the system of [Blanche et al. (Biochem. Biophys-Acta, 665; 408-419
(1981))]. Samples containing 10-20 micrograms of protein in
0.008-0.010 ml aliquots were electrophoresed for 24 hours at 130
volts (constant voltage) in 4-30 percent acrylamide gradient slab
gels. The high molecular weight calibration kit (Pharmacia) was
used for molecular weight determinations. The gels were fixed,
stained and destained, and where appropriate, visualized by
autoradiography.
D. Generation of Monoclonal Antibodies
The four monoclonal antibodies were obtained from three separate
fusions of splenocytes from immunized Balb/c mice (Scripps Clinic
and Research Foundation Vivarium, La Jolla, Calif.), using standard
fusion protocols discussed herein. Culture supernatants were
collected and screened by either solid-phase or fluid-phase
radioimmunoassay as described below. All hybridomas were cloned at
least twice by limiting dilution, and were stored frozen in liquid
nitrogen.
Briefly, Balb/c mice were immunized intraperitoneally with native
human HDL or apo-VLDL as immunogen in complete Freund's adjuvant. A
booster injection of immunogen in incomplete Freund's adjuvant was
administered approximately 3 to 4 weeks following the first
injection. Three days prior to harvesting of the mouse spleen, a
final booster of immunogen in normal saline was injected
intravenously.
The animals so treated were sacrificed, and the spleen of each
mouse was harvested. A spleen cell suspension was then prepared.
Spleen cells were then extracted from the spleen cell suspension by
centrifugation for about 10 minutes at 1000 r.p.m., at 23 degrees
C. Following removal of supernatant, the cell pellet was
resuspended in 5 ml. cold NH.sub.4 Cl lysing buffer, and was
incubated for about 10 minutes.
To the lysed cell suspension were added 10 ml Dulbecco's Modified
Eagle Medium (DMEM) (Gibco) and HEPES
[4-(2-hydroxyethyl)-1-piperidineethanesulfonic acid]buffer, and
that admixture was centrifuged for about 10 minutes at 1000 r.p.m.
at 23 degrees C.
The supernatant was decanted, the pellet resuspended in 15 ml of
DMEM and HEPES, and was centrifuged for about 10 minutes at 1000
r.p.m. at 23 degrees C. The immediately preceding procedure was
repeated.
The pellet was then resuspended in 5 ml DMEM and HEPES. An aliquot
of the spleen cell suspension was then removed for counting.
Fusions were accomplished in the following manner using mouse
myeloma cell line P3.times.63Ag8 for ATCC HB 8744 and line
P3.times.63Ag8.653 for the remaining hybridomas. Using a myeloma to
spleen cell ratio of about 1 to 10 or about 1 to 5 (the most
preferred myeloma to spleen cell ratio being 1 to 5), a sufficient
quantity of myeloma cells were centrifuged to a pellet, washed once
in 15 ml DMEM and HEPES, and centrifuged for 10 minutes at 1000
r.p.m. at 23 degrees C. Spleen cells and myeloma cells were
combined in round bottom 15 ml tubes (Falcon). The cell mixture was
centrifuged for 7 minutes at 800 r.p.m. at 23 degrees C., and the
supernatant was removed by aspiration. The remaining cell pellet
was then gently broken into large chunks. Thereafter, 200
microliters of 30 percent aqueous polyethylene glycol (w/v) (PEG)
(ATCC Baltimore, Md.) at about 16 degrees C. were added, and the
mixture was gently mixed for between 15 and 30 seconds. The cell
mixture was centrifuged 4 minutes at 600 r.p.m. At about 8 minutes
from the time of adding the PEG, the supernatant was removed.
Then 5 ml DMEM plus HEPES buffer was added to the pellet, allowed
to set for 5 minutes, and was followed by gently breaking the
pellet into large chunks. This mixture was centrifuged 7 minutes at
600 r.p.m. The supernatant was decanted, 5 ml of HT
(hypothanthine/thymidine) media were added to the pellet and left
undisturbed for 5 minutes. The pellet was then broken into large
chunks and the final cell suspension was placed into T75 flasks
(2.5 ml per flask) into which 7.5 ml HT media had been placed
previously. The resulting cell suspension was incubated at 37
degrees C. to grow the fused cells. Three days after fusion the
fused cells were plated out and treated as described below.
In an alternate procedure, the spleens of the two mice were
removed, suspended in complete HT medium containing 0.1 millimolar
azaguanine [formulated according to Kennett et al., Curr. Top.
Microbiol. Immunol., 81, 77 (1978)], pooled to yield
3.2.times.10.sup.8 total cells, and fused with mouse myeloma cells
in the presence of a fusion promoter [e.g., 30 percent (weight per
volume) polyethylene glycol-1000 to about 4000; ATCC]at a ratio of
10 myeloma cells per spleen cell as described in Curtiss et al., J.
Biol. Chem., 257, 15213-15221 (1982).
Three days after fusion, viable cells were plated out in 96-well
tissue culture plates at 2.times.10.sup.4 viable cells per well
(768 total wells) in HAT (hypothanthine, aminopterin, thymidine)
buffer medium as described in Kennett et al., supra). The cells
were fed seven days after fusion with HT medium and at
approximately 4-5 day intervals thereafter as needed. Growth was
followed microscopically and culture supernatants that contained
antibodies were collected on day 14 for assay of antigen-specific
antibody production by solid phase radioimmunoassay (RIA).
The hybridomas so prepared were screened, assayed, and their
viabilities were determined.
The hybridomas were given the following designations for reference
purposes and were deposited on Mar. 5, 1985 with the American Type
Culture Collection, Rockville, Md. under the following ATCC of
accession numbers.
______________________________________ Hybridoma ATCC Accession No.
______________________________________ HA62 HA227A2.7D3 HB 8741
HA61 H112F3.1A11 HB 8743 HA60 HA22GF.5F8 HB 8745 611 AV63C2.111 HB
8744 ______________________________________
Immunoglubolin heavy and light chains of the antibodies secreted by
the cloned hybridomas were typed using the Mono AB-ID EIA Kit A
(Zymed Labs Inc., San Francisco, Calif.). The assays were performed
with hybridoma culture supernatants as described by the
manufacturer. Those results were as shown below.
______________________________________ ATCC Accession No. Isotype
______________________________________ HB 8741 IgG.sub.1 kappa HB
8743 IgM kappa HB 8745 IgG.sub.1 kappa HB 8744 IgG.sub.1 kappa
______________________________________
E. Monoclonal Antibody Production
Once the desired hybridoma had been selected and cloned, the
resultant monoclonal antibody (receptor) was produced in one of two
ways. The more pure monoclonal antibody is produced by in vitro
culturing of the desired hybridoma in a suitable medium for a
suitable length of time, followed by recovery of the desired
antibody from the supernatant. Suitable media and length of
culturing time are well known in the art, and may be readily
determined. The in vitro technique produces essentially
monospecific monoclonal antibodies that are substantially free from
other specific antibodies. There is often a small amount of other
antibodies present since usual media contain exogenous serum (e.g.,
fetal calf serum). However, this in vitro method may not produce a
sufficient quantity or concentration of antibody for some
purposes.
To produce a much greater concentration of slightly less pure
monoclonal antibody, the desired hybridoma may be injected into
mice, preferably syngenic or semi-syngenic mice as described
hereinbelow. The hybridoma causes formation of antibody-producing
tumors after a suitable incubation time, which result in a
relatively high concentration of the desired antibody in the
bloodstream and peritoneal exudate (ascites) of the host mouse.
Although these host mice also have normal antibodies in their blood
and ascites, the concentration of these normal antibodies is
typically only about 5 percent of the monoclonal antibody
concentration.
Ascites fluids containing the antibodies were obtained from
10-week-old Balb/c mice (Scripps Clinic and Research Foundation),
which had been primed with 0.3 ml of mineral oil and injected
intraperitoneally with 3-50.times.10.sup.5 hybridoma cells.
Alternatively, antibodies were produced by injecting Balb/c mice
intraperitoneally with 0.3 ml Pristane (2,6,10,14
tetramethylpentadecane) (Sigma Chemical Co., St. Louis Mo.;
hereinafter Sigma). Seven to ten days later, 1-5.times.10.sup.6
hybridoma cells in log phase growth were injected intraperitoneally
into the same mice. Following a 7-14 day incubation period, ascites
fluid was removed from the mice. The concentration of antibody in
the ascites fluid was within the range of about 1 to about 10
mg/ml.
F. A-I Vesicle Formation
Lipid-protein complexes were prepared from cholesterol. The
lipid-protein complexes were formed into vesicles, purified by Bio
Gel P-4 chromatography. Large particles or vesicles were collected
for the radioimmunoassay setforth hereinbelow.
The reagents for this procedure were prepared in accordance with
the method set forth by Selinger and Lapidot, J. Lipid Res., 7, 174
(1966). Vesicle formation, the formation of a liquid protein
complex, was performed in accordance with the method of Pownall et
al., Biochem. Biophys. Acta, 713, 494-503 (1982). The lipid protein
complex was utilized in the isolation of various densities of
liquid proteins including VLDL, density of less than 1.006 g/ml;
LDL, density equal to 1.019 to 1.063 g/ml; and HDL, density equal
to 1.063 to 1.21 g/ml.
G. Enzymatic Cholesterol Assay
The enzymatic cholesterol assay was used to obtain a standard
against which the efficacy of the assay was tested. A free
cholesterol standard was prepared by serial dilution utilizing
cholesterol (U.S.P.) at original concentration of 1 mg/ml, and
diluted in 95 percent ethanol to give a final 6 standard points
ranging from 1000 ng/15 mCi to 31.25 ng/mCi.
A cholesteryl oleate standard was prepared in the same manner as
the free cholesterol standard (Gibco).
Assay solutions were prepared, and the assay of the plasma samples
obtained was performed against the above standards utilizing a
fluorometer at 325 nanometers, in accordance with the method set
forth by Gamble et al., J. Lipid Res., 19,1068-1070 (1978) and
Heider and Boyett, J. Lipid Res., 19, 514-581 (1978).
H. Chromatofocusing
Chromatofocusing was performed as a technique to separate HDL from
the admixture of lipoproteins found in the plasma pool analyzed in
accordance with the method of this invention. Chromatofocusing was
performed in accordance with the following method.
The HDL was isolated by ultracentrifugation and dialyzed into 25 mM
piperazine hydrochloride having a pH value of 5.8. Forty milligrams
of protein was applied to a 1.6 by 30 centimeter column of
Polybuffer exchanger 94 (Pharmacia), equilibrated with 25 mM
piperazine HCl having a pH value of 5.8, and the HDL was eluted
with Polybuffer 74 (Pharmacia) diluted 1 to 15 with an aqueous
solution having a pH value of 4.0. Six HDL subpopulations
corresponding to those described by Nestrock et al., Biochem.
Biophys. Acta, 753, 65-73 (1980) in eluting a pH maxima value of
5.0, 4.9, 4.8, 4.7, 4.5, and 4.4, respectively were collected and
desalted by chromatography on Sephadex G-75 (Pharmacia) that had
been equilibrated with LLB.
I. Iodination of Immunoaffinity Purified Goat Anti-Mouse
Immunoqlobulin
Iodination was performed utilizing the Enzymobead iodination
procedure and Enzymobeads obtained from Biorad, (Burlingame,
Calif.). The Enzymobead iodination was utilized to characterize the
antigens and antibodies for the solid phase radioimmunoassay as
discussed later herein.
The solid phase radioimmunoassay was performed utilizing a
quantitative aliquot of dilute antibodies.
The antibody dilution curve was prepared by the following method.
In a series of glass disposable tubes, the following were added in
0.100 ml aliquots: I.sup.125 antigen plus 9 percent BSA in barbital
buffer; competitor in barbital buffer; and first antibody in a 1:40
diluted normal mouse serum or optimum dilution in barbital buffer;
1:40 normal mouse serum in barbital without antibody was added to
control tubes. The aliquots were admixed and incubated for four
hours at four degrees C.
The tubes were placed on ice and 0.100 ml. of second antibody and
barbital buffer, normal goat serum or 100 percent TCA was added.
0.100 Ml of 100 percent trichloroacetic acid were placed in the
control tubes in lieu of normal goat serum. The admixture was then
incubated on ice for four hours and 2.0 ml of barbital buffer were
added at 4 degrees C. The admixtures were then spun for 30 minutes
at 2700 r.p.m. (1500 g) at 4 degrees C. The supernatant was
aspirated and discarded and counts of the I.sup.125 gamma emissions
were measured. Values for the ratio of bound antibodies to maximum
available antibody binding (B/Bo) were calculated as: ##EQU1##
where X is the iodinated sample; PPT is the protein precipitate;
TCA is the maximum trichloroacetic acid precipitated radioactivity;
and CPM is counts per minute.
J. Solvent Delipidization of Lipoproteins
The lipoprotein to be analyzed was dialyzed against 0.01 percent
EDTA having a pH value of 7.5 overnight (approximately 18
hours).
The resulting sample was dialyzed against 0.003 percent EDTA for
approximately 12 hours, and was then lyophilized at 10 to 20
milligrams of protein per tube. To each tube was added 35 ml of 1:1
absolute ethanol:anhydrous ether at 4 degrees C. This solution was
mixed.
Following mixture, the solution was incubated for 20 minutes at -20
degrees C. The solutions were then spun for 30 minutes at 2000
r.p.m. at 0 degrees C., and the supernatant was poured off.
The ethanol ether extraction as described above was performed twice
for a total of three extractions. Then 35 ml anhydrous ether at 4
degrees C. was added to the sample and incubated for 30 minutes at
-20 degrees C. The sample was spun at 2000 r.p.m. for 30 minutes at
-20 degrees centigrade, and the supernatant poured off and
discarded. Pellets were dried using nitrogen gas.
K. Protein Transfers
Proteins were transferred from the polyacrylamide gel (Biorad)
using a transfer cassette. Proteins were electrophoresed from the
polyacrylamide gel to nitrocellulose (Biorad) . The process
employed utilized a 2-hour electrophoreses at 400 milliamperes.
Following the transfer, active sites were blocked utlizing a
blocking buffer solution of 24 mM Tris, 192 mM glycine and 20
percent methanol. Incubation of the protein was performed in a two
step incubation at 4 degrees C.; one incubation of about 6 hours
and the second incubation of about 18 hours.
The proteins were then stained according to manufacturers
directions using Coomassie Brilliant Blue-250 (Sigma), destained
with 10 percent acetic acid, and dried.
A dilution of antibody was then prepared. Antibody was diluted in
blocking buffer (as prepared above). Gel membrane fragments,
prepared by the transfer process set forth hereinabove, were then
incubated with antibody dilutions for 6 hours at 4 degrees C. The
incubated gel membrane fragments were washed in a solution of 0.05
percent Tween-20 [polyoxyethylene (20) sorbitan
monolaurate](Sigma), 3.0 percent BSA (Sigma), 3 percent normal goat
serum (Sigma) in phosphate buffered-saline for about 30 minutes
followed by a LiCl-SDS wash in a solution of 0.5 M LiCl, 0.1
percent SDS in water, for about 10 minutes. This was followed with
another wash in the Tween-20 wash solution (as set forth above) for
about 30 minutes and blocking with the above blocking buffer
additionally containing Tween-20 (Tween Blocking Buffer) for about
18 hours.
Then, a 0.5 micro Ci/ml dilution of I.sup.125 immuno-purified goat
anti-mouse IgG was prepared in Tween-Blocking Buffer. The membranes
were incubated in this solution at minimum volume for 4 hours at 4
degrees C. on a horizontal rotator as follows: Tween-20 wash for
about 30 minutes; LiCl-SDS wash for 5-10 minutes at 20-22 degrees
C.; Tween-20 wash for 30 to 60 minutes; incubation in
Tween-Blocking Buffer at minimum volume for about 18 hours and
Tween-20 wash for about one hour. Membranes were then air dried on
absorbent paper for at least two hours.
L. Radioimmunoassays (RIA)
Solid-phase RIAs were performed in polyvinyl chloride microtiter
plates (Falcon, Becton-Dickenson Rutherford, N.J.) as solid
supports. The plates were coated with antigen at about 1 microgram
per well in 50 microliter aqueous solutions in phosphate-buffered
saline (PBS) at pH 7.3. The plates were then maintained for 3 hours
at 37 degrees C. The solution was removed, and the wells were
washed 3-4 times with PBS. Non-specific binding sites were then
blocked.
The antigen-coated plates were admixed with 50 microliter dilutions
of mouse serum, hybridoma culture supernatants or ascites fluids
and the admixtures were maintained for about 16-18 hours at 4
degrees C. The solid and liquid admixtures were separated, and the
wells were rinsed. Antibody binding was detected by a second
admixture following a maintenance period of about 4 hours at 4
degrees C. using 10 ng/well of .sup.125 I-goat anti-mouse Ig (4-4
micro-Ci/microgram as the indicating means.
Fluid-phase RIAs were performed in triplicate in 12.times.75-mm
glass tubes. To 0.1 ml of radioiodinated antigen (human HDL,
apo-A-I, or apo-A-II) were admixed 0.1 ml of buffer or competing
antigen if present, and 0.1 ml of varying dilutions of mouse
hybridoma antibody diluted in 1:60 normal mouse serum. All buffers
also contained 5 percent dextran (MW, 40,000). The admixtures were
maintained for a time period of 18 hours at 4 or 24 degrees C., at
the end of which time 0.1 ml of precipitating second antibody (goat
anti-mouse Ig serum) was added. The second antibody was diluted to
give a slight antibody excess and complete precipitation of mouse
immunoglobulin. That admixture was maintained for a time period of
4 hours, after which time, 2 ml of cold borate buffer was added,
and the tubes were centrifuged at 2000.times.g for 30 minutes.
Supernatants were removed by aspiration, and the .sup.125 I content
of the pellet was determined in a gamma radiation counter.
Maximum precipitable radioactivity was determined by replacing the
goat anti-mouse Ig serum with 100 percent trichloroacetic acid. The
minimum precipitable radioactivity or zero binding control (B) was
determined by replacing the specific hybridoma antibody with an
irrelevant hybridoma antibody of the same heavy chain class.
The minimum precipitable radioactivity or zero binding control (B)
was determined by replacing the specific hybridoma antibody with an
irrelevant hybridoma antibody of the same heavy chain class.
Data were calculated as either total counts bound or as percent of
.sup.125 I-antigen bound = ##EQU2## where X =mean radioactivity
precipitated in the presence of a given amount of specific
antibody, and TCA is the maximum trichloroacetic acid-precipitable
radioactivity. Competitive radioimmunoassays were analyzed by
logit-transformation to compare qualitative and quantitative
epitope expression. The variance of the slopes of the competitive
inhibitition dose titration regression lines was compared using the
Student's t test.
M. Radioiodination
Radioiodination of HDL, apo-A-I, apo-A-II, and immunochemically
purified goat anti-mouse Ig was performed enzymatically using
immobilized lactoperoxidase and glucose oxidase Enzymobeads,
(Biorad). For selected studies, HDL was labeled also with the
Bolton-Hunter reagent. The specific activity of .sup.125 I in each
preparation of .sup.125 I-HDL was trichloroacetic
acid-precipitable, and 5 percent of the radioactivity was
extractable into organic solvent. Greater than 99 percent of the
radioactivity of .sup.125 I-apo-A-I, .sup.125 I-apo-A-II ranged
from 20.9 to 25.5 micro-Ci/microgram.
The preceding description of the invention is set forth by way of
example and not of limitation. Others skilled in the art may
discern additional applications of the invention that are fully
within the scope and spirit of the invention set forth herein.
* * * * *