U.S. patent application number 10/005710 was filed with the patent office on 2003-05-29 for saliva immunoassay for detection of antibodies for cardiovascular disease.
Invention is credited to Vojdani, Aristo.
Application Number | 20030100036 10/005710 |
Document ID | / |
Family ID | 21717302 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030100036 |
Kind Code |
A1 |
Vojdani, Aristo |
May 29, 2003 |
Saliva immunoassay for detection of antibodies for cardiovascular
disease
Abstract
A method for diagnosing the likelihood and severity of
cardiovascular disease in a patient is disclosed. The method
determines the levels of antibodies against autoantigens, including
myosin, oxidized LDL, .beta.-2-glycoprotein, heat shock protein-60,
platelet glycoprotein, and immune complexes. It then compares the
results to normal levels to determine the likelihood and severity
of cardiovascular disease.
Inventors: |
Vojdani, Aristo; (Los
Angeles, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
21717302 |
Appl. No.: |
10/005710 |
Filed: |
November 8, 2001 |
Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
G01N 33/564 20130101;
G01N 33/6893 20130101; Y10S 436/811 20130101; G01N 2800/32
20130101 |
Class at
Publication: |
435/7.92 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543 |
Claims
What is claimed is:
1. A method for diagnosing the likelihood and severity of
cardiovascular disease in a patient, comprising the steps of: a)
determining a level of antibodies against an autoantigen or a
corresponding recombinant antigen or synthetic peptide for
cardiovascular disease in a sample from said patient; and b)
comparing the level of antibodies determined in step a) with normal
levels of said antibodies, wherein (i) normal levels of autoantigen
antibodies for cardiovascular disease indicate optimal conditions;
and (ii) higher than normal levels of autoantigen antibodies for
cardiovascular disease indicate ongoing pathology or prediction of
early pathogenic reaction for cardiovascular disease.
2. The method according to claim 1, wherein the autoantigen for
cardiovascular disease is selected from the group consisting of
myosin, oxidized LDL, heat shock protein-60,
.beta.-2-glycoprotein-1, platelet glycoprotein, and immune
complexes.
3. The method according to claim 1, wherein determining the level
of antibodies in steps a) and b) is accomplished using an
immunoassay.
4. The method according to claim 3, wherein the immunoassay is an
ELISA test.
5. The method according to claim 1, wherein the antibodies in steps
a) and b) is measured from saliva.
6. The method according to claim 5, wherein the measured antibodies
are IgA.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to a saliva immunoassay for detection
of antibodies for cardiovascular disease.
[0003] 2. Description of the Related Art
[0004] Cardiovascular disease is predicted to be the most common
cause of death worldwide by the year 2020. Half of heart disease
patients lack established risk factors such as elevated lipids,
hypertension, tobacco abuse, and positive family history.
Additionally, these risk factors are generally associated with the
disease, and the exact mechanism by which they may contribute to
the development of atherosclerosis is not clear. However, previous
and recent studies point to a linkage between infection with
different bacteria and heart disease in the other 50% of observed
incidences. Pathogenesis of the disease induced by infectious
agents is described by three different mechanisms of action:
release of toxins or superantigens, induction of inflammation, and
molecular mimicry or cross-reactivity. This may result in plaque
formation or antimyosin cellular and humoral immunity and
subsequently, to myocarditis or other autoimmune diseases.
[0005] Through the years, many reports have incriminated various
infectious agents in the pathogenesis of autoimmune disease.
Moreover, the American College of Cardiology has issued a list of
harmful pathogens as possible links to heart disease.
[0006] Traditionally, it is assumed that infectious agents induce
disease by direct tissue damage via secretion of toxins or
different antigens, particularly myosin. These toxins may directly
or indirectly induce tissue damage and cause release of tissue
antigens.
[0007] An infectious agent can be taken up by macrophages and
transferred to the bloodstream and arteries. When a macrophage
burrows into the wall of a blood vessel to take in irritants such
as LDL and oxidized LDL, it transfers the infectious agent into the
neighboring arterial cells. Infected arterial cells then attract
more macrophages and other inflammatory responses, such as
platelets, and then die. If this vicious cycle of inflammation
continues, it can result in fibrous lesions or plaque formation.
When pieces of the plaque break loose, they can start blood clots
and cause heart attack.
[0008] Another mechanism by which infectious agents can cause
autoimmune disease is molecular mimicry. Molecular mimicry is
defined as structural similarity between antigens coded by
different genes. Antigenic cross-reactivity between host and
bacteria is exemplified by blood group substances and bacterial
polysaccharides; cardiac tissue and streptococcal proteins; and
kidney tissue and E. coli polysaccharides. Viruses may also induce
autoimmune responses through shared determinants on molecules
notably present on host cells, by altering the host immune system,
or by causing the expression or release of "normally sequestered"
self antigens.
[0009] Harmful pathogens may be the cause of many human diseases.
These pathogens may induce their pathologic response through one of
the above-mentioned mechanisms of action.
[0010] Many viruses, bacteria, and even parasites are claimed to
affect atherosclerosis plaque deposition. Among them, Chlamydia
pneumoniae probably has the strongest association with
atherosclerosis. There is a close relationship between C.
pneumoniae infection, IgG and IgM titers, and increased evidence of
MI, CVA, and peripheral vascular disease (PVD). C. pneumoniae
antigens are found in atherosclerosis plaques, and T-cell reactions
to these antigens have been demonstrated. Experimental models
illustrate the pathogenic role of C. pneumoniae and the unique heat
shock protein (HSP)-60. Other major atherosclerosis-associated
pathogens are Helicobacter pylori, Epstein-Barr virus and
cytomegalovirus. For some pathogens, interfering pathogenic
mechanisms have been described, such as cytomegalovirus
gene-induced proliferation of smooth-muscle cells. From data
showing a correlation between increased atherosclerosis incidence
and chronic bronchitis, as well as periodontitis, it has been
suggested that any infectious agent, and especially multiple
chronic infections, could result in accelerated atherosclerosis
formation. This multiplicity was confirmed recently in experimental
animal models. There is no doubt therefore, that chronic infections
with specific or nonspecific infectious agents can contribute to
the acceleration of atherosclerosis development, either by
nonspecific mechanisms [hypercoagulation and increased adhesion
molecule and elevated C-reactive protein (CRP) levels] or by more
specific mechanisms, such as induction of HSP-60 expression and
eventually pathogenic anti-HSP-60 antibody production.
[0011] Autoantibodies are frequently found in the sera of
virus-infected individuals, both during and after infection. For
example, after infection with Epstein-Barr virus (EBV), antibodies
reacting with intermediate filaments of cells, immunoglobulin or
thyroglobulin were detected (Oldstone, J. Autoimmunity,
2(Suppl.):187-194, 1989; Srinivasappa et al., J. Virology,
57:397-401, 1986; Talal et al., J Clin. Invest., 85:1886-1871).
Myosin Antibody
[0012] Myosin accounts for over 50% of muscle proteins. Along with
actin, myosin is involved in muscle contraction. Myosin is one of
the largest proteins in the body, with a molecular mass of 500 kDa.
Due to its large mass, antigenic mimicry between infectious agents
and myosin molecules is highly probable.
[0013] It is now well-known fact that infectious agents are
associated with human myocarditis. The development of autoimmunity
to myocardial antigens has been widely recognized after myocardial
infarction or after cardiac surgery. Autoantibodies to heart tissue
in patients with rheumatic carditis, post-myocardial infarction and
post-pericardiotomy syndromes have been described. Antibodies
against heart tissue can also occur in patients with post-infection
myocariditis, dilated cardiomyopathy, rheumatic carditis, Chagas
disease, and adriamycin cardiotoxicity. It has also been observed
that serum from patients with myocarditis reacted specifically with
sarcolemmal and cytoplasmic heart antigens. Moreover, serum samples
containing circulating heart antibodies also induced
complement-mediated myocyte lysis and antibody-dependent
cell-mediated cytotoxic reactions in vitro, suggesting that they
may be pathogenic in myocarditis.
[0014] It is now clear that some patients with active myocarditis
or cardiomyopathy carry antibodies to the mitochondrial adenine
nucleotide translocator. Such patients, whose serum inhibit in
vitro ADP-ATP translocator activity, have reduced cardiac function
relative to their counterparts without these antibodies. The
existence of multiple heart-reactive antibodies in autoimmune heart
disease is consistent with the presence of multiple tissue- and
organelle-specific antibodies in both systemic lupus and autoimmune
thyroiditis.
[0015] For years it has been known that Chlamydia can induce
cardiovascular disease in experimental animals. This
Chlamydia-mediated heart disease in mice can be induced by
antigenic mimicry of a heart muscle-specific protein, thus
providing a molecular link between Chlamydia infections and heart
disease. Since many infectious agents have been implicated in heart
disease, it is not surprising that organisms other than Chlamydia
can also supply mimicking epitopes. Indeed, Machmaier, K. et al.,
in a study published in Nature Medicine in August 2000, screened
public databases for proteins sharing the pathogenic mouse
M7A.alpha. peptide MA'ST motif (whose amino acid sequence is as
follows: SLKLMATLFSTYASA). This motif is found in proteins from a
multitude of viruses, bacteria, fungi, and protozoa, which are
involved in cardiovascular disease.
[0016] Cross-reactive antibodies appear to be quite common in
patients with rheumatic fever. Some of these autoantibodies could
be absorbed by certain streptococcal strains, and some reacted
specifically with cardiolipin and tropomyosin. Group A and mutant
streptococci share a common epitope with cardiac myosin, which may
be associated with the heavy meromyosin region of the molecule. In
Chagas disease--caused by the protozoan parasite Trypanosoma
cruzi--heart autoantibodies react with laminin, while Chagasic
cardiomyophathy may be due to recognition of the
calcium-sequestering ATPase in the sarcoplasmic reticulum.
[0017] Oxidized LDL Antibody
[0018] Oxidized Low Density Lipoprotein (oLDL), the prime candidate
for an autoantigen, plays a critical role in the development and
progression of atherosclerosis and other vascular diseases. It is
incriminated in foam cell generation through uptake by the
unregulated scavenger receptors on macrophages.
[0019] Recent evidence suggest that autoantibodies against
oxidatively modified LDL can be used as a parameter that
consistently mirrors the occurrence of oxidation processes taking
place in vivo. In fact, elevated levels of autoantibodies against
oLDL have been detected in the bloodstream of patients with
coronary artery disease. Moreover, recent studies indicate a
correlation between autoantibodies against oLDL and the progression
of carotid atherosclerosis. Increased serum concentrations of oLDL
have also been described in various diseases such as pre-eclampsia
and systemic lupus erythematosus (SLE).
[0020] Heat Shock Protein 60 (HSP60) Antibody
[0021] Heat Shock Protein 60 (HSP60), also known as CPN60, is an
abundant protein synthesized constitutively in the cell that is
induced to a higher concentration after brief cell stress or shock.
It is present in all species analyzed so far and exhibits a
remarkable sequence homology among various counterparts in
bacteria, plants, and mammals: more than half of the residues are
identical between bacterial and mammalian HSP60. The ubiquitous
occurrence and remarkable evolutionary conservation suggests that
HSP60 may play an essential role in the cell. It is now believed
that HSP60, which is localized in mitochondrial matrix in
eukaryotes, interacts with multiple proteins during translocation
and/or folding. E. coli HSP60 (GroEL) has been shown to catalyze
folding of many proteins in vitro and is involved in the assembly
of bacteriophage lambda proteins during infections. TCP-I, a member
of the HSP60 family, has similar functions to HSP60 but is
localized within the cytoplasm. Bacterial HSP60 proteins are major
targets of immune responses during infection, and the highly
conserved nature of bacterial and mammalian HSP60 has led to
speculation that immune reactivity to these stress proteins may be
a component of certain autoimmune diseases and atherosclerosis. In
fact, G. Wick (Innsbruck, Austria) first claimed that HSP60 is
involved in atherosclerosis. Anti-HSP60 antibody titers correlate
with the degree of atherosclerosis in carotid ultrasound studies.
The increase in anti-HSP60 antibody levels could result from direct
turbulence damage to bifurcated arteries or could be caused by
infectious agents (e.g. C. pneumoniae) releasing HSP60, which
becomes immunogenic. T-cell lines cultured from the atherosclerosis
plaque proliferate when exposed to HSP60 and both the
autoantibodies, as well as the autoantigen can be found in the
plaque. Finally, active immunization of rabbits and
apolipoprotein-E or low-density lipoprotein (LDL)-receptor knockout
mice with HSP60 leads to accelerated formation of atherosclerosis
plaques.
[0022] Anti-.beta.2-Glycoprotein-1
[0023] .beta.2-Glycoprotein-1 (.beta.2GP1) is a normal glycoprotein
synthesized by the liver that behaves as an anti-coagulant and is
also an anti-atherogenic agent. This glycoprotein, also known as
apolipoprotein-H, is a human plasma glycoprotein that consists of a
single polypeptide of 326 amino acids with a molecular weight of 50
kDa.
[0024] It is now widely accepted that P2GP1 is an absolute
requirement for the binding of "antiphospholipid" (aPL) Abs
purified from patients with autoimmune disease when assayed using
anionic phopholipid ELISAs. These autoantibodies are of
considerable clinical importance because of their association with
arterial and venous thrombosis, recurrent fetal loss, and
thromobocytopenia. The interaction of autoantibodies with
.beta.2GP1 may be important in relation to the pathogenesis of
thrombosis in vivo. .beta.2GP1 is known to bind to negatively
charged surfaces as well as to activated platelets and to act as an
inhibitor of the intrinsic blood coagulation pathway in vitro.
[0025] .beta.2GP1 also binds to oLDL. This binding of .beta.2GP1 to
oLDL reduces the uptake of oLDL by scavenger receptors on
macrophages. In fact, .beta.2GP1 is found in the atherosclerosis
plaque and is the target antigen in antiphospholipid syndrome
(APS). Antibody titer to .beta.2GP1 correlates with
atherosclerosis. In in vitro conditions, these antibodies enhance
uptake of oLDL by macrophages.
[0026] Recently, in a classical study, accelerated atherosclerosis
plaque formation was induced in LDL-receptor-deficient mice by the
passive transfer of lymphocytes from the lymph nodes and spleens of
mice actively immunized with .beta.2GP1.
[0027] Anti-Platelet Glycoproteins
[0028] A number of diseases and syndromes are thought to involve
antibody, or immune complex-mediated platelet destruction. Among
these are both the acute and chronic forms of idiopathic
thrombocytopenic purpura; the closely related thrombocytopenia of
systemic lupus erythematosus; quinidine, apronalide, and other
drug-induced thrombocytopenias; post-transfusion purpura; neonatal
isoimmune thromobocytopenia; and the alloimmunization that renders
multi-transfused patients refractory to random platelet
transfusion.
[0029] Platelet function and number can both be affected in
immune-mediated diseases; however, thrombocytopenia is by far the
more common finding. Abnormalities of platelet number and function
can occur via any of several immune mechanisms. Both humoral and
cell-mediated immune mechanisms can produce thrombocytopenia. The
most commonly considered, although by no means the most commonly
noted, immune mechanism for thrombocytopenia is the formation of
specific antiplatelet autoantibodies. Platelets have a large number
of immunogenic structures on their surface, with the glycoprotein
IIb/IIIa (GP IIb/IIIa) complex being the most numerous. It is not
surprising, therefore, that autoantibodies directed against
epitopes on the GP IIb/IIIa complex are the most frequent when the
specificity of the autoantibodies have been determined in blood.
Platelet autoantibodies are usually of the IgG immunoglobulin
class, although IgA, IgD, and IgM autoantibodies have been
demonstrated occasionally. Complement has also been found on
surface of platelets in clinical syndromes consistent with
increased immune-mediated platelet destruction. However, most
autoantibodies are not complement fixing, and removal of the
immunoglobulin-coated platelets occurs in the spleen and other
sites of reticuloendothelial tissue.
[0030] Immune Complexes
[0031] Immune complexes are formed when antigens bind with
antibodies. Antigen-antibody complexes can activate the complement
cascade and bind the C1q component of complement and form
pathologic complexes.
[0032] Both exogenous and endogenous antigens can trigger
pathogenic immune responses that result in immune complex (IC)
disease. Because circulating IC's play such an important part in
many diseases, including autoimmunity, neoplasms, infectious
diseases due to bacteria, viruses, and parasites, and other
unclassified disorders, the demonstration of IC's in tissues and
biological fluids has achieved rising prominence.
[0033] There are a number of cases in which immune complexes assays
are helpful in the diagnosis and monitoring of disease activity,
for example, lupus and arthritis.
[0034] The fact that SLE is considered the prototype of human
immune complex disease has led to studies of SLE with almost every
type of immune complex assay developed. A high incidence of
positive tests and disease activity has been uniformly reported.
There is considerable evidence that DNA-anti-DNA complexes are
involved in the pathogenesis of SLE. Immune complex determinations
coupled with detection of serum antibodies to native DNA and
determinations of levels of hemolytic complement (CH50) in serum
are useful diagnostic tests. Most studies have found a correlation
between positive immune complex assays and antibodies to native
DNA, which is the most important laboratory marker of lupus.
Several serial studies have indicated that the C1q solid-phase
assay correlates better with disease activity than do other immune
complex tests.
[0035] The role of circulating immune complexes (CIC) in cancer is
of particular interest because tumors express antigens that elicit
both cellular and humoral immune responses. CMI in tumor-bearing
host is blocked by CIC or "blocking factors" in circulation.
Antigen-antibody complexes are formed by noncovalent hydrophobic
coulombic hydrogen bonds. The nature and quantity of CIC detected
in circulation is dependent upon the dynamics of formation,
clearance, and tissue deposition of immune complexes. Immune
complexes cause tissue injury through the terminal lytic component
of activated complement system. Since activated complement
components are strong chemotactic agents, leukocytoclastic
vasculitis is seen in cancer patients with high levels of CIC.
[0036] Manifestation of Antibodies
[0037] The deposition of antigens in the gut has been shown to lead
to the production of IgA antibodies in secretions at sites distant
from the gut, such as colostrums, lacrimal and salivary secretions
in man and salivary secretions in rhesus monkeys and in rats.
[0038] A general conclusion therefore is that the secretory immune
system can be stimulated centrally and that precursors of
IgA-producing cells migrate from the gut-associated lymphoid tissue
to several secretory sites in addition to the lamina propria of the
gut itself. Therefore, if antigens are injected into the submucosal
tissues, they are likely to induce serum IgG antibodies as well as
secretory IgA antibodies in saliva. However, if it is applied
topically to the skin or to the intraepiethelial tissue, secretory
IgA is the main product which is detected in saliva. The role of
topically applied antigen in the localization and persistence of
IgA responses has been demonstrated in several secretory sites,
including the respiratory tract, oral cavity, gut, and vagina.
[0039] The evidence that cells migrate from the gut to various
secretory tissues, and that immunization in the gut leads to
antibodies at various secretory sites has led to the concept of a
common mucosal system. However, this concept may be an
oversimplification, since although immunization in the lung may
lead to antibodies in distant secretory sites, such as salivary
glands and immunization in the lacrimal glands has also been shown
to lead to the production of antibodies in saliva. Thus, with firm
evidence that antigen deposition in the gut may lead to antibodies
not only in the gut but also in saliva, lungs, lacrimal secretions
and genitourinary tract, it is probably more correct to designate
the system as an enteromucosal system.
[0040] Saliva is a source of body fluid for detection of an immune
response to bacterial, food, and other antigens present in the oral
cavity and gastrointestinal tract. Indeed, salivary antibody
induction has been widely used as a model system to study secretory
responses to ingested material, primarily because saliva is an easy
secretion to collect and analyze. It seems to be a general feature
that salivary IgA antibodies can be induced in a variety of species
in the absence of serum antibodies. This has been demonstrated
after immunization with particulate bacterial antigens in human
could selectively induce an immune response to Streptococcus mutans
by oral administration of the antigen. This route of administration
resulted only in antibody production in saliva and not in serum.
Similar mucosal immune response in the form of saliva IgA did occur
in monkeys, rabbits, rats, and mice after oral administration of
Streptococcus mutans or other bacteria.
[0041] This lack of production of IgG, but IgA production in saliva
after oral or intragastric administration of bacterial antigens is
shown in the following table.
1TABLE 1 Induction of salivary IgA antibody after stimulation of
gut associated lymphoid tissue Route of Salivary IgA Serum Antibody
Species Antigen Administration Production Production Human
Streptococcus Mutans Oral ++ - Monkeys Streptococcus Mutans
Intragastric ++ - Rabbits Penumococcus or BGG Intragastric ++ -
Rats Streptococcus Mutans Oral ++ - Mice Streptococcus Mutans or
Intragastric ++ - Ovalbumin
[0042] As indicated in this table, oral or intragastric
administration of dietary soluble proteins such as bovine
gammaglobulin (BGG) and ovalbumin or eggalbumin resulted in
salivary IgA production but not in any antibody production in
serum. For these reasons, saliva has been selected not only because
of its relevance in oral disease, but mainly because it is an
accessible fluid, easy to collect, and is thought to show
representative responses in secretions after central or
intragastric immunization. However, if both saliva IgA and serum
IgG antibodies are detected in the same patient, it means that this
individual has been primed with the antigen orally as well as
systematically.
[0043] This IgA production in saliva and IgG production in serum is
dependent upon antigen dosage as well as the integrity of the gut.
For example, a single intragastric immunization with 1 mg of
eggalbumin led to oral tolerance but did not lead to detectable
secretory IgA antibodies, whereas 10 mg of ovalbumin led to
systemic tolerance, but to a significant level of salivary IgA
antibodies. Thus, detection of high levels of antibody in saliva is
an indication of the body's exposure to significant levels of
antigenic stimulation.
[0044] While this concept of oral tolerance to high doses of
soluble antigen may be correct, certain conditions--such as
overloading of the GI tract with bacterial toxins--may not lead to
oral tolerance. This is due to the fact that bacterial toxins will
cause the opening of tight junctions, which will in turn lead to
the absorption of ingested proteins and bacterial antigens from the
gut in significant amounts. This excessive uptake of bacterial,
fungal, viral, and dietary proteins into the circulation may induce
immune response first in the form of IgM, and thereafter in the
form of IgG and IgA antibodies in the serum, all of which may lead
to different clinical conditions.
SUMMARY OF THE INVENTION
[0045] One aspect of the preferred embodiment is a method for
diagnosing the likelihood and severity of cardiovascular disease in
a patient. This method includes (a) determining a level of
antibodies against an autoantigen or a corresponding recombinant
antigen or synthetic peptide for cardiovascular disease in a sample
from the patient and (b) comparing the level of antibodies
determined in step (a) with normal levels of the same
antibodies.
[0046] Possible outcomes for the comparison include (i) normal
levels of autoantigen antibodies for cardiovascular disease
indicate optimal conditions; and (ii) higher than normal levels of
autoantigen antibodies for cardiovascular disease indicate ongoing
pathology or prediction of early pathogenic reaction for
cardiovascular disease.
[0047] In one embodiment, autoantigens are myosin, oLDL, HSP60,
.beta.2GP1, platelet glycoprotein, or immune complexes.
[0048] In one embodiment, an ELISA test is used to determine the
levels of antibodies.
[0049] In one embodiment, the antibodies, preferably IgA
antibodies, are measured from saliva.
[0050] Further objects, features and other advantages of the
preferred embodiments become apparent from the ensuing detailed
description, considered together with the appended figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 is a graph showing saliva IgA antibodies against
infectious agents, specific and non-specific autoantigens involved
in cardiovascular disease and autoimmune disease expressed by
O.D.'s from patients with possible cardiovascular disease.
[0052] FIG. 2 is a graph showing saliva IgA antibodies against
infectious agents, specific and non-specific autoantigens involved
in cardiovascular disease and autoimmune disease expressed by
O.D.'s from healthy controls.
[0053] FIG. 3 is a graph showing the mean and standard deviation of
thirty saliva samples of IgA antibody levels against myosin.
[0054] FIG. 4 is a graph showing the mean and standard deviation of
thirty saliva samples of IgA antibody levels against oLDL,
.beta.-2-Glycoprotein, and HSP-60.
[0055] FIG. 5 is a graph showing the mean and standard deviation of
thirty saliva samples of IgA antibody levels against immune
complex.
[0056] FIG. 6 is a table showing the correlation of reactivity of
saliva IgA antibody against infectious agents and autoantigens to
cardiovascular disease.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0057] The inventor has developed a single test that will
accurately inform the physician of important clinical conditions
required to diagnosing in patients the likelihood and severity of
cardiovascular disease. The test utilizes a highly sensitive and
accurate ELISA test method that measures saliva IgA specific
antibody titers to the purified antigens or a corresponding
recombinant antigen or synthetic peptide from autoantigens.
[0058] Such quantitative and comparative test results allow the
physician to determine cardiovascular autoimmune disease. The test
thus helps the clinical investigator to evaluate and treat patients
by using immunological responses as indications of cardiovascular
autoimmune disease.
[0059] The test involves determining the level of antibodies
against an autoantigen or a corresponding recombinant antigen or
synthetic peptide for cardiovascular disease. The level of
antibodies against autoantigens for cardiovascular disease is
compared between test samples of a patient and normal controls. A
higher than normal level of antibodies against autoantigens for
cardiovascular disease, such as myosin peptides, oLDL, HSP60,
.beta.2GP1, platelet glycoprotein, and immune complexes, indicate a
presence or possibility of cardiovascular disease.
[0060] Defined autoantigens involved in atherosclerosis include the
following: myosin, oxidized LDL (oLDL), Heat Shock Protein-60
(HSP60), .beta.-2-Glycoprotein-1 (.beta.2GP1), cardiolipin,
platelet glycoproteins, and immune complexes.
[0061] Chronic infections and the resulting production of
antibodies against them can accelerate atherosclerosis by means of
specific and non-specific mechanisms. Myosin, HSP60, oLDL, and
.beta.2GP1 are defined autoantigens involved in specific mechanisms
for the induction of cardiovascular disease. Platelet
glycoproteins, immune complexes, endothelial cell antigens and
intracellular adhesion molecules are indirect factors involved in
non-specific mechanisms for the induction of cardiovascular
disease.
[0062] The detection of specific biomarkers, such as myosin, oLDL,
.beta.2GP1, and HSP60 antibodies in saliva along with non-specific
markers, such as platelet glycoprotein antibodies, elevated immune
complexes, endothelial cell antibodies and intracellular adhesion
molecules may detect ongoing pathology or predict early pathogenic
reaction. Because of this, preventive measures may be taken to
reverse the course of action of the disease. As compared to healthy
controls, patients with elevated lipid profile profile
(Cholesterol/HDL ratio of>7 and a blood pressure>140/80) have
a much higher level of antibodies against the defined autoantigens
(myosin, HSP60, oLDL, and .beta.2GP1). There may also possibly be
elevated levels of immune complexes, C-reactive proteins, and
intracellular adhesion molecules.
[0063] Although other materials and methods similar or equivalent
to those described herein can be used in the practice or testing of
the preferred embodiments, the preferred method and materials are
now described.
EXAMPLE 1
General Procedures for Immunity Panel for Cardiovascular
Disease
[0064] For the test, about 2 ml of patient saliva was collected.
Saliva specimen was kept at -20.degree. C. until the performance of
the assays.
[0065] The purified antigens were immobilized by attachment to a
solid surface, such as a microtiter plate. The saliva sample was
added to the plate followed by incubation and washing. Antibody
bound to antigen was revealed by adding enzyme labeled monoclonal
antibody directed against the first immunoglobulin. After addition
of substrate, color development was measured by microtiter reader
at 405 nm. The intensity of the color was directly related to the
concentration of antibodies to these antigens present in patient's
specimen.
[0066] Saliva samples were collected in the morning, before
brushing teeth, smoking, or drinking. 2 ml of saliva was collected.
Saliva was collected after a gentle chewing action in a test tube
containing 0.1 ml of preservative. Saliva specimen was kept at
-20.degree. C. until the performance of the assays.
[0067] Calibrator samples I, II, III as well as positive and
negative controls were used.
[0068] The wash buffer was made as follows: in a 500 ml graduated
cylinder, 450 ml of water was added to 50 ml of 10.times.wash
buffer. It was mixed and transferred to a 500 ml squeeze bottle and
stored at 2-8.degree. C. until used.
[0069] Substrate buffer and Stop Solution were ready for use.
(CAUTION: Both solutions are caustic: avoid contact with skin and
eyes, rinse with copious amounts of water in event of contact.)
[0070] The substrate solution was prepared only immediately before
use. For 1-5 strips, 5 ml of substrate buffer were pipeted into the
empty substrate reconstitution bottle and 1 substrate tablet was
dropped in. The bottle was shaken to dissolve the tablet. The
buffer was used within an hour after reconstitution as
recommended.
[0071] Reagent and specimen were prepared as follows. All strips to
be used, reagents, controls, and patient's specimen were
equilibrated to room temperature (22-25.degree. C.). Patient's
specimen was diluted 1:100 with specimen diluent buffer: 20 .mu.l
specimen+2.0 ml buffer. Specimen dilutions were made in tubes prior
to addition to wells and thoroughly mixed before dispensing. Only
one well per test was necessary. For every determination, six
strips (1-6) of eight wells were needed to run blank calibrators
and four patient's samples.
[0072] Well Identification: 6 antigen-coated strips were used. Each
was divided into 8 equal-sized squares. The top 6 squares were
labeled "BLANK", the next 3 were "CALIBRATOR I, CALIBRATOR II, and
CALIBRATOR III". The last 4 were labeled "SPECIMEN I, SPECIMEN II,
SPECIMEN III and SPECIMEN IV". Note: Blank and calibrators may need
to be positioned differently if specified by the instrument
manufacturer. For each test performance the following wells were
used: One blank well (reagent blank), one well each for Calibrator
I, II and III, and one well each for patient specimens.
[0073] The assay procedure was as follows: 100 .mu.l of specimen
diluent buffer was pipeted into all eight wells of strip# 1, 2, 3,
4, 5, and 6. The contents were discarded and the addition of
specimen diluent buffer to the same wells was repeated. Then, 100
.mu.l of each calibrator or patient specimen dilutions were
pipetted into identified wells; being careful to avoid splashing
and air bubbles because cross-contamination between the wells may
cause erroneous results. Then, 100 .mu.l of specimen diluent buffer
was pipeted into a blank well. The reagents were dispensed slowly
to avoid splashing and air bubbles. If large air bubbles occurred,
they were aspirated or the plate was gently shaken. The plate was
covered and incubated for 60 minutes at room temperature
(22-25.degree. C.). Specimen was shaken from the wells into a
container containing disinfectant solution or aspirated with a
vacuum device. All wells were empty prior to filling with
1.times.wash buffer and allowing a 10-20 second soak time. The
wells were emptied by shaking into a disposal container or
aspirated. Washing was repeated three more times. The inverted
plate was tapped onto a paper towel to completely remove all
residual liquid. Then, 100 .mu.l of anti IgA conjugate was added to
the tested strips. The plate was covered and incubated for 60
minutes at room temperature (22-25.degree. C.). The liquid was
shaken or aspirated from all the wells and washed four times. Then,
100 .mu.l of p-NPP substrate was added to all the wells at timed
intervals that corresponded to the reading time of the instrument
used to read the reactions. The 45-minute incubation time was
started as substrate was added to the first well. The plate was
covered and incubated 45 minutes at 22-25.degree. C. (The assay may
be incubated for less than 45 minutes if incubation temperature is
higher than 25.degree. C.). Then, 50 .mu.l of 3N NaOH was pipeted
into all the wells at the same timed intervals that the p-NPP was
added. The plate was shaken for 1-2 minutes by hand or on a shaker,
avoiding splashing. The bottom of the wells was wiped with a
non-abrasive paper towel and the instrument was zeroed on the blank
well. The OD was read at 405.+-.5 nm within 30 minutes, and
reactions recorded.
[0074] The ELISA values for the calibrators used in this test
system were according to the samples used in the test.
[0075] ELISA values for each test specimen were determined using
the following formula:
ELISA values of test specimen=Values of calibrator.times.Absorbance
of test specimen/ Absorbance of calibrator
EXAMPLE 2
Test for Myosin Antibody
[0076] Myosin pathogenic peptide "SLKLMATLFSTYASA" was synthesized
by a robotic multiple peptide synthesizer and resin was used as
solid support. Peptide was characterized by reversed-phase HPLC and
electrospray mass-spectrometry with purity greater than 80%. This
peptide was bound to bovine serum albumin and used for coating
microtiter plates.
[0077] Each well of microtiter plate was coated with 3 .mu.g
peptide in 0.1 M carbonate buffer pH 9.5. After 24 hours incubation
and washing, 200 ml of 2% BSA was added and incubated for an
additional 2 hours. Plates were washed, dried, and used for
measurement of myosin antibodies. The test specimen was added to
the plate followed by incubation and washing. The procedure in
Example 1 was followed to measure for the myosin antibodies.
[0078] The ELISA values for the calibrators used in this test
system were as follows: Calibrator I=7.5, Calibrator II=15, and
Calibrator III=30.
[0079] The ELISA values for each test specimen were determined
using the formula in Example 1.
EXAMPLE 3
Test for Oxidized LDL Antibody
[0080] Wells of microtiter polystryrene plate were coated with 3
.mu.g of oLDL in 100 .mu.l of 0.1 M carbonate buffer pH 9.6 and
were kept overnight at 4.degree. C. The plates were then washed
with PBS and blocked with 2% BSA for 2 hours at room temperature.
Plates were washed, dried, and used for detection of antibodies
against oLDL. The test specimen was added to the plate followed by
incubation and washing. The procedure in Example 1 was followed to
measure for oLDL antibodies.
[0081] The ELISA values for the calibrators used in this test
system were as follows: Calibrator I=37, Calibrator II=75,
Calibrator III=300, and Calibrator IV =1200.
[0082] The ELISA values for each test specimen were determined
using the formula in Example 1.
EXAMPLE 4
Test for Heat Shock Protein 60 Antibody
[0083] Human HSP60 Peptide "AMTIAKNAGEGSLIVEKIM" was synthesized by
a robotic multiple peptide synthesizer and resin was used as solid
support. Peptide was characterized by reversed-phase HPLC and
electrospray mass-spectrometry with purity greater than 80%. This
peptide was bound to bovine serum albumin and used for coating
microtiter plates.
[0084] Each well of microtiter plate was coated with 3 .mu.g of
peptide in 0.1 M carbonate buffer pH 9.5. After 24 hours of
incubation and washing, 200 .mu.l of 2% BSA was added and incubated
for an additional 2 hours. Plates were washed, dried, and used for
measurement of HSP60 antibodies. The test specimen was added to the
plate followed by incubation and washing. The procedure in Example
1 was followed to measure for HSP60 antibodies.
[0085] The ELISA values for the calibrators used in this test
system were as follows: Calibrator I=10, Calibrator II=20,
Calibrator III=40.
[0086] The ELISA values for each test specimen were determined
using the formula in Example 1.
EXAMPLE 5
Test for Anti-.beta.-2-Glycoprotein-1
[0087] Wells of microtiter polystryrene plate were coated with 3
.mu.g of .beta.2GP1 in 100 .mu.l of 0.1 M carbonate buffer pH 9.6
and were kept overnight at 4.degree. C. The plates were then washed
with PBS and blocked with 2% BSA for 2 hours at room temperature.
Plates were washed, dried, and used for detection of antibodies
against .beta.2GP1. The test specimen was added to the plate
followed by incubation and washing. The procedure in Example 1 was
followed to measure for .beta.2GP1 antibodies.
[0088] The ELISA values for the calibrators used in this test
system were as follows: Calibrator I=25, Calibrator II=75, and
Calibrator III=150.
[0089] The ELISA values for each test specimen were determined
using the formula in Example 1.
EXAMPLE 6
Test for Anti-Platelet Glycoproteins
[0090] Well of microtiter plate were coated with 3 .mu.g of
platelet glycoprotein in 100 .mu.l of 0.1M carbonate buffer pH 9.6
and were kept overnight at 4.degree. C. The plates were then washed
with PBS and blocked with 2% BSA for 2 hours at room temperature.
Plates were washed, dried, and used for detection of antibodies
against platelet glycoproteins. The test specimen was added to the
plate followed by incubation and washing. The procedure in Example
1 was followed to measure for platelet glycoprotein antibodies.
[0091] The ELISA values for the calibrators used in this test
system were as follows: Calibrator I=10, Calibrator II=20, and
Calibrator III=40.
[0092] The ELISA values for each test specimen were determined
using the formula in Example 1.
EXAMPLE 7
Test for Immune Complexes
[0093] Wells of microtiter plates were coated with 3 .mu.g of
purified C1q in 100 .mu.l of 0.1M carbonate buffer pH 9.6 and were
kept overnight at 4.degree. C. The plates were then washed with PBS
and blocked with 2% BSA for 2 hours at room temperature. Plates
were washed, dried, and used for detection of immune complexes. The
test specimen was added to the plate followed by incubation and
washing. The procedure in Example 1 was followed to measure for
immune complexes.
[0094] The ELISA values for the calibrators used in this test
system were as follows: Calibrator I=7, Calibrator II=15, and
Calibrator III=30.
[0095] The ELISA values for each test specimen were determined
using the formula in Example 1.
EXAMPLE 8
Analysis of Results
[0096] The results are analyzed as a panel. The values for myosin;
oLDL; .beta.2GP1; HSP60; and immune complexes were obtained from a
set of healthy controls.
[0097] Thirty patients (15 men and 15 women) with known risk
factors for cardiovascular disease were tested. These patients have
a blood pressure greater than 140/80 and cholesterol/HDL level
greater than 7.
[0098] The assays for antibodies were performed according to the
preceding Examples. The results summarized in FIGS. 1-5 are
expressed based on optical densities, which are easily converted to
ELISA units.
[0099] Tables 2 and 3 and FIGS. 1-5 summarize the saliva IgA
antibody levels against infectious agents as well as human tissue
target antigens or epitopes in patients with possible
cardiovascular disease and healthy control subjects.
2TABLE 2 Saliva IgA Antibodies Against Specific and Non-Specific
Autoantigens Involved in Cardiovascular Disease Expressed by O.D.'s
From Patients With Possible Cardiovascular Disease SUB- IMMUNE
JECTS MYOSIN O-LDL B-2-GP1 HSP-60 COMPLEX 1 0.57 0.45 0.15 1.1 0.66
2 0.66 0.51 0.45 0.65 0.67 3 0.31 0.2 0.5 0.64 0.4 4 0.15 0.16 0.02
0.05 0.1 5 0.4 0.46 0.2 0.68 0.8 6 1.2 1.1 1.3 1.6 1.4 7 0.01 0.07
0.01 0.01 0.01 8 0.93 1.2 0.48 1.3 0.93 9 1.6 0.8 1.25 1.4 0.85 10
1.5 1.6 1.6 1 2 11 0.45 0.4 0.8 1.2 1.1 12 0.4 1.2 0.72 1.7 0.95 13
0.06 0.08 0.1 0.1 0.1 14 0.05 0.1 0.01 0.05 0.01 15 0.55 0.48 0.01
0.7 1.1 16 0.9 1.8 0.56 0.75 1.25 17 0.1 0.1 0.62 0.1 0.07 18 0.1
0.05 0.06 0.01 0.06 19 0.05 0.1 0.1 0.02 0.07 20 0.1 0.1 0.1 0.1
0.1 21 1.2 0.85 1.3 1.9 2.3 22 1.8 1.6 2.1 1.7 1.6 23 0.96 1.2 1.4
2.1 2.1 24 0.53 0.66 0.82 0.8 1.5 25 0.1 0.05 0.1 0.1 0.01 26 1.6
1.3 1.5 1.8 1.6 27 1.3 1.1 1.6 1.2 0.9 28 0.1 0.1 0.08 0.5 0.1 29
1.5 1.2 1.1 1.6 1.7 30 0.82 0.55 0.95 0.9 0.85 Mean +/- 0.52 +/-
0.65 +/- 0.67 +/- 0.85 +/- 0.85 +/- S.D. 0.55 0.54 0.60 0.67
0.69
[0100]
3TABLE 3 Saliva IgA Antibodies Against Specific and Non-Specific
Autoantigens Involved in Cardiovascular Disease Expressed by O.D.'s
From Healthy Controls SUB- IMMUNE JECTS MYOSIN O-LDL B-2-GP1 HSP-60
COMPLEX 1 0.1 0.15 0.1 0.1 0.2 2 0.1 0.1 0.1 0.15 0.1 3 0.31 0.21
0.2 0.18 0.39 4 0.1 0.1 0.1 0.05 0.1 5 0.1 0.1 0.1 0.1 0.1 6 0.37
0.4 0.31 0.36 0.45 7 0.01 0.01 0.01 0.1 0.1 8 0.15 0.1 0.1 0.1 0.3
9 0.1 0.15 0.1 0.1 0.2 10 0.1 0.1 0.1 0.1 0.15 11 0.21 0.18 0.23
0.15 0.32 12 0.12 0.61 0.55 0.52 0.85 13 0.01 0.15 0.1 0.1 0.17 14
0.1 0.1 0.1 0.1 0.23 15 0.39 0.36 0.41 0.53 0.41 16 0.18 0.2 0.1
0.16 0.3 17 0.24 0.22 0.31 0.25 0.1 18 0.95 0.76 0.82 0.98 0.88 19
0.1 0.1 0.1 0.1 0.1 20 0.01 0.05 0.01 0.01 0.15 21 0.12 0.1 0.19
0.22 0.29 22 0.1 0.1 0.1 0.1 0.1 23 0.1 0.1 0.1 0.1 0.15 24 0.38
0.85 0.65 0.59 1.8 25 0.1 0.2 0.1 0.1 0.3 26 0.1 0.1 0.1 0.15 0.1
27 0.15 0.26 0.31 0.39 0.35 28 0.1 0.1 0.1 0.1 0.26 29 0.45 0.38
0.3 0.51 0.43 30 0.1 0.1 0.1 0.1 0.15 Mean +/- 0.18 +/- 0.21 +/-
0.19 +/- 0.22 +/- 0.31 +/- S.D. 0.18 0.20 0.18 0.21 0.33
[0101] FIGS. 1 and 2 illustrate each optical density as well as the
mean of saliva IgA antibody level against 12 antigens. FIGS. 3-5
illustrate the mean and standard deviation of saliva IgA antibody
levels from healthy controls and patients with cardiovascular
disease.
[0102] Detection of IgA antibody in saliva against antigens of
tissue antigens would help in early detection and prevention of
cardiovascular disease. FIG. 6 shows data interpretation of
antibody levels to infectious agents and human target tissue
antigens relating to the possibility or presence of cardiovascular
disease. The detection of above normal levels of saliva IgA
antibody against the antigens listed in FIG. 6 can help to diagnose
possible atherosclerosis. A normal level of antibody is defined as
an average level of antibody taken from a set of healthy control
individuals. For instance, the average levels are shown as the big
squares on FIGS. 1 and 2.
[0103] The results of the test panels shown in combination with
other clinical data and evaluation by the clinician allows for a
faster and more accurate diagnosis of the above indications.
* * * * *