U.S. patent number RE43,914 [Application Number 13/333,849] was granted by the patent office on 2013-01-08 for method for detecting syphilis using synthetic antigens.
This patent grant is currently assigned to N/A, The United States of America as represented by the Secretary of the Department of Health and Human Services, Centers for Disease. Invention is credited to Arnold R. Castro.
United States Patent |
RE43,914 |
Castro |
January 8, 2013 |
**Please see images for:
( Certificate of Correction ) ** |
Method for detecting syphilis using synthetic antigens
Abstract
An antigen composition and method for the detection of
antibodies to Treponema pallidum and the diagnosis of syphilis are
described. The antigen composition contains synthetic cardiolipin
and synthetic lecithin. The antigen composition may additionally
contain cholesterol and an alcohol. The antigen composition is
useful as an immunoreagent in immunoassays for the detection of
antibodies associated with T. pallidum infection. The methods are
sensitive and specific for T. pallidum infection.
Inventors: |
Castro; Arnold R. (Monroe,
GA) |
Assignee: |
The United States of America as
represented by the Secretary of the Department of Health and Human
Services, Centers for Disease Control and Prevention
(Washington, DC)
N/A (N/A)
|
Family
ID: |
33312744 |
Appl.
No.: |
13/333,849 |
Filed: |
June 8, 2000 |
PCT
Filed: |
June 08, 2000 |
PCT No.: |
PCT/US00/15828 |
371(c)(1),(2),(4) Date: |
December 05, 2001 |
PCT
Pub. No.: |
WO00/75666 |
PCT
Pub. Date: |
December 14, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60138192 |
Jun 9, 1999 |
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Reissue of: |
10009698 |
Dec 5, 2001 |
6815173 |
Nov 9, 2004 |
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Current U.S.
Class: |
435/7.36;
424/262.1; 435/69.3; 436/518 |
Current CPC
Class: |
A61K
39/00 (20130101); G01N 33/571 (20130101); G01N
33/92 (20130101); G01N 2333/20 (20130101) |
Current International
Class: |
G01N
33/571 (20060101); G01N 33/543 (20060101); C12N
15/09 (20060101); A61K 39/02 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1053504 |
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Mar 1964 |
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GB |
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74046051 |
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Jul 1974 |
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JP |
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74046051 |
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Dec 1974 |
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JP |
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05312808 |
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Nov 1993 |
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JP |
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10239315 |
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Sep 1998 |
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JP |
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629927 |
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Oct 1978 |
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SE |
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629927 |
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Oct 1978 |
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SU |
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WO 2004/040311 |
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May 2004 |
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WO |
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Other References
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Antigen Used by the Venereal Disease Research Laboratory Test for
Serodiagnosis of Syphilis," Clin. Diag. Lab. Immunol. 7:658-661,
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Doxorubicin in Liposomes: Pharmacological, Toxicological and
Therapeutic Evaluation," British Journal of Cancer, vol. 74, No. 1,
pp. 43-48 (1996). cited by other .
Inoue, K. et al., "Immunochemical Studies of Phospholipids--IV: The
Reactivities of Antisera Against Natural Cardiolipin and Synthetic
Cardiolipin Analogues-Containing Antigens," Chemistry and Physics
of Lipids, vol. 3, No. 1, pp. 70-77 (1969). cited by other .
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Lecithin Kline Test," Technical Bulletin of the Registry of Medical
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cited by other.
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Primary Examiner: Duffy; Patricia A
Attorney, Agent or Firm: Klarquist Sparkman, LLP
Parent Case Text
PRIORITY CLAIM
This is a .sctn.371 U.S. National Stage of PCT/US00/15828, filed
Jun. 8, 2000, which was published in English under PCT Article
21(2), which claims the benefit of U.S. Provisional Application
60/138,192, filed Jun. 9, 1999.
Claims
We claim:
1. An antigen composition comprising .Iadd.between 0.01 and 0.05%
by volume .Iaddend.tetramyristoyl cardiolipin and .Iadd.between
0.11 and 0.16% by volume
.Iaddend.1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.
2. The antigen composition of claim 1, further comprising
cholesterol.
3. The composition of claim 2, wherein the .Iadd.volume
.Iaddend.concentration of cholesterol is 0.9%.
4. The composition of claim 2, further comprising an alcohol.
5. The composition of claim 1, wherein the concentration of
cardiolipin is between 0.02 and 0.04%.
6. The composition of claim 5, wherein the .Iadd.volume
.Iaddend.concentration of cardiolipin is 0.03%.
.[.7. The composition of claim 1, wherein the concentration of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine is between 0.11
and 0.16%..].
8. The composition of claim .[.7.]. .Iadd.1.Iaddend., wherein the
.Iadd.volume .Iaddend.concentration of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine is 0.14%.
9. The composition of claim 4, wherein the alcohol is ethanol.
10. A method for detecting anti-lipoidal antibodies in a human
comprising combining a biological sample from the human with a
composition comprising tetramyristoyl cardiolipin and
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and detecting an
immunocomplex formed between an anti-lipoidal antibody in the
biological sample and the composition.
11. The method of claim 10, wherein the composition further
comprises cholesterol and an alcohol.
12. The method of claim 11, wherein the .Iadd.volume
.Iaddend.concentration of cholesterol in the composition is
0.9%.
13. The method of claim 10, wherein the alcohol is ethanol.
14. The method of claim 10, wherein the .Iadd.volume
.Iaddend.concentration of tetramyristoyl cardiolipin in the
composition is between 0.01 and 0.05%.
15. The method of claim 10, wherein the .Iadd.volume
.Iaddend.concentration of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the composition
is between 0.11 and 0.16%.
16. The method of claim 10, wherein the detection of an
immunocomplex is used to diagnose syphilis in the human.
17. The method of claim 10, wherein the immunocomplex is detected
using a flocculation or agglutination test.
.[.18. The antigen composition of claim 1 comprising between 0.02
and 0.04% tetramyristoyl cardiolipin, and between 0.11 and 0.16%
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine..].
19. An antigen composition comprising between 0.02 and 0.04%
.Iadd.by volume .Iaddend.tetramyristoyl cardiolipin, between 0.11
and 0.16% .Iadd.by volume
.Iaddend.1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 0.9%
.Iadd.by volume .Iaddend.cholesterol, and ethanol to volume.
20. An antigen composition comprising 0.03% .Iadd.by volume
.Iaddend.tetramyristoyl cardiolipin, between 0.11 and 0.16%
.Iadd.by volume
.Iaddend.1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 0.9%
.Iadd.by volume .Iaddend.natural cholesterol in absolute ethanol to
volume.
21. A method for detecting anti-lipoidal antibodies in a human
comprising: (a) obtaining a biological sample from a human; (b)
combining the biological sample with a composition comprising
between 0.02 and 0.04% .Iadd.by volume .Iaddend.tetramyristoyl
cardiolipin, between 0.11 and 0.16% .Iadd.by volume
.Iaddend.1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 0.9%
.Iadd.by volume .Iaddend.cholesterol, and ethanol to volume; and
(c) detecting an immunocomplex formed between an antibody in the
biological sample and the composition.
22. The method of claim 21, wherein the detection of the
immunocomplex is used to diagnose syphilis in the human.
23. The method of claim 10, wherein the .Iadd.volume
.Iaddend.concentration of tetramyristoyl cardiolipin in the
composition is between 0.02 and 0.04%.
.Iadd.24. An antigen composition consisting of tetramyristoyl
cardiolipin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine,
cholesterol, and an alcohol..Iaddend.
.Iadd.25. The composition of claim 24, wherein the volume
concentration of tetramyristoyl cardiolipin is between 0.01 and
0.05%, the volume concentration of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine is between 0.11
and 0.16%, and the volume concentration of cholesterol is
0.9%..Iaddend.
.Iadd.26. The composition of claim 25, wherein the volume
concentration of tetramyristoyl cardiolipin is between 0.02 and
0.04%..Iaddend.
Description
FIELD OF THE INVENTION
The present invention relates to the fields of microbiology and
immunology and more specifically relates to compositions and
methods for detecting, diagnosing and monitoring the treatment of
syphilis. In particular, the invention pertains to synthetic
cardiolipin and lecithin antigen compositions and their use in
immunoassays.
BACKGROUND OF THE INVENTION
Syphilis is a sexually transmitted disease (STD) caused by the
bacterium Treponema pallidum. Over 100,000 cases of adult syphilis
are reported worldwide each year. The disease is also transmitted
congenitally, affecting 3000 or more infants annually. Failure to
obtain antibiotic treatment in the early stages of the disease
allows progression of the disease throughout the body, often
resulting in irreversible damage to organs, insanity, blindness, or
death. The spread of the human immunodeficiency virus (HIV) around
the world has greatly amplified the severity of syphilis as a
health problem because genital ulcers produced during the early
stages of syphilis infection facilitate the sexual transmission of
HIV.
The course of syphilis has been divided into stages; primary,
secondary, latent, neurosyphilis and tertiary (late). An infected
individual may infect others during the first two stages.
Transmission occurs when bacteria are spread from the ulcer of an
infected person to the skin or mucous membranes of the genital
area, mouth, or anus of a sexual partner. T. pallidum organisms can
also pass through broken skin on other parts of the body. In
tertiary syphilis and neurosyphilis, the bacterial infection is not
contagious, but the invasion of the organism into the organs,
tissues, and brain can have fatal consequences such as serious
cardiovascular abnormalities or neurologic disease.
Vertical or transplacental syphilis infection can occur during the
first four years a pregnant woman is infected and not treated.
Although adequate treatment of the mother usually prevents
congenital syphilis, approximately 25% of human fetuses that have
been exposed to T. pallidum infection in utero are reported as
stillbirth deaths. Some infants with congenital syphilis have
symptoms at birth, but most develop symptoms two to three months
post partum. These symptoms include skin sores, rashes, fever,
swollen liver and spleen, jaundice, anemia, and various
deformities. As infected infants mature, they may develop the
symptoms of late-stage syphilis including irreversible damage to
bones, teeth, eyes, ears, and brain.
The first symptom of primary syphilis is an ulcer, or chancre. The
chancre appears within ten days to three months after exposure and
is usually found on the part of the body that was exposed to the
ulcer of an infected sexual partner, such as the penis, vulva,
vagina, cervix, rectum, tongue, or lip. Because the chancre lasts
only a few weeks and may be painless or occur inside the body, it
may go unnoticed. The chancre disappears with or without treatment.
In persons who are untreated, secondary symptoms will appear
approximately nine weeks after the appearance of the primary
lesion.
Secondary syphilis is often marked by a skin rash that is
characterized by brown sores approximately the size of a penny.
Because active bacteria are present in these sores, any physical
contact, sexual or non-sexual, with the broken skin of an infected
individual may spread the infection at this stage. Other symptoms
include mild fever, fatigue, headache, sore throat, patchy hair
loss, and swollen lymph glands. These symptoms may be mild and,
like the chancre of primary syphilis, will disappear with or
without treatment. If untreated, the infected person then enters a
period of latency.
Latent syphilis is characterized by the absence of clinical signs
or abnormal findings in cerebrospinal fluid (CSF) in conjunction
with positive results of serologic tests. Early latent syphilis,
which occurs within one year of infection, is potentially
transmissible and relapses may occur, while late latent syphilis is
associated with immunity to relapse and resistance to
re-infection.
During the early stages of syphilis infection, the bacteria may
invade the nervous system. If left untreated, neurosyphilis may
develop. Progression of the disease to neurosyphilis may take up to
twenty years, and some individuals having neurosyphilis fail to
develop recognizable symptoms, making diagnosis very difficult.
Those who do present symptoms may complain of headache, stiff neck,
or fever, which result from an inflammation of the lining of the
brain. Seizures and symptoms of stroke such as numbness, weakness,
or visual problems may also afflict neurosyphilis patients.
Although approximately two-thirds of T. pallidum-infected
individuals who fail to obtain treatment will suffer no further
consequences of the disease, approximately one-third of those with
untreated latent syphilis develop the complications of late, or
tertiary, syphilis. In the tertiary stage of syphilis, the bacteria
damage the heart, eyes, brain, nervous system, bones, joints, or
almost any other part of the body. The tertiary stage can last for
years, or even decades. Late syphilis commonly results in
cardiovascular disease, mental illness, blindness, or even
death.
Due to the sometimes serious and life threatening effects of
syphilis infection, and the risk of transmitting or contracting
HIV, specific and early diagnosis of the infection is essential.
Syphilis, however, has sometimes been referred to as "the great
imitator" because its early symptoms are similar to those of many
other diseases. Therefore, a physician usually does not depend
solely on a recognition of the signs and symptoms of syphilis, but
relies on the results of clinical tests including the microscopic
identification of syphilis bacteria and analytical tests for
manifestations of syphilis infection in biological samples.
Diagnosis of syphilis by microscopic identification of the bacteria
is performed generally as follows. A scraping is taken from the
surface of the ulcer or chancre and is examined under a special
"dark-field" microscope to detect the organism. Dark-field
microscopy requires considerable skill and is prone to
misinterpretation.
For these reasons, most cases of syphilis are first diagnosed
serologically using non-treponemal assays. Non-treponemal tests
detect substances, such as antibodies, that are produced in the
presence of a T. pallidum infection. The currently available
non-treponemal assays most often used to detect evidence of a
syphilis infection are the Venereal Disease Research Laboratory
(VDRL) test and the rapid plasma reagin (RPR) test. The VDRL test
employs lipids obtained from naturally-occurring sources, to detect
anti-lipoidal antibodies that are generated upon infection by T.
pallidum. These antibodies are generated against the cardiolipin of
the T. pallidum organism by the immune system of the individual
infected with T. pallidum and may be found in the serum or
cerebrospinal fluid of the individual.
One disadvantage to the presently available non-treponemal tests is
poor specificity. Many medical conditions, including mycoplasma
infection, pneumonia, malaria, acute bacterial and viral
infections, and autoimmune disease can cause false positive test
results in presently available tests for syphilis. For example,
intravenous drug use or autoimmune disease causes tissue damage,
which results in the release of cardiolipin and the production of
anti-cardiolipin antibodies. Detection of these anti-cardiolipin
antibodies in a non-treponemal test would therefore produce a false
positive result. Successful diagnosis is particularly problematic
for the detection of neurosyphilis.
Due to the occurrence of false positive and false negative results
when using these existing tests, confirmation using an alternative
method of analysis, such as microscopy or a treponemal-based
serological test, is normally required. Standard treponemal-based
tests include the fluorescent treponemal antibody-absorption
(FTA-ABS) test and the FTA-ABS double staining test (FTA-ABS DS).
Although treponemal-based assays may be used to confirm a positive
test result, these tests are often expensive, complicated, and time
consuming, and may require the use of highly sophisticated
scientific instrumentation and trained scientific personnel. In
addition, treponemal assays cannot be used as tests to monitor the
success of antibiotic therapy because, due to the continued
presence of anti-T. pallidum antibodies after cure, the tests
results remain positive even after eradication of the infection for
approximately 85% of successfully treated individuals.
Therefore, a single assay for the sensitive and specific detection
of T. pallidum infection in a sample for the diagnosis of early
stage syphilis or neurosyphilis is needed. Also needed is a simple,
inexpensive assay that can be used to monitor the success of
syphilis treatment.
SUMMARY OF THE INVENTION
An antigen composition and method for detecting Treponema pallidum
infection and thereby diagnosing syphilis are provided. The antigen
composition contains a combination or mixture of synthetic
cardiolipin and synthetic lecithin. The preferred antigen
composition also contains a cholesterol. The preferred antigen
composition further includes an alcohol. The alcohol solubilizes
the lipids in the antigen composition to form a suspension. The
antigen composition is useful as a reagent in assays for the
detection of antibodies associated with T. pallidum infection in a
biological sample, particularly a body fluid such as serum or
cerebrospinal fluid. Preferably, the antigen composition is an
immunoassay reagent for the detection or measurement of antibodies
associated with T. pallidum infection.
The more preferred antigen composition contains purified, synthetic
cardiolipin, synthetic lecithin, natural or non-synthetic
cholesterol, and an alcohol. The optimal purity of the synthetic
cardiolipin and lecithin in the composition is 99% or greater. The
optimal purity of the cholesterol is 98% or greater, or is ash
free. Most preferably, the antigen composition contains
tetramyristoyl cardiolipin,
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, cholesterol, and
absolute (100%) ethanol. The preferred concentration by volume of
synthetic cardiolipin in the composition is between about
0.01-0.05%, or between approximately 0.02-0.04%, or more preferably
0.03%. The preferred concentration by volume of synthetic lecithin
in the composition is between approximately 0.11-0.16%, more
preferably 0.14%. The preferred concentration by volume of
cholesterol in the composition is approximately 0.9%, and the
remainder of the composition is the alcohol.
The antigen composition provided herein is also useful in general
as an in vitro research tool for studying syphilis. More
particularly, the composition is useful in assays or diagnostic
kits to detect the presence of T. pallidum infection, which is
diagnostic or prognostic for the occurrence or recurrence of
syphilis disease.
The preferred method provided herein is an immunoassay for the
detection of cardiolipin antibodies in a biological sample such as
serum or cerebrospinal fluid. In accordance with the method, the
antigen composition described herein is combined with the
biological sample for a sufficient amount of time under conditions
that facilitate the binding of anti-lipoidal antibodies in the
sample to a synthetic cardiolipin-lecithin matrix, to form an
antibody-antigen complex. This complex is then detected using
methods well known to those skilled in the art such as flocculation
or microflocculation tests, or the like.
Accordingly, it is an object of the present invention to provide a
method for detecting carriers of T. pallidum infection and thus
prevent the spread of T. pallidum from one host to another.
It is another object of the present invention to provide a
sensitive method for the diagnosis of early or latent syphilis or
neurosyphilis.
It is yet another object of the present invention to provide a
rapid, simple, and inexpensive assay for the accurate detection of
T. pallidum.
It is a further object of the present invention to provide an
inexpensively produced antigen composition for the reproducible
measurement or detection of T. pallidum.
It is another object of the present invention to provide a test for
the detection of T. pallidum offers advantages in the
standardization and stability of the VDRL antigen.
These and other objects, features, and advantages of the present
invention will become apparent after a review of the following
detailed description of the disclosed embodiments and the appended
claims.
DETAILED DESCRIPTION
An antigen composition and method for the detection of Treponema
pallidum are described herein. The antigen composition contains a
mixture or combination of synthetic cardiolipin and synthetic
lecithin. Cardiolipin is a 1,3-bis (phophatidyl)glycerol having
antigenic properties. Lecithin is a phospholipid. The antigen
composition preferably also includes a non-synthetic (natural)
cholesterol. An alcohol is also a component of the preferred
antigen composition. The alcohol solubilizes the lipids, thereby
forming a suspension. The synthetic cardiolipin and lecithin have
an optimal purity of 99% or greater. The cholesterol has an optimal
purity of 98% or greater, or is ash free. The antigen composition
is useful as a reagent in assays for the detection of antibodies
associated with T. pallidum infection in a biological sample.
Preferably, the antigen composition is an immunoassay reagent for
the detection or measurement of antibodies generated in a patient
infected with T. pallidum, which is diagnostic or prognostic for
the occurrence or recurrence of syphilis.
The method described herein is an assay for the detection or
quantitation of antibodies associated with infection of a patient
by T. pallidum in a biological sample, particularly a body fluid
sample such as serum or cerebrospinal fluid. The method permits
detection of circulating antibodies associated with T. pallidum
infection in order to detect or monitor a T. pallidum infection. A
preferred method provided herein is an immunoassay. In accordance
with the preferred method, the antigen composition is combined with
the biological sample for a sufficient amount of time under
conditions that facilitate the binding of anti-lipoidal antibodies
in the sample to the cardiolipin in the antigen composition, to
form antibody-antigen complexes. These antibody-antigen complexes
are then detected using methods well known to those skilled in the
art such as the VDRL flocculation or microflocculation test, which
is read microscopically.
Definitions
The terms "a", "an" and "the" as used herein are defined to mean
"one or more" and include the plural unless the context is
inappropriate.
The term "antibodies" as used herein includes monoclonal
antibodies, polyclonal, chimeric, single chain, bispecific,
simianized, and humanized antibodies as well as Fab fragments,
including the products of an Fab immunoglobulin expression
library.
The phrases "specifically binds to" or "specifically immunoreactive
with", when referring to an antibody, refers to a binding reaction
which is determinative of the presence of the antigen of interest
in the presence of a heterogeneous population of peptides,
proteins, lipids and other biologics. Thus, under designated
immunoassay conditions, the specified antigen or antigens bind
preferentially to particular antibodies and do not bind in a
significant amount to other antibodies present in the sample.
Specific binding under such conditions requires an antigen that is
selected for its specificity for a particular antibody. A variety
of immunoassay formats may be used to select antigens specifically
immunoreactive with a particular antibody. For example, solid-phase
ELISA immunoassays are routinely used to select an antigen
specifically immunoreactive with an antibody. See, Harlow and Lane
(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York, for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity.
The term "antigen" refers to an entity or fragment thereof which
can induce an immune response in a mammal. The term includes
immunogens and regions responsible for antigenicity or antigenic
determinants. The term "antigen composition" as used herein refers
to a composition containing synthetic cardiolipin and synthetic
lecithin. "Antigenic determinant," as used herein, refers to a
region of an antigen that is recognized by an antibody.
As used herein, the terms "detecting" or "detection" refers to
qualitatively or quantitatively determining the presence of the
biomolecule under investigation.
By "isolated" is meant a biological molecule free from at least
some of the components with which it naturally occurs.
Antigen Compositions
The composition provided herein contains a combination, suspension
or physical mixture of one or more synthetic cardiolipins and
lecithins. A preferred composition contains synthetic cardiolipin,
synthetic lecithin, and synthetic or non-synthetic (naturally
occurring) cholesterol. The more preferred composition contains
synthetic cardiolipin, synthetic lecithin, natural cholesterol, and
an alcohol.
The preferred concentration of synthetic cardiolipin in the
composition is between about 0.01-0.05% by volume, or between
approximately 0.02-0.04% by volume, or more preferably 0.03% by
volume. The preferred concentration of synthetic lecithin in the
composition is between approximately 0.11 and 0.16% by volume, more
preferably 14% by volume. The preferred concentration of natural
cholesterol in the composition is approximately 0.9% by volume, and
the remainder of the composition is alcohol, preferably ethanol,
most preferably absolute (100%) ethanol.
The synthetic cardiolipin may be synthesized from semi-synthetic
lipid precursors originating from plant sources. In a most
preferred embodiment, the cardiolipin is tetramyristoyl
cardiolipin, which is commercially available from sources such as
Avanti Polar Lipids (Alabaster, Ala.).
The synthetic lecithin may be derived from soybeans or egg. In a
preferred embodiment, the lecithin is a 16:0, 18:1 lecithin also
described as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or
3-sn-phosphatidylcholine (sn means stereospecifically numbered),
which is also available commercially from sources such as Avanti
Polar Lipids (Alabaster, Ala.).
In a most preferred embodiment, the composition is a suspension
containing approximately 0.03% tetramyristoyl cardiolipin,
0.11-0.16% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and
0.9% natural cholesterol in absolute ethanol. The cholesterol is
also available from commercial sources such as Avanti Polar Lipids
(Alabaster, Ala.). The alcohol can be purchased from chemical
suppliers such as Sigma Chemical Company (St. Louis, Mo.).
When combined together with the alcohol, the cardiolipin, lecithin,
and cholesterol form a lipid matrix or micelle. As described in
more detail below, antibodies associated with the presence of a
human T. pallidum infection, referred to herein as anti-cardiolipin
antibodies, bind to these lipid micelles and form antibody-antigen
complexes. Therefore, the detection or measurement of these
antibody-antigen complexes can be used to diagnose a T. pallidum
infection. Although not wishing to be bound by the following
hypothesis, it is believed that the anti-cardiolipin matrix
antibodies bind to the synthetic antigen composition described
herein with greater specificity and higher avidity than they bind
to naturally occurring cardiolipin and lecithin. The synthetic
antigen composition thereby provides a more efficient, more
sensitive, and more specific means to detect antibodies associated
with a syphilis infection.
It will be understood by those skilled in the art that one or more
components of the antigen composition can be labelled with a
detectable label to facilitate the direct measurement or detection
of antibody-antigen complex formation. Various types of labels and
methods of conjugating the labels to the antigen composition are
well known to those skilled in the art.
Also, the antigen composition may be employed as a laboratory
research tool to generate, isolate or purify anti-cardiolipin
antibodies, and the antibodies can be used to study syphilis in
general. Therefore, the antigen composition is useful for purposes
such as in vivo and in vitro diagnostics and laboratory
research.
Preparation of the VDRL Antigen
The VDRL antigen can be prepared, for example, by preparing an
ethanolic solution of tetramyristoyl cardiolipin at a concentration
by volume ranging from about 0.01-0.05%, or 0.02 to 0.04%, or more
preferably 0.03%. An ethanolic solution of synthetic lecithin
having a concentration by volume of approximately 0.11 to 0.16%,
more preferably 0.14%, and an ethanolic solution of natural
cholesterol of 0.9% are added to the cardiolipin solution. The
components are added in the following sequence: cardiolipin,
lecithin, cholesterol, and ethanol to volume. The antigen is
solubilized and stored at room temperature over-night before
testing.
Detection of Anti-Cardiolipin Antibodies
The method provided herein includes diagnostic and prognostic
methods to detect and quantify antibodies capable of binding to the
antigen composition described above. These methods permit detection
of circulating antibodies to the cardiohpin-lecithin matrix in
order to indicate the presence of T. pallidum infection and thereby
diagnose infection or monitor the progress of an antibiotic in
treating a T. pallidum infection.
There are many techniques known in the art for detecting or
measuring antibody-antigen complexes, also referred to herein as
immunocomplexes. Classical methods involve reacting a sample
containing the antibody with a known excess amount of the antigen
specific for the antibody, separating bound from free antigen, and
determining the amount of bound antigen or free antigen. If free
antigen is measured, the amount of bound antigen can be calculated
by subtracting the amount of free antigen from the known starting
amount. Often the antigen is directly or indirectly labeled with a
reporter group or detectable label to aid in the determination of
the amount of antibody-antigen complex as described herein. The
reporter group or "label" is commonly a fluorescent or radioactive
group or an enzyme. The label is then detected using methods well
known to those skilled in the art such as spectrophotometry,
scintillation counting, or flow cytometry.
Alternatively, the antigen can be conjugated to a solid phase bead
or particle that is filtered, centrifuged, or otherwise removed
from the mixture, such as by magnetically removing a metallic or
magnetized particle. Attaching the antigen to solid phase beads,
such as latex beads, provides a new more sensitive and rapid slide
agglutination test.
In a preferred embodiment, the antigen is attached to the beads
through the cardiolipin molecule. One method of attaching the
antigen to the beads is to modify the cardiolipin molecule so that
it can be covalently bonded to the beads. For example, an amine
group can be attached to the terminal methyl groups of the fatty
acid chains of the cardiolipin. Cardiolipin modified in this manner
can be attached to carboxylated or aminealated latex beads.
A preferred immunoassay for the detection of anti-cardiolipin
antibodies in a sample is performed as follows. A sample is
collected or obtained using methods well known to those skilled in
the art. The sample containing the anti-cardiolipin antibodies to
be detected is obtained from a biological source. The sample is
preferably obtained from a biological fluid, such as, but not
limited to, whole blood, blood serum, blood plasma, saliva,
cerebrospinal fluid, and the like. Optimal diagnostic results are
obtained when the sample is serum or spinal fluid. The sample may
be filtered or otherwise manipulated prior to immunoassay to
optimize the immunoassay results.
The sample is then incubated with the antigen composition described
herein to form an antibody-antigen immuno-complex. The
antibody-antigen complex is then detected using methods well known
to those skilled in the art. The term "detecting" or "detected" as
used herein means using known techniques for detection of biologic
molecules such as immunochemical or histological methods. Such
methods include immunological techniques employing monoclonal or
polyclonal antibodies to the lipids, such as enzyme linked
immunosorbant assays (ELISA), sandwich assays, flow cytometric
assays, radioimmunoassays, or other types of assays involving
antibodies known to those skilled in the art.
In a preferred embodiment of the method, anti-cardiolipin
antibodies in a sample are detected by employing the synthetic
antigen composition described herein in a flocculation assay.
Examples of known flocculation assays include the unheated serum
reagin test (USR), the rapid plasma reagin 10 mm circle card test
(RPR), the toluidine red unheated serum test (TRUST) and the VDRL
slide assay. These assays are all flocculation tests. It will be
understood by those skilled in the art that the anti-cardiolipin
antibodies are also detectable using the antigen composition
described herein in agglutination tests, which further employ
carrier particles. In a more preferred embodiment of the method,
the synthetic antigen composition is used in a VDRL slide assay as
described briefly below and in more particularly in Venereal
Disease Research Laboratory (VDRL) Slide Test, Kennedy, E. J. Jr.
and Creighton, E. T., 157-78 (1998) in A Manual of Tests for
Syphilis, 9.sup.th ed., Larsen, S. A., Pope, V., Johnson, R. E. and
Kennedy, E. J. Jr. (Eds.), American Public Health Association,
Washington, D.C., which is incorporated by reference herein.
The VDRL slide assay is performed as follows. VDRL-buffered saline
containing formaldehyde, Na.sub.2HPO.sub.4, KH.sub.2 PO.sub.4 ,
NaCl and distilled water, is placed in a vessel. The antigen
composition is added slowly to the saline at a constant rate while
rotating the vessel and the mixture is then agitated to thoroughly
combine the contents and form a suspension. The sample, such as
serum, is placed into a ring of a paraffin or ceramic-ringed slide
and one drop of the antigen composition suspension is added. The
slide is rotated to mix the sample and antigen composition and is
then read microscopically. The presence of clumps, clumping or
roughness indicates antibody-antigen formation. Serial dilutions of
the antigen suspension can be used for a qualitative measurement of
antibody in the sample. Quantitative determinations can be made by
performing the assay with standard concentrations of antibody and
comparing the results of the sample with the results generated by
the standards.
It is to be understood that the assay methods are contemplated to
include the use of synthetic antigen compositions as described
above and synthetic derivatives of the antigen compositions
described herein provided that the derivatives retain antigenic
activity or display an equivalent antigenic activity and have
specificity for anti-cardiolipin antibodies.
Kit for Detecting the Presence of T. pallidum
A kit for diagnosing or otherwise evaluating a syphilis infection
by detecting the presence or quantity of anti-cardiolipin
antibodies is also provided. The kit can be in any configuration
well known to those of ordinary skill in the art and is useful for
performing one or more of the methods described herein for the
detection of anti-cardiolipin-lecithin matrix antibodies in
biological samples or for the detection or monitoring of T.
pallidum infection in a patient or carrier. The kits are convenient
in that they supply many if not all of the essential reagents for
conducting an assay for the detection of syphilis antibodies in a
biological sample. The reagents may be pre-measured and contained
in a stable form in vessels or on a solid phase in or on which the
assay may be performed, thereby minimizing the number of
manipulations carried out by the individual conducting the assay.
In addition, the assay may be performed simultaneously with a
standard that is included with the kit, such as a predetermined
amount of antibody, so that the results of the test can be
validated or measured.
The kit preferably contains the antigen composition described
herein, which can be used for the detection of cardiolipin
antibodies associated with T. pallidum infection. The kit also
preferably contains cholesterol and can additionally contain the
appropriate reagents that aid in detecting antibody-antigen
complexes. The kit may additionally contain equipment for safely
obtaining the sample, a vessel for containing the reagents, a
buffer for diluting the sample or reagents, and circle cards such
as the 10 mm slides or 18 mm circle cards used in VDRL, RPR and
TRUST assays.
The assay kit includes but is not limited to reagents to be
employed in the following techniques; flocculation tests such as
USR, RPR and TRUST; agglutination assays; and sandwich or ELISA
assays. Materials used in conjunction with these techniques
include, but are not limited to, micro-titer plates,
antibody-coated strips or dipsticks for rapid monitoring of
biological fluids. For each kit, the range, sensitivity, precision,
reliability, specificity and reproducibility of the assay are
established. Standardization may be achieved using reference
control sera and titering the sera to endpoint or a panel of sera
may be utilized.
In a more preferred embodiment, the assay kit uses VDRL slide
techniques and provides instructions and the antigen composition
described above. The kit is useful for the measurement of T.
pallidum infection, and more specifically, for the measurement of
antibodies directed toward cardiolipin in biological fluids of
humans exhibiting symptoms of syphilis or those at risk for
syphilis infection.
This invention is further illustrated by the following examples,
which are not to be construed in any way as imposing limitations
upon the scope thereof. On the contrary, it is to be clearly
understood that resort may be had to various other embodiments,
modifications, and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the present invention
and/or the scope of the appended claims.
EXAMPLE 1
Preparation of a Synthetic Cardiolipin and Lecithin Composition
Tetramyristoyl cardiolipin, purified by silica gel chromatography
to approximately 99% purity, was obtained in powder form from
Avanti Polar Lipids (Alabaster, Ala.). The final concentration of
sodium salt was tested for purity by thin layer chromatography and
high-pressure liquid chromatography. The sample was stored at
-20.degree. C. The tetramyristoyl cardiolipin was originally
synthesized from semi-synthetic lipid precursors that originated
from a plant source.
Lecithin (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) powder,
purified by silica gel chromatography to a purity of approximately
99%, was also obtained from Avanti Polar Lipids. The lecithin was
originally isolated from soybeans.
A 1.2% solution of cholesterol (Avanti Polar Lipids) in absolute
ethanol was prepared and filtered with alcohol-rinsed filter paper
#560. The cholesterol was originally derived from wool grease and
purified by re-crystallization, and the crystals were stored at
-20.degree. C.
An antigen composition was prepared by combining the synthetic
cardiolipin with the synthetic lecithin, the cholesterol solution
and the ethanol, in that order. The final concentration of
synthetic cardiolipin was 0.02-0.03% by volume. The final
concentration of synthetic lecithin was 0.11-0.16% by volume. The
final concentration of cholesterol was 0.9% by volume and the
remainder of the antigen composition was the ethanol.
EXAMPLE 2
Comparative Analysis of Synthetic VDRL Slide Assay versus
Conventional VDRL Slide Assay
The sensitivity of the VDRL slide assay using the synthetic
cardiolipin and lecithin composition described in Example 1 was
compared with the sensitivity of the conventional VDRL slide assay
as described in A Manual of Tests for Syphilis, 9.sup.th ed.,
159-77, Larsen, S. A., Pope, V., Johnson, R. E. and Kennedy, E. J.
Jr. (Eds.), American Public Health Association, Washington, D.C.
Briefly, 0.4 ml VDRL-buffered saline (formaldehyde,
Na.sub.2HPO.sub.4, KH.sub.2PO.sub.4, NaCl and distilled water) was
added to the bottom of a round 30 ml glass-stoppered bottle with a
flat inner-bottom surface or a 25 ml glass-stoppered Erlenmeyer
flask. Subsequently, 0.5 ml of the antigen composition suspension
was added directly to the saline at a rate of 6 seconds/0.5 ml of
antigen suspension while rotating the bottle continuously. Rotation
continued for ten seconds until 4.1 ml of buffered saline was
added. The bottle was tightly capped and shaken from bottom to top
approximately thirty times in ten seconds. The antigen suspension
was used within eight hours.
The qualitative tests were performed by placing 50 .mu.l of serum
into one ring of a paraffin or ceramic-ringed slide using a safety
pipetting device. The antigen suspension was gently re-suspended
and one free-falling drop (17 .mu.l) was added. The slide was
placed on a mechanical rotator for four minutes at 180.+-.2 rpm.
The slide was immediately removed and read microscopically using
10.times. oculars and a 10.times. objective. Results were reported
as follows: reactive--medium or large clumps, weakly or minimally
reactive--small clumps, nonreactive--no clumping or a very slight
roughness. Quantitative tests were similarly performed with serial
two-fold dilutions of the serum.
The results, set forth below in Table 1, indicated that the test
using the synthetic antigen composition was more sensitive than the
test using the standard VDRL antigen, which was made using natural
cardiolipin and lecithin.
TABLE-US-00001 TABLE 1 Comparison of Sensitivities of Natural VDRL
versus Synthetic VDRL Assays Sensitivity Test Primary Secondary
Latent Natural VDRL 80% 100% 85% Synthetic VDRL 84% 100% 88%
The sensitivities of the VDRL slide assay using the synthetic
cardiolipin and lecithin antigen composition described in Example 1
and the conventional VDRL slide assay were also compared against
the conventional RPR slide assay. As shown in Tables 2 and 3 below,
the assay using the synthetic VDRL antigen composition was more
reactive with samples that tested positive using the RPR test than
the assay using the non-synthetic VDRL antigen.
TABLE-US-00002 TABLE 2 RPR versus Synthetic VDRL Synthetic VDRL RPR
Reactive Nonreactive Reactive* 41 13 Nonreactive 1 5 *All RPR
reactives were minimally reactive
TABLE-US-00003 TABLE 3 RPR versus Natural VDRL Natural VDRL RPR
Reactive Nonreactive Reactive* 13 41 Nonreactive 0 6 *All RPR
reactives were minimally reactive
EXAMPLE 3
Comparative Analysis of Synthetic VDRL antigen and Natural VDRL
antigen (Qualitative Test)
Samples from 100 frozen banked sera, reactive by the nontreponemal
(RPR) test, were used to compare the CDC synthetic VDRL antigen and
a reference VDRL antigen (Natural VDRL antigen). The serum samples
were heat inactivated for 30 minutes at 56.degree. C. Fifty
microliters of each serum sample was placed into a corresponding
paraffin or ceramic-ringed slide. A drop (17 .mu.L) of each of the
antigens was placed in the corresponding rings of the slide. The
slides were placed in a mechanical rotator and rotated for 4
minutes at 180 rpm and then read microscopically. The degree of
flocculation of the two antigens was observed and recorded.
As reported in Table 4 (undocumented) all of the sera (100%),
reactive by RPR, were reactive with the CDC synthetic VDRL antigen
while only 88% were reactive with the natural VDRL antigen.
Additionally, the synthetic VDRL antigen and the natural VDRL
antigen were compared in the same test using 100 samples from
documented syphilis cases. The results of this test are also shown
in Table 4 (documented). All of the results from these tests were
confirmed by the SERODIA Treponema pallidum particle agglutination
test (TP-PA) (Fujirebio America, Inc., Fairfield, N.J.).
TABLE-US-00004 TABLE 4 Number of serum samples reactive with Number
of Synthetic Natural Serum VDRL VDRL Syphilis Category Samples
antigen antigen TP-PA Undocumented 100 100 88 99 Documented
Untreated Primary 9 9 9 8 Secondary 20 20 20 20 Latent 6 5 5 6
Treated Primary 15 12 11 13 Secondary 30 30 30 30 Latent 20 18 17
19 Total 200 194 180 195
EXAMPLE 4
Comparative Analysis of Synthetic VDRL Antigen and Natural VDRL
Antigen (Quantitative Test)
Samples from 100 frozen banked sera, reactive by the nontreponemal
(RPR) test, were used to compare the CDC synthetic VDRL antigen and
a reference VDRL antigen (Natural VDRL antigen). The serum samples
were diluted two-fold in a test tube with 0.9% saline. Fifty
microliters of each of the tube dilutions was transferred to the
corresponding rings of a ceramic or paraffin-ringed slide. A drop
(17 .mu.L) of each of the antigens was placed in the corresponding
rings of the slide. The slides were placed in a mechanical rotator
and rotated for 4 minutes at 180 rpm. The endpoint titer of each of
the serum dilutions was read microscopically. One doubling dilution
difference was defined as an endpoint either (R) from one antigen
or (N) for the other antigen.
As seen in Table 5 (undocumented), this test showed that 85% of the
frozen banked sera reactive in the RPR test had end-point titers of
one half or one dilution greater with CDC synthetic VDRL antigen
than with natural VDRL antigen. In 15% of the cases, the end-point
titer obtained with the CDC synthetic VDRL antigen was equal to
that obtained with the natural VDRL antigen. In none of the samples
tested was the end-point titer greater with the natural antigen
than with the CDC synthetic antigen.
This test was repeated using 100 samples from documented syphilis
cases. As seen in Table 5 (documented), 84% of the sera from
documented syphilis cases had end-point titers of one half or one
dilution greater with CDC synthetic VDRL antigen than with natural
VDRL antigen. In 7% of the cases, the end-point titer obtained with
the CDC synthetic VDRL antigen was equal to that obtained with the
natural VDRL antigen, while in 3% of the cases the end-point titer
of the natural VDRL antigen was one half or one dilution greater
than that of the CDC synthetic VDRL antigen. The results of these
tests were confirmed by TP-PA test.
TABLE-US-00005 TABLE 5 CDC CDC synthetic Synthetic Natural and
Natural VDRL VDRL VDRL Number of antigen antigen antigens equal
Syphilis Category Specimens higher higher endpoints Undocu- 100 85
0 0 mented Doc- umented Untreated Primary 9 3 2 4 Secondary 20 20 0
0 Latent 6 4 0 1 Treated Primary 15 10 0 2 Secondary 30 30 0 0
Latent 20 17 1 0 Total 200 169 3 7
EXAMPLE 5
Comparative Analysis of Synthetic VDRL Antigen and Natural VDRL
Antigen (Qualitative Test) in Patients Having Diseases Other than
Syphilis
Samples from 100 patients having diseases other than syphilis were
qualitatively tested using the procedure of Example 3. These tests
were confirmed by the TP-PA test and the FTA-ABS test. The results
of these tests are reported in Table 3 which shows that all of the
samples were nonreactive with both the CDC synthetic VDRL antigen
and the natural VDRL antigen. Four of the samples were reactive in
the TP-PA test, but nonreactive in the FTA-ABS test.
TABLE-US-00006 TABLE 6 Number of serum samples reactive and
nonreactive CDC Becton Synthetic Dickinson Number VDRL VDRL
Category of of antigen antigen TP-PA FTA-ABS Sample Specimens R N R
N R N R N Rheumatic 27 0 27 0 27 4 23 0 27 Fever Coronary 9 0 9 0 9
0 9 ND Arterial Disease Hypertension 6 0 6 0 6 0 6 ND Diabetes 4 0
4 0 4 0 4 ND Parkinson's 2 0 2 0 2 0 2 ND Obesity 2 0 2 0 2 0 2 ND
Angina 2 0 2 0 2 0 2 ND Miscellaneous 48 0 48 0 48 0 48 ND Category
Total Number 100 0 100 0 100 4 96 R = reactive; N = nonreactive
EXAMPLE 6
Comparative Analysis of Synthetic VDRL Antigen and Natural VDRL
Antigen (Qualitative Test) in Biological False Positive Samples
Samples were obtained from 50 individuals that were originally
classified as biological false positives (BFP). These individuals
tested nontreponemal test reactive and treponemal test nonreactive.
These samples were tested using the procedure of Example 3. The
results of this test is shown in Table 7. Four of the serum samples
that were originally misclassified as BFP were found reactive with
the CDC synthetic VDRL antigen, the TP-PA, and the FTA-ABS tests.
Three of these four samples were also reactive with the natural
VDRL antigen.
TABLE-US-00007 TABLE 7 CDC synthetic Natural VDRL Reactivity of
Sample TP-PA VDRL antigen antigen Reactive 4 28 27 Nonreactive 46
22 23 R = reactive; N = nonreactive
EXAMPLE 7
Comparative Analysis of Synthetic VDRL Antigen and Natural VDRL
Antigen (Qualitative Test) in Unknown Samples
495 samples with no patient identifiers were tested with CDC
synthetic VDRL antigen and natural VDRL antigen using the procedure
in Example 3 above. Reactive specimens were confirmed with the
TP-PA test, the ELISA test for syphilis IgG antibody, or the
FTA-ABS test. Thirty-eight of the samples were reactive in one of
the treponemal tests and 457 were nonreactive. As can be seen in
Table 8, all of the samples that were treponemal reactive were
reactive with the CDC synthetic VDRL antigen and 36 were reactive
with the natural VDRL antigen. Of the 457 serum samples that were
treponemal nonreactive, 452 were nonreactive with the CDC synthetic
VDRL antigen and 450 were nonreactive with the natural VDRL
antigen.
TABLE-US-00008 TABLE 8 CDC synthetic Natural VDRL Number of VDRL
antigen antigen Tests R N R N Reactive 38 38 0 36 2 Nonreactive 457
5 452 7 450 R = reactive; N = nonreactive
* * * * *