Title:  Method for detecting syphilis using synthetic antigens

United States Patent:  6,815,173

Issued:  November 9, 2004

Inventors:  Pope; Victoria (Atlanta, GA); Castro; Arnold R. (Atlanta, GA); Morrill; William E. (Snellville, GA)

Assignee:  The United States of America as represented by the Department of Health and (Washington, DC); Centers for Disease Control & Prevention (Atlanta, GA)

Appl. No.:  009698

Filed:  December 5, 2001

PCT Filed:  June 8, 2000

PCT NO:  PCT/US00/15828

PCT PUB.NO.:  WO00/75666

PCT PUB. Date:  December 14, 2000

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.

Description of the Invention

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 air 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 OF THE INVENTION

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.

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 overnight 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 immunocomplex. 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^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, Na₂ HPO₄, KH2PO4, 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, microtiter 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.

Claim 1 of 23 Claims

We claim:

1. An antigen composition comprising tetramyristoyl cardiolipin and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

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