Antigenic composition and method for detection of presence of treponema pallidum in human

FIELD: medicine, infectious diseases.

SUBSTANCE: invention relates to antigenic composition and a method for detection of antibodies raised to Treponema pallidum in syphilis diagnosis. The antigenic composition comprises synthetic cardiolipin and synthetic lecithin (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and can comprise additionally cholesterol and alcohol. The antigenic composition can be used as an immunoreagent in immune analysis for detection of antibodies associated with the T. pallidum infection. The proposed method shows the enhanced sensitivity and specificity with respect to the T. pallidum infection.

EFFECT: improved detecting method.

23 cl, 8 tbl, 7 ex

 

The invention made by the Centre for Control and Prevention of Diseases, CDC, office of the Government of the United States.

The scope of this invention

The invention relates to the fields of Microbiology and immunology, and more specifically relates to compositions and methods for detecting, diagnosing and monitoring treatment of syphilis. The invention in particular relates to the antigenic compositions of synthetic cardiolipin and synthetic lecithin and their application in immunologicheskikh analyses.

The premise of this invention

Syphilis is a sexually transmitted disease (STD)caused by the bacterium Treponema pallidum. Reported every year more than 100,000 cases of syphilis in adults worldwide. The disease is also hereditary, affecting 3,000 or more babies every year. The inability to receive treatment with antibiotics in the early stages of the disease allows the progression of the disease throughout the body, often leading to irreversible damage of organs, psychosis, blindness or death. The spread of human immunodeficiency virus (HIV) (HIV) worldwide is extremely increased the severity of syphilis as health problems as ulcers of the genital organs, obtained during the early stages of syphilis infection, facilitate the transmission of HIV infection by sexual contact.

Uchenie syphilis has been divided into stages: primary, secondary, latent, and tertiary neurosyphilis (late). An infected individual can infect others during the first two stages. Transmission occurs when bacteria spread from the sores of an infected person to the skin or mucous membranes of the genitals, mouth or anus of a sexual partner. Organisms T.pallidum can also penetrate through the damaged skin on other parts of the body. During tertiary syphilis and neurosyphilis bacterial infection is not contagious, however, invasion (intrusion) of the microorganism in organs, tissues and the brain can have fatal consequences, as for example, serious cardio-vascular disorders or neurological disorder.

Vertical or transplacental syphilis infection can occur within the first four years, if a pregnant woman is infected and not treated. Although appropriate treatment of the mother usually prevents congenital syphilis, it was reported that approximately 25% of human embryos that have been exposed to infection T.pallidum in utero, were stillborn. Some babies with congenital syphilis have symptoms at birth, but most develop symptoms in the postpartum two to three months. These symptoms include sores on the skin, rash, fever, swollen liver and spleen, jaundice, anemia and different is cnie malformations. As infected infants develop, they may develop symptoms of late stage syphilis, including irreversible lesion of bones, teeth, eyes, ears and brain.

The first symptom of primary syphilis is an ulcer or chancre. The chancre appears within from ten days to three months after exposure and is usually found on the side of the body, which was unprotected from the sores of an infected sexual partner, such as the penis, the outer female genitals, vagina, cervix, rectum, tongue or lip. Because the chancre is saved only a few weeks and may be painless or may occur inside the body, it can go unnoticed. The chancre disappears with treatment or without treatment. In persons who do not treat secondary symptoms will appear after about nine weeks after manifestations the primary lesion.

Secondary syphilis often there is a skin rash that is characterized by brown sores about the size of a penny. As active bacteria are present in these ulcers, any physical contact, sexual or asexual, and affected skin of the infected individual can spread the infection at this stage. Other symptoms include mild fever, fatigue, headache, sore throat, alopecia baldness and opusi the lymph nodes. These symptoms may be weak and, like the chancre of primary syphilis will disappear with treatment or without treatment. Then, in the absence of treatment, an infected person enters a latent period.

Latent syphilis is characterized by the absence of clinical signs or pathological indicators in the cerebrospinal fluid (CSF) together with the positive results of serological tests. Early latent syphilis, which occurs during the first year of infection is potentially transmissible, and can cause relapses, while late latent syphilis is associated with immunity to relapse and resistance to re-infection.

During the early stages of infection with syphilis bacteria can affect the nervous system. In the absence of treatment, may develop neurosyphilis. Disease progression in the direction of neurosyphilis can last up to twenty years and in some individuals with neurosyphilis, unable to detect recognizable symptoms, making diagnosis very difficult. Those who really find your symptoms may complain of headache, Krivoshey or heat, which are the result of inflammation of the lining of the brain. The seizures and signs of a stroke such as numbness, weakness, or vision problems, can also affect patients with neurosyphilis.

Although PR is approximately two-thirds of persons infected T.pallidum, who were unable to get treatment, do not suffer further consequences of the disease, approximately one-third of persons with untreated latent syphilis detected complications of late, or tertiary, syphilis. During the tertiary stage of syphilis bacteria infect the heart, eyes, brain, nervous system, bones, joints, or almost any other part of the body. The tertiary stage may last for years or even decades. Late syphilis usually leads to cardiovascular disease, mental disorder, blindness, or even death.

Because of sometimes serious or life-threatening results of syphilis infection and the risk of transmitting or Contracting HIV accurate and early diagnosis of the infection is necessary. Syphilis, however, was sometimes called "the great imitator"because its early symptoms are similar to symptoms of many other diseases. Therefore, the physician usually does not rely only on the recognition of signs and symptoms of syphilis, but is based on the results of clinical tests, including microscopic identification of bacteria of syphilis and analytical tests on manifestations of syphilis infection in a biological sample.

Diagnosis of syphilis by microscopic identification of bacteria are usually performed as follows. Take a scraping from the surface of Zvi or chancre and examined under a special "dark-field" microscope, to detect the microorganism. Microscopy dark-field requires considerable skill and prone to incorrect interpretation.

For these reasons, the majority of syphilis cases first diagnosed serologically, using a non-treponemal tests. Non-treponemal tests detect substances, such as antibodies, which are formed in the presence of infection T.pallidum. Currently available non-treponemal tests most commonly used to detect evidence of infection with syphilis, are the test of the research laboratory for the study of venereal disease (VDRL) and rapid test for the reagents in the plasma (RPR). VDRL test uses lipids obtained from natural sources, to detect antilipogenic antibodies that are generated after infection T.pallidaum. These antibodies are produced against cardiolipin T.pallidum-organisms the immune system of an individual infected with T.pallidum, and can be detected in serum or cerebrospinal fluid of the individual.

One drawback of the currently available non-treponemal reactions is their low specificity. Many medical conditions, including mycoplasmal infections, pneumonia, malaria, acute bacterial and viral infections and autoimmune disease, can cause false-positive p the results of the tests in the currently existing tests for syphilis.

For example, the use of intravenous drugs or autoimmune disease cause tissue damage, which leads to the separation of cardiolipin and production anticardiolipin antibodies. The discovery of these anticardiolipin antibodies in non-treponemal test may therefore give a false positive result. Successful diagnosis is extremely problematic for the detection of neurosyphilis.

Due to the presence of false-positive and false-negative results when using these existing tests to confirm typically require an alternate method of analysis, such as microscopy or serological test based on Treponema. Normal treponemal tests include fluorescent test for treponemal antibody absorption (FTA-ABS) test FTA-ABS double staining (FTA-ABS DS). Although tests based on Treponema can be used to confirm a positive test result, these tests are often costly, complex and time consuming and can require highly sophisticated scientific equipment and trained scientific personnel. Moreover, treponemal tests cannot be used as tests for monitoring the success of antibiotic treatment, because, due to continuing after treatment with antibodies to T.pallidum, the results of the TES the s remain positive even after the destruction of the infection in approximately 85% of successfully treated patients.

So necessary a single test for sensitive and specific detection of infection T.pallidum in the sample for the diagnosis of early stages of syphilis or neurosyphilis. Also need a simple, cheap analysis, which could be used to monitor success of treatment of syphilis.

The invention

Provided antigenic composition and method for the detection of infection Therapeia pallidum and, therefore, the diagnosis of syphilis. The antigenic composition comprises a combination or mixture of synthetic cardiolipin and synthetic lecithin. Preferred antigenic composition also contains cholesterol. Preferred antigenic composition also includes alcohol. Alcohol solubilities lipids in antigenic composition, forming a suspension. Antigenic composition applicable as a reagent in tests for detection of antibodies associated with infection T.pallidum, in a biological sample, in particular in body fluids such as serum or cerebrospinal fluid. Preferably, the antigenic composition is a reagent immunoassay for the detection and titration of antibodies associated with infection T.pallidum.

Preferred antigenic composition comprises a purified, synthetic cardiolipin, synthetic lecithin, natural or non-synthetic, cholesterol and alcohol. The optimal h is state synthetic cardiolipin and lecithin in the composition is 99% or higher. Optimum purity cholesterol is 98% or higher, or he is ashless. Most preferably, the antigenic composition comprises terminator-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 0.02 and 0.04%, more preferably of 0.03%. The preferred concentration by volume of synthetic lecithin in the composition is between about 0.11 and 0.16 per cent, more preferably about 0.14%. The preferred concentration by volume of cholesterol in the composition is about 0.9% and the remaining part of the composition is alcohol.

Antigenic composition provided here is also generally useful as a research tool for the study of syphilis in vitro. More specifically, the composition is particularly useful in tests or diagnostic kits to detect the presence of infection T.pallidum, which is diagnostic or prognostic in respect of the occurrence or recurrence of the disease syphilis.

The preferred method presented here is an immunological assay for detection cardiolipin antibodies in a biological sample, such as serum or cerebrospinal fluid. In accordance with this method, an is yannou composition, described here, combined with biological breakdown for sufficient time under conditions that promote the binding antilipogenic antibodies in the sample with synthetic cardiolipin-lecithin matrix with the formation of the complex antigen-antibody. This complex then discover using methods well known to specialists in this field, as, for example, tests flocculation or microflocculation, or the like, Therefore, the object of the present invention is providing a method for the detection of carriers of the infection T.pallidum and, thus, prevent the spread of T.pallidum from one host to another.

Another object of the present invention is to provide a sensitive method for the diagnosis of early or latent syphilis or neurosyphilis.

Another objective of the present invention is to provide a fast, simple and low-cost analysis for accurate detection of T.pallidum.

A further object of the present invention is the provision of cheap manufactured antigenic compositions for reproducible measurement or detection of T.pallidum.

The next task of the present invention is the provision of a test for the detection of T.pallidum, which gives advantages in standardization and stability VDRL antigen.

These and other objectives, features and advantages which estva of the present invention will become apparent after reviewing the following detailed description in its expanded options and the accompanying claims.

DETAILED DESCRIPTION

Here antigenic composition and method detection Therapeia pallidum. The antigenic composition comprises a mixture or a combination of synthetic cardiolipin and synthetic lecithin. Cardiolipin is a 1,3-bis(phophatidyl)glycerol, possessing antigenic properties. Lecithin is a phospholipid. Preferably, the antigenic composition also contains a non-synthetic (natural) cholesterol. Alcohol is also a component of the preferred antigenic composition. Alcohol solubilities lipids, thus forming a suspension. Synthetic cardiolipin and lecithin are of the optimum purity 99% or higher. Cholesterol is optimal purity of 98% or higher and is ashless. Antigenic composition applicable as a reagent in assays for the detection of antibodies associated with infection T.pallidum, in a biological sample. Preferably, the antigenic composition is a reagent immunoassay for the detection or quantitative determination of antibodies generated by a patient infected T.pallidum, reagent, which is diagnostic or prognostic in relation to the presence or recurrence of syphilis.

The described method is a reaction for the detection or quantitative determination of antibodies associated with infection p is they T.pallidum, in a biological sample, in particular in a sample of body fluid such as serum or cerebrospinal fluid. The method allows to detect circulating antibodies associated with infection T.pallidum, for the detection or monitoring of infection T.pallidum. The preferred method provided here is immunological analysis. In accordance with this preferred method, the antigenic composition combined with biological breakdown for the required period of time under conditions that promote the binding antilipogenic antibodies in the sample with cardiolipin in antigenic composition with the formation of complexes of antigen-antibody. These complexes antigen-antibody is then detected using methods well known to specialists in this field, such as the VDRL test-flocculation or microflocculation in which results are obtained with the aid of the microscope.

DEFINITION

The terms “a”, “an” and “the”, which is used here, means “one or more” and include the plural as long, yet allow the context.

The term “antibody”used here includes monoclonal antibodies, polyclonal, chimeric, single-chain, bespecifically, monkey and human antibodies as well as Fab fragments, including the products of the libraries expressing Fab immunoglobulins.

The phrase “specifically SV is called with” or “specifically immunoreactive with”, when they refer to the antibody, indicate a binding reaction which is determinative of the presence of interest antigen in the presence of a heterogeneous population of peptides, proteins, lipids and other biological molecules. Thus, under these conditions, immunological analysis, the antigen or antigens are associated primarily with specific antibodies and does not bind in significant quantities with other antibodies present in the sample. Specific binding under these conditions requires the presence of antigen, which is selected for its specificity in relation to specific antibodies. Various forms of immunological analysis can be applied for the selection of antigens that are specifically immunoreactive with a specific antibody. For example, solid-phase immunoassay ELISA is used routinely for screening antigen specifically immunoreactive with the antibody. See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, in the description of the forms and conditions of immunological analysis, which can be used to determine specific immunoreactivity.

The term “antigen” refers to an object or its fragment, which can induce an immune response in mammals. The term includes immunogen and areas responsible for antigenicity, or antigenic deter inanti. The term “antigenic composition” used here refers to compositions containing synthetic cardiolipin and synthetic lecithin. The term “antigenic determinant”as used here refers to a part of the antigen that is recognized by the antibody.

Used here, the terms “detecting” or “detection” refers to a qualitative or a quantitative determination of the presence of the studied biomolecules.

The term “isolated” refers to a biological molecule that does not contain at least some components with which it is natural met.

ANTIGENIC COMPOSITION

The composition provided here contains a combination of, the suspension or physical mixture of one or more synthetic cardiolipin and lecithins. The preferred composition contains a synthetic cardiolipin, synthetic lecithin and synthetic or non-synthetic (natural occurring) cholesterol. A more preferred composition contains a synthetic cardiolipin, synthetic lecithin, natural cholesterol and alcohol.

The preferred concentration of synthetic cardiolipin in the composition is equal to approximately 0,02-0,04% by volume, more preferably of 0.03% by volume. The preferred concentration of synthetic lecithin in the composition is between priblizitelen is of 0.11 and 0.16% by volume, more preferably equal to 0.14% by volume. The preferred concentration of natural cholesterol in the composition is about 0.9 percent by volume, and the remainder of the composition is an alcohol, preferably ethanol, preferably absolute (100%) ethanol.

Synthetic cardiolipin can be synthesized from synthetic lipid precursors originating from plant sources. In the most preferred embodiment, cardiolipin is terminatorsalvation, which is commercially available from companies, for example, Avanti Polar Lipids (Alabaster, AL).

Synthetic lecithin can be derived from soy or eggs. In a preferred embodiment, the lecithin is a 16:0, 18:1 lecithin, described as 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or 3-sn-phosphatidylcholine (sn denotes stereospecific numbered), which is also commercially available from companies, for example, Avanti Polar Lipids (Alabaster, AL).

In the most preferred embodiment, the composition is a suspension containing approximately 0.03% terminatorsalvation, 0,11-0,16% 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 0.9% natural cholesterol in absolute ethanol. Cholesterol is also available from commercial sources, e.g., Avanti Polar Lipids (Alabaster, AL). Alcohol can be purchased from chemical suppliers is of such drugs, as for example, Sigma Chemical Company (St.Louis, MO).

When combined with alcohol cardiolipin, lecithin and cholesterol form a lipid matrix or micelle. As described in more detail below, antibodies, associated with the presence of infection T.pallidum in man, called here anticardiolipin antibodies that bind to these lipid micelles and form complexes antigen-antibody. Thus, the detection or quantification of these complexes antigen-antibody can be used to diagnose infection T.pallidum. While not wishing to be bound by the following hypothesis, the authors still believe that antibodies to cardiolipin matrix associated with synthetic antigenic composition described here, with much greater specificity and higher avidity than they are associated with natural occurring cardiolipin and lecithin. Thus, synthetic antigenic composition provides a more effective, more sensitive and specific method of detecting antibodies associated with syphilis infection.

Specialists in this field will understand that one or more components of the antigenic compositions can be labeled with the detectable label to facilitate direct measure or detect the formation of complex antigen-ant the body. Specialists in this area is well known for various types of labels and methods of conjugating these labels with antigenic composition.

The antigenic composition may be applied as a laboratory research tool for induction, separation or purification anticardiolipin antibodies, and these antibodies can be used to study syphilis at all. Thus, the antigenic composition is useful for purposes such as diagnostics in vivo and in vitro and laboratory studies.

GETTING VDRL ANTIGEN

VDRL antigen can be obtained, for example, by preparation of a solution terminatorsalvation in ethanol at a concentration by volume in the range of 0.02 to 0.04%, more preferably of 0.03%. The solution of the synthetic lecithin in ethanol having a concentration of approximately from 0.11 to 0.16%, more preferably of 0.14%, and the natural solution of 0.9%cholesterol in ethanol are added to a solution of cardiolipin. These components add in the following sequence: cardiolipin, lecithin, cholesterol and ethanol to volume. Before testing the antigen solubilizers and stored at room temperature over night.

DETECTION ANTICARDIOLIPIN ANTIBODIES

Provided here is the method includes diagnostic and prognostic methods for the detection and the quantitative determination of antibodies, the ability to communicate with antigenic composition described above. These methods allow to detect circulating antibodies to cardiolipin-lecithin matrix to indicate the presence of infection T.pallidum and thus to diagnose the infection, or to monitor the success of antibiotics in the treatment of infections T.pallidum.

In this area there are many methods for measuring or detecting complexes of the antibody-antigen, referred to here as well as immune complexes.

Classical methods include Simocatta sample containing the antibody with a known excess amount of antigen-specific antibodies, the separation of the bound antigen from free and determining the amount of bound antigen or free antigen. If you measure the amount of free antigen, the amount of bound antigen can be calculated by subtracting the amount of free antigen from a known initial amount. As described here, often the antigen is directly or indirectly labeled reporter group or a detectable label, to assist in determining the amount of a complex of antibody-antigen. Reporter group or label is usually a fluorescent or radioactive group, or an enzyme. The label then find, using well-known to specialists in this is blasti ways such as spectrophotometry, scintillation account or flow cytometry.

Alternatively, the antigen may be anywhereman with solid pellet or particle, which is filtered, centrifuged or removed from a mixture of other means, as for example, removing the metal magnetic or magnetized particles. Attaching the antigen to the solid-phase granules, for example, latex beads, provides a new, more sensitive and rapid test agglutination on a slide.

In a preferred embodiment, the antigen is attached to the granules through the cardiolipin molecule. One method of attachment of the antigen to the granules is in the modification of the molecules of cardiolipin so that she could be covalently linked to the beads. For example, the amino group can be attached to the terminal methyl groups of the fatty acid chains of cardiolipin. This modification of cardiolipin can be attached to carboxypropanoyl or laminirovannyy latex granules.

A preferred immunoassay for the detection anticardiolipin antibodies in the sample perform the following way. Sample collect or receive, using methods well-known to specialists with expertise in this area. Sample containing anticardiolipin antibodies, which should be detected, is obtained from b the ideological 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, etc. Optimum diagnostic results are obtained when the breakdown is serum or cerebral spinal fluid. The sample may be filtered or subjected to other manipulations before immunoassay for best results, immunological analysis.

Then, the sample is incubated with the antigenic composition described herein, to form a complex of the antibody-antigen. The complex of antibody-antigen is then detected using methods well known to specialists in this field. The term “detection” or “detected”, in the application here, means the use of known methods for detecting biological molecules, such as immunohistochemistry or histological methods. Such methods are known in the field, including immunological methods using monoclonal or polyclonal antibodies to lipids, such as enzyme-linked immunosorbent assay (ELISA), “sandwich” assays, flow-cytometrical assays, radioimmunoassay analyses or other types of analyses using antibodies known to specialists in this field.

In a preferred variant of the method anticardiolipin and is tetela in the sample find using synthetic antigenic composition described here in flocculation analysis. Examples of well-known flocculation tests include serological test reagents in normal serum (USR), fast Microtest on the reagents in plasma using a 10-millimeter round cards (RPR), serological test in normal serum samples using toluidine red (TRUST) and VDRL test on a glass slide. All these tests are flocculation reactions. Specialists in this field will be clear that anticardiolipin antibodies can also be detected using antigenic composition described here, in the reactions of agglutination, which additionally used in particle media. In a more preferred variant of the method of synthetic antigenic composition used in VDRL reaction on a glass slide, as described briefly below and in more detail in Veneral Desease Research Laboratory (VDRL) Slide Test, Kennedy.E.J.Jr. and Creighton, E.T, 157-78 (1998) A Manual of Tests for Syphilis, 9thed., Larsen, S.A., Pope, V., Johnson, R.E and Kennedy, E.J.Jr. (eds.), American Public Health Association, Washington, D.C., which is incorporated herein by reference.

VDRL reaction on a slide performed as follows.

VDRL-buffered saline solution containing formaldehyde, Na2HPO4KN2RHO4NaCl and distilled water, placed in the vessel. Antigenic the composition is added slowly to the salt solution at a constant speed during rotation of the vessel and the mixture is then thoroughly mixed, to connect content and to form a suspension. The sample, for example serum, placed inside a paraffin or a ceramic material ringed wells on a glass slide and add one drop of the suspension antigenic composition. The slide rotate to mix the sample with the antigenic composition, and then the slide examined microscopically. The presence of aggregates, sticky or rough, indicates the formation of a complex of antibody-antigen. Serial dilution of antigen suspension can be used for qualitative measurement of antibodies in the sample. Quantitative determination can be made by performing this analysis with standard concentrations of antibodies and comparison of test results with the results obtained with standards.

As should be clear, it is assumed that the methods of analysis include the use of synthetic antigenic compositions described above, and synthetic derivatives of antigenic compositions described herein, provided that these derivatives retain the antigenic activity or demonstrate equivalent antigenic activity and have specificity against anticardiolipin antibodies.

A KIT FOR DETECTING the presence of T.pallidum

Provided with the kit for diagnosing, or, in other words, the evaluation of C is eliticism infection by detecting the presence or quantity anticardiolipin antibodies. The set can be in any arrangement, known to experts in this field, and is useful for performing one or more of the methods described here for the detection of antibodies to cardiolipin-lecithin matrix in a biological sample or for the detection or monitoring of infection T.pallidum patient or carrier. The data sets are convenient for the reason that they supply most, if not all, of the necessary reagents for analysis to detect sifelani antibodies in a biological sample. The reagents can be pre-measured and maintained in a stable form in containers or on a solid phase, in which or on which the analysis can be performed, thus minimizing the number of manipulations performed by the person conducting the analysis. In addition, the analysis can be performed simultaneously with the standard, which is placed in the set, such as a predetermined number of antibodies to the results of the analysis could be alidibirov or measured.

The kit preferably contains antigenic composition described herein, which can be applied to detect cardiolipin antibodies associated with infection T.pallidum. This kit also preferably contains cholesterol, and may additionally contain the appropriate reagents, which help in diagnosing the AI complexes antigen-antibody. The kit may optionally contain equipment for safe receipt of the sample, the vessel contained a reagent, a buffer for diluting the sample or reagents, and round cards, such as 10 mm slides or 18 mm round cards used in tests VDRL, RPR and TRUST.

The test kit contains, but not limited to, reagents, which must be used in the following tests: flocculation reactions, such as USR, RPR and TRUST; the reactions of agglutination and “sandwich” or ELISA-assays. The materials used with these methods include, but are not limited to, microtiter plates, strips, coated with antibodies or probes for rapid monitoring of biological fluids. For each set range, sensitivity, precision, accuracy, specificity and reproducibility of the analysis. Standardization can be achieved using standard control serum and titration to the end point serum or can be used sera panel.

In a more preferred embodiment, the test kit uses the method VDRL reaction on a glass slide and provides instructions and antigenic composition described above. The set is suitable for assessing infection T.pallidum and, more specifically, for the quantitative determination of antibodies directed at lilipin in biological fluids of humans, manifesting the symptoms of syphilis, or the people who run the risk of Contracting syphilis.

The invention is additionally illustrated by the following examples, which should not be construed in any way as imposing limitations on the scope of the present invention. On the contrary, it must be clearly understood that the application may have various other alternatives, modifications and equivalents of the present invention that after reading this description we can offer ourselves experts in this field, without departing from the idea of the present invention and/or scope of the attached claims.

EXAMPLE 1

Preparation of compositions of synthetic cardiolipin and synthetic lecithin.

Tetrameristaceae, purified by chromatography on silica gel to almost 99% purity, was obtained in powder form from Avanti Polar Lipids (Alabaster, AL). The final concentration of sodium salt was tested for purity by thin-layer chromatography and high performance liquid chromatography. The sample was stored at -20° C. Tetrameristaceae was originally synthesized from synthetic lipid precursors of plant origin.

Powder lecithin (1-Palmitoyl-2-oleoyl-sn-glycerophosphocholine), purified by chromatography on silica gel to approximately 99% purity, was obtained from Avanti Polar Lipids. Lezi the Institute was originally isolated from soybean.

Preparing a 1.2%solution of cholesterol (Avanti Polar Lipids) in absolute ethanol and filtered using washed with alcohol filter paper #560. Cholesterol was originally derived from wool grease (grease) and was purified by recrystallization and the crystals were stored at -20° C.

Antigenic composition was prepared by combining synthetic cardiolipin with synthetic lecithin, a solution of cholesterol and ethanol in this order. The final concentration of synthetic cardiolipin was a 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 antigenic composition was ethanol.

EXAMPLE 2

Comparative analysis VDRL reaction on a glass slide with synthetic antigens in comparison with traditional VDRL-reaction on the glass

Compared the sensitivity VDRL reaction on a glass slide using a composition of synthetic cardiolipin and lecithin, as described in example 1, with a sensitivity of traditional VDRL reaction at the glass described in the Manual of tests for Syphilis, 9thed., 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 of VDRL-buffered salt solution (formaldehyde, Na2HPO4KH2PO4NaCl and distilled water) was applied to the bottom 30 round-illimitably flask with a glass sealing plug with the flat inner surface of the bottom or in 25-ml Erlenmeyer flask with ground glass stopper. Then 0.5 ml of the suspension antigenic composition was added directly to the electrolyte solution at a rate of 0.5 ml antigen suspension for 6 seconds, continuously rotating the flask. Rotation was continued for 10 seconds until until it was added to 4.1 ml of saline buffer. The flask was tightly closed and shaken, starting from the bottom to the top, approximately thirty times within 10 seconds. Antigenic suspension used within eight hours.

Qualitative reactions were performed by placing 50 μl of serum inside wax or ceramic ringed wells on a glass slide using a secure device for pipetting. Antigenic suspension gently resuspendable and added one free-fall drop (17 μl). A glass slide was placed on a mechanical rotator and rotated for 4 minutes at 180±2 rpm glass slide immediately removed and studied under a microscope using 10X-10X eyepieces and lens. The results were described as follows: reactive - medium or large aggregates, poorly or minimally reactive small units, directionspanel - without aggregates or very light roughness. Quantitative reactions were performed in a similar manner with serial twofold dilutions of serum.

The results set forth below in Table 1, showed that this re the Ktsia, using synthetic antigenic composition was more sensitive than the response, which uses the standard VDRL antigen, which is manufactured using natural cardiolipin and lecithin.

Table 1

Comparison of sensitivity VDRL reaction with natural antigens with VDRL-reaction with synthetic antigens
Sensitivity
ReactionPrimarySecondaryHidden
Natural VDRL antigen80%100%85%
Synthetic VDRL antigen84%100%88%

Compare also the sensitivity VDRL reaction on a glass slide using antigenic composition of synthetic cardiolipin and lecithin, as described in example 1, and the traditional VDRL reaction at the glass with traditional RPR-reaction on the glass. As shown in tables 2 and 3 below, the test using synthetic antigenic composition VDRL was more reactive with samples that tested positive in RPR-response than the test using no synthetic VDRL antigen.

Table 2

RR in comparison with synthetic VDRL
Synthetic VDRL
RPRReactiveDirectionspanel
Reactive*4113
Directionspanel*15
5All RPR-reactivity were minimally reactive

Table 3

RPR in comparison with natural VDRL
Natural VDRL
RPRReactiveDirectionspanel
Reactive*1341
Directionspanel*06
6All RPR-reactivity were minimally reactive

EXAMPLE C

Comparative analysis of synthetic VDRL antigen and natural VDRL antigen (qualitative test)

Samples of 100 frozen stored sets of serum samples reactive in non-treponemal (RPR) test was used for comparison of synthetic VDRL antigen CDC and control VDRL antigen (natural VDRL antigen). Serum samples iactiveaware by heating for 30 minutes pri° C. Fifty microlitres each sample of serum was placed in the corresponding paraffin or ceramic ringed hole on the slide. Drop (17 μl) of each antigen was placed in appropriate wells of the slide. Slides were placed in a mechanical rotator and rotated for 4 minutes at 180 Rev/min and then were read microscopically. Observed and recorded the degree of flocculation of the two antigens.

As described in Table 4 (undocumented), all serum (100%), according reactive RPR was reactive with synthetic VDRL-antigen CDC, while only 88% were reactive with natural VDRL-antigen.

Additionally, in the same test comparing synthetic VDRL antigen and natural VDRL antigen, using 100 samples of documented cases of syphilis. The results of this test are also shown in table 4 (documented). All results of these tests were confirmed by SERODIA-reaction on the agglutination of particles Treponema pallidum (TP-PA) (Fujirebio America, Inc., Fairfield, NJ).

Table 4
 The number of samples of sera reactive with, 
Categories of syphilisThe number of samples sivaram is to Synthetic VDRL-antigenNatural VDRL-antigenTR-RA
Undocumented1001008899
Documented    
without treatment, the Primary9998
Secondary20202020
Latent6556
Treatment Primary15121113
Secondary30303030
Latent20181719
Only200194180195

EXAMPLE 4

Comparative analysis of synthetic VDRL antigen and natural VDRL antigen (quantitative test)

Serum samples from 100 frozen stored serum, by reactive non-treponemal (RPR) reaction was used to compare the synthetic VDRL antigen CDC and control VDRL antigen (natural VDRL antigen) serum Samples were diluted two times in a test-tube with 0.9% saline solution. Fifty microlitres each test-tube dilutions was transferred into a corresponding ceramic material or paraffin ringed holes on the slide. Drop (17 μl) of each of the antigens was placed in appropriate wells on a glass slide. Slides were placed in a mechanical rotator and rotated for 4 minutes at 180 rpm Titer endpoint titration of each of the serum dilution were read microscopically. One difference from the double dilution was defined as the endpoint, or (R) for a single antigen, or (N) for a different antigen.

As can be seen from table 5 (undocumented), this reaction showed that 85% of the frozen stored serum RPR reactive in the reaction had a final titles on half or one dilution higher with synthetic VDRL-antigen CDC than with natural VDRL-antigen. In 15% of cases, the final titer obtained with synthetic VDRL-antigen CDC, was equal to that obtained with natural VDRL-antigen. None of the tested samples titer endpoint was not above the natural antigen than with a synthetic antigen CDC.

This reaction was repeated using 100 samples of documented cases of syphilis. As can be seen from table 5 (documented), 84% of sera from documented cases of syphilis had titles endpoint half or what but breeding is higher with synthetic VDRL-antigen CDC. In 7% of cases titer endpoint obtained with synthetic VGRL-antigen CDC, was equal to the titer obtained with natural VDRL-antigen, while in 3% of cases titer endpoint natural VDRL antigen was half or one dilution higher than the titer of such synthetic VDRL-antigen CDC. The results of these reactions confirmed the TR-RA-response.

Table 5
Category SyphilisThe number of samplesSynthetic VDRL antigen CDCNatural VDRL antigen aboveSynthetic CDC and natural VDRL-antigens, equal endpoints
Undocumented1008500
Documented    
Without treatment, the primary9324
secondary202000
latent6401
Treatment primary151002
secondary30 3000
latent201710
Total20016937

EXAMPLE 5

Comparative analysis of synthetic VDRL antigen and natural VDRL antigen (qualitative test) in patients having other than syphilis, diseases

Samples from 100 patients with other diseases than syphilis, were analyzed qualitatively using the procedure of example 3. These tests were confirmed by TR-RA-response and FTA-ABS-reaction. The results of these reactions are reported in Table 6, which shows that all the drugs were directionspanel as with synthetic VDRL-antigen CDC and natural VDRL-antigen. Four drugs were reactive in TR-RA-reaction, but non-reactive in the FTA-ABS-reaction.

Table 6
The number of serum samples reactive and directionspublic
Category sampleThe number of samplesSynthetic VDRL antigen CDCVDRL antigen Becton-DickinsonTR-RARTA-ABS
   RNRNRNRN
Rheumatic fever27027027423027
Coronary heart disease, defeat artery9090909ND
Hypertension6060606ND
Diabetes4040404ND
Parkinson's disease20202about2ND
Obesity2020202ND
Angina2020202Mixed category48048048048ND
The total number of10001000100496 
R=reactive; N=directionspanel

EXAMPLE 6

Comparative analysis of synthetic VDRL antigen and natural VDRL antigen (qualitative test) in a biological false-positive samples

Samples were obtained from 50 people who the source was classified as biologically (bacteriologically) false-positive (BFP). These people had a positive result in non-treponemal test and a negative result in a treponemal test. These samples were analyzed using the procedure of example 3. The results of this test are shown in Table 7. Four serum samples that source were misclassified as BFP, were found reactive with synthetic VDRL-antigen CDC, TR-RA - and FTA-ABS-reactions. Three of these four samples were also reactive with natural VDRL-antigen.

Table 7
The reactivity of the robe TR-RASynthetic VDRL antigen CDCNatural VDRL antigen
Reactive42827
Directionspanel462223
R=reactive; N=directionspanel

EXAMPLE 7

Comparative analysis of synthetic VDRL antigen and natural VDRL antigen (qualitative test) in unknown samples

495 samples of unidentified patients analyzed with synthetic VDRL-antigen CDC and natural VDRL-antigen using the procedure of the above example 3. Reactive samples were confirmed TR-RA-reaction, ELISA analysis for IgG antibodies syphilis or FTA-ABS - reaction. 38 of these samples were reactive in one of treponemal reactions and 457 were directionspanel. As can be seen from Table 8, all samples that were reactive against Treponema were reactive with synthetic VDRL-antigen CDC, and 36 samples were reactive with the natural VDRL-antigen. From 457 serum samples that were directionspanel against Treponema, 452 samples were directionspanel with synthetic VDRL-antigen CDC and 450 samples were directionspanel with natural VDRL-ant the genome.

Table 8
 The number of testsSynthetic VDRL antigen CDCNatural VDRL antigen
  RNRN
Reactive38380362
    
Directionspanel45754527450
R=reactive; N=directionspanel

1. Antigenic composition for detecting the presence of Treponema pallidum containing synthetic cardiolipin and 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

2. The composition according to claim 1, characterized in that it further comprises cholesterol.

3. The composition according to claim 2, characterized in that the concentration of cholesterol in the composition is approximately 0.9 percent.

4. The composition according to claim 2, characterized in that it further comprises alcohol.

5. The composition according to claim 1, characterized in that the concentration of cardiolipin in the composition is approximately the nutrient 0,02-0,04%.

6. The composition according to claim 5, characterized in that the concentration of cardiolipin in the composition is approximately 0.03%.

7. The composition according to claim 1, characterized in that the concentration of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine approximately 0,11-0,16%.

8. The composition according to claim 7, characterized in that the concentration of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine is approximately 0.14%.

9. The composition according to claim 1, wherein the cardiolipin is terminatorsalvation.

10. The composition according to claim 4, characterized in that the alcohol is ethanol.

11. The antigenic composition of claim 10, characterized in that it contains a synthetic cardiolipin, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, cholesterol and ethanol, the concentration of cardiolipin is approximately 0,02-0,04%, the concentration of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine approximately 0,11-0,16%and the cholesterol concentration of approximately 0.9 percent.

12. The composition according to claim 11, characterized in that it contains approximately 0,02-0,04% terminatorsalvation, approximately 0,11-0,16% 1-Palmitoyl-oleoyl-sn-glycero-3-phosphocholine and approximately 0.9% cholesterol and ethanol up to full volume.

13. The composition according to claim 11, characterized in that it contains approximately 0.03% terminatorsalvation, approximately 0,11-0,16% 1-Palmitoyl-oleoyl-sn-3-phosphocholine and approximately 0.9% of natural cholesterol in absolute ethanol to volume.

14. The method of detecting the presence of Treponema pallidum in human, comprising combining a biological sample from a human with a composition comprising a synthetic cardiolipin and 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and detecting the complex formed between the antibody in a biological sample and composition.

15. The method according to 14, characterized in that the composition additionally contains cholesterol and alcohol.

16. The method according to item 15, wherein the concentration of cholesterol in the composition is approximately 0.9 percent.

17. The method according to item 15, wherein the alcohol is ethanol.

18. The method according to 14, characterized in that the concentration of cardiolipin in the composition is about 0,02-0,04%.

19. The method according to 14, characterized in that the concentration of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the composition is approximately 0,11-0,16%.

20. The method according to 14, wherein the cardiolipin in the composition is terminatorsalvation.

21. The method according to 14, characterized in that the detection of the immunocomplex is used for diagnosis of syphilis in humans.

22. The method according to 14, characterized in that immunocomplex find using the flocculation reaction or agglutination reaction.

23. The method according to 14, characterized in that it envisages

(a) obtaining Biol the environmental sample from a person;

(b) combining a biological sample with a composition containing approximately 0,02-0,04% terminatorsalvation, approximately 0,11-0,16% 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, approximately 0.9% cholesterol and ethanol to volume, and

(c) detecting the immunocomplex formed between the antibody in a biological sample and composition.



 

Same patents:
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The invention relates to medicine, namely to venereology
The invention relates to medicine, namely to microbiological research methods
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The invention relates to medicine, in particular to Microbiology, and in particular to methods of predicting the course of infectious-inflammatory diseases of the urogenital tract

The invention relates to medicine, namely, venereology, and can be used for differential diagnosis of false-positive serological reactions blood for syphilis and early latent syphilis
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The invention relates to medicine, in particular to immunology, and can be used in clinical and laboratory practice

FIELD: medicine, infectious diseases.

SUBSTANCE: invention relates to antigenic composition and a method for detection of antibodies raised to Treponema pallidum in syphilis diagnosis. The antigenic composition comprises synthetic cardiolipin and synthetic lecithin (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and can comprise additionally cholesterol and alcohol. The antigenic composition can be used as an immunoreagent in immune analysis for detection of antibodies associated with the T. pallidum infection. The proposed method shows the enhanced sensitivity and specificity with respect to the T. pallidum infection.

EFFECT: improved detecting method.

23 cl, 8 tbl, 7 ex

FIELD: medicine, dermatology.

SUBSTANCE: before carrying out separate IEA for detecting anti-Treponema antibodies one should additionally treat patient's blood serum with a magnetic sorbent with an immobilized protein A of staphylococcus that sorbs IgG1 and IgG4. The innovation provides higher specificity and simplicity of the method conducted.

EFFECT: higher accuracy of diagnostics.

3 ex, 1 tbl

FIELD: biotechnology, immunology, in particular production of serum panels to control quality of test systems for lues serotological diagnosis.

SUBSTANCE: claimed panel contains set of samples with standardized content of IgG class antibodies to T. palladium p17 and p41 antigens: namely sample with prevalent content of antibodies to p17 antigen, samples with prevalent content of antibodies to p41 antigen, samples containing antibodies to p17 antigen only, samples containing antibodies to p41 antigen only, and samples containing mixture antibodies to h17 and p41 in equal concentrations. Claimed method includes sampling of positive serums based on ratio (K) of reverse titers of antibodies to T. palladium antigens with mol.w. of 17 and 41 kD (K = T-1p17/T-1p41) and sampling of original serum samples with K>=2, K<=1|2 and K = 1. Said samples are diluted with non-immune donated serum to produce necessary concentration of specific antibodies.

EFFECT: improved method for examination of test system specificity, sensitivity, and other characteristics.

7 cl, 2 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns development of a diagnostic test system in immunochip format and a method of simultaneous and differential detection of reaginic antibodies and antibody spectrum to diagnostically significant mmunologically relevant proteins Treponema pallidum of G (IgG) and M (IgM) classes. The diagnostic test system in immunochip format for differential serum diagnostics of syphilis consists of an immunosorbent with separately immobilised antigens Treponema pallidum Tp15, Tp17, TmpA, Tp47, conjugate and reactants required to detect an antigen-antibody complex. Cardiolipin is additionally immobilised on the immunosorbent; antigens Treponema pallidum and cardiolipin are immobilised at least, in two repetitions, and conjugate consists of mixed antispecies human IgG antibodies and human IgM antibodies modified by two phosphors with different spectral characteristics. On the immunosorbent, there can be additionally immobilised at least, one antigen and/or peptide Treponema pallidum specified e.g. of Tp39, Tp41, Tp42, Tp44.5, Tp92, Tp0453 at least in two repetitions. Cardiolipin can represent an oxidised cardiolipin derivative bounded with protein. The differential serum syphilis diagnostic technique with using the diagnostic test system in immunochip format involves application of human IgM modified by two phosphors with different spectral characteristics. On the immunosorbent, there can be additionally immobilised at least one antigen and/or peptide Treponema pallidum, specified e.g. of Tp39, Tp41, Tp42, Tp44.5, Tp92, Tp0453 at least in two repetitions. Cardiolipin can represent an oxidised cardiolipin derivative bounded with protein. The differential serum syphilis diagnostic technique with using the diagnostic test system in immunochip format implying that a cultivation solution for check and test samples is introduced on the immunosorbent with separately immobilised antigens Treponema pallidum and cardiolipin; the check and test samples are introduced; the prepared mixture is incubated at temperature 20-42°C for 15-60 min to prepare the antigen-antibody complex; the immunosorbent is rinsed; then a conjugate solution of mixed human IgG antibodies and human IgM antibodies modified by two phosphors with different spectral characteristics is introduced on the immunosorbent; it is followed with incubation at temperature 20-42°C for 15-60 min; the immunosorbent is rinsed, dried to detect the prepared antigen-antibody complex.

EFFECT: improved diagnostic accuracy.

20 cl, 5 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to preparing diagnostic products. The method is implemented by intratesticular infections of rabbits producers of 3.0-3.5 kg with pathogenic Nichols strain T. Pallidum pallidum. 7-8 days after the infection, the animals are additionally twice intravenously immunised with purified treponema proteins ("ОПТ"-antigen) recovered from "КСТ"-antigen of cultural Treponema Palladium in dosage 0.023 g, every 7-8 days. It is followed with exsanguination 30 days after the infection. The prepared serums are analysed for anticardiolipin antibodies titre in quantitative cardiolipin antigen microprecipitation test.

EFFECT: method allows increasing anticardiolipin antibodies titre, standardising a procedure of quantitative cardiolipin microprecipitation test for syphilis, and halving the findings analysis time.

2 ex

FIELD: medicine.

SUBSTANCE: what is involved is biostimulation accompanied with suppression of pathogenic microflora by the local introduction of 0.5-5% aqueous solution of the biologically active preparation Tomed-Aqua in tampone. It is followed by smear microscopy for detection of trichomonads taken from three points of the urogenital mucosa starting from the 4-7th day after the beginning of the preparation administration.

EFFECT: use of the technique enables higher diagnostic effectiveness of urogenital trichomoniasis ensured by considerable reduction of length of diagnosis, extended spectrum of suppressed bacterial agents and reduced risk of side effects.

2 ex

FIELD: medicine.

SUBSTANCE: genital pathology is diagnosed by microscopy of a clinical material of genital secretion; questionnaire survey is conducted, and total value is calculated. It is added by determining a patient's infectivity index (PII) by formula wherein: cL is a relation of one-field leukocyte count at a certain moment, cL1, cL2, cL3 are average three-field leukocyte counts at a certain moment. It is followed by calculation of a quality of life index by formula: wherein OI is the observed incidence of the given pathology, absolute numbers calculated by formula wherein PII is the patient's infectivity index, N is total number of the patients with specific smear cells, CI is a correction for the incidence (equal to 0.1), OSA is observed sexual activity (number of intercourse partners), CSA is a correction for sexual activity (equal to 0.001), k is total quality of life index, points. The HIQLSTi values within the range [0-0.15] enables diagnosing a very low quality of life index; the value HIQLsti [0.16-0.35] shows a low quality of life index; the value HIQLSTI [0.36-0.85] presents an average quality of life index; and a high quality of life index is shown by the value HIQLsti [0.86≥1.0].

EFFECT: method improvement.

3 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method involves immobilising the number of mixed proteins on a working surface of an immunosorbent; the above proteins are able to bind markers of the number of relevant infectious diseases; the markers of at least one infectious disease bound to the working surface of the immunosorbent in the form of immune complexes are detected with the above markers being detected by measuring total enzymatic activity of the immunosorbent.

EFFECT: method is easy to implement, it requires no special equipment and enables reducing time and labour content for making a complex diagnosis of infectious diseases in many times.

12 cl, 1 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: diagnostic puncture is preceded by measuring blood antitreponemal titres in flocculation tests; if the titres are not less than 1:32, a high probability of neurosyphilis is stated; if the titres are less than 1:32, blood treponema-specific titres are determined in haemagglutination test, and if the values are not less than 1:40960, a high probability of neurosyphilis is also stated.

EFFECT: method is accessible and simple, and enables achieving the result in a relatively short time.

1 tbl, 3 ex

FIELD: biochemistry.

SUBSTANCE: described is structure triplet of Treponema pallidum antigen, which includes three antigens to Treponema pallidum (TP15, TP17 and TP47), as well as leader sequence of ten amino acids (mark 261) and copper-zinc containing human superoxide dismutase (hSOD). This design is optimized for diagnosing syphilitic infection in vitro. Described also is plasmid containing DNA coding triplet antigen, host cells, methods of producing, methods for detection and sets.

EFFECT: invention extends range of agents and methods for determination of syphilis.

45 cl, 19 dwg, 8 tbl, 4 ex

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