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Method for detecting syphilis using synthetic antigens

a technology of synthetic antigens and syphilis, applied in the field of microbiology and immunology, can solve the problems of inability to detect, diagnose and monitor the treatment of syphilis, inability to detect, or die, and inability to detect the presence of syphilis, so as to prevent the spread of

Inactive Publication Date: 2013-01-08
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention aims to provide a method for detecting carriers of T. pallidum infection to prevent its spread from one host to another. The invention offers a rapid, simple, and inexpensive assay for accurately detecting T. pallidum. It also provides an inexpensively produced antigen composition for reproducible measurement or detection of T. pallidum. Furthermore, the invention offers a test for detecting T. pallidum that offers advantages in standardization and stability of the VDRL antigen.

Problems solved by technology

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.
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.
Because the chancre lasts only a few weeks and may be painless or occur inside the body, it may go unnoticed.
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.
Late syphilis commonly results in cardiovascular disease, mental illness, blindness, or even death.
Dark-field microscopy requires considerable skill and is prone to misinterpretation.
One disadvantage to the presently available non-treponemal tests is poor specificity.
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.
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.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of a Synthetic Cardiolipin and Lecithin Composition

[0058]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° C. The tetramyristoyl cardiolipin was originally synthesized from semi-synthetic lipid precursors that originated from a plant source.

[0059]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.

[0060]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-crystall...

example 2

Comparative Analysis of Synthetic VDRL Slide Assay versus Conventional VDRL Slide Assay

[0062]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, 9th 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, Na2HPO4, KH2PO4, 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 ca...

example 3

Comparative Analysis of Synthetic VDRL antigen and Natural VDRL antigen (Qualitative Test)

[0069]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° C. Fifty microliters of each serum sample was placed into a corresponding paraffin or ceramic-ringed slide. A drop (17 μL) 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.

[0070]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.

[0071]Additionally, the synthetic VDRL antigen and the natural VDRL antige...

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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

PRIORITY CLAIM[0001]This is a §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.FIELD OF THE INVENTION [0002]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[0003]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...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): G01N33/571G01N33/543C12N15/09A61K39/02G01N33/92
CPCG01N33/571G01N33/92G01N2333/20A61K39/00
Inventor CASTRO, ARNOLD R.
Owner UNITED STATES OF AMERICA
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