Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-03-30
2002-11-12
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007320, C435S007360, C435S007920, C435S007100, C435S007710, C435S071100, C435S252300, C435S252100, C435S260000, C435S253600, C536S023700
Reexamination Certificate
active
06479248
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method and agent for the detection of antibodies to
Treponema pallidum.
2. Related Art
Treponema pallidum
is the causative agent of syphilis. Diagnosis of infections with this pathogen is important not only when infection is suspected, but it is also of great significance to exclude syphilis among pregnant women and blood donors. In prenatal care, the purpose of such diagnosis is to prevent congenital syphilis, i.e. a transfer of syphilis to the neonate, and any consequent harm to the child. Negative serologic results for syphilis constitute one selection criterion for blood donors.
Syphilis is primarily diagnosed serologically, i.e. by the detection of antibodies to
Treponema pallidum
and/or cardiolipin. Specific IgM antibodies are detectable already 14 days after infection and IgG antibodies at the latest from 4 weeks after infection. Only before that time is direct detection of the causal agent from infected tissue of the primary lesion is another decisive diagnostic criterion.
Diagnosis by culture, as is conventional for many other bacterial pathogens, is not possible as
Treponema pallidum
has not hitherto successfully been cultured in vitro, with the exception of propagation in cell cultures (U.S. Pat. No. 5,264,360).
Serologic detection methods may be divided into three groups depending upon the nature of antibody detected:
1. Nontreponemal tests. Tests such as the cardiolipin microflocculation test (CMT), which is known in the English-speaking world as the Venereal Disease Research Laboratory Test (VDRL test), the rapid plasma reagin test (RPR test) and the cardiolipin complement binding reaction (cardiolipin CBR) are based on the detection of antibodies to cardiolipin. These tests reveal positive results 3-5 weeks after infection or approx. 7-10 days after appearance of the primary lesion. Sensitivity is 60 to 87% in the primary stage and may be as high as 100% in secondary syphilis. Sensitivity does, however, fall in the later stages of the disease, such that up to 30% of the late stages are no longer reactive. When the VDRL test is performed quantitatively, titre may be correlated to the activity of the disease. The disadvantage of this test is the large proportion of 0.3-0.9% of false positive test results when screening blood donors and the occurrence of false negative results in sera having an elevated titre due to the prozone phenomenon, which may be observed in 1-2% of cases in the VDRL test in secondary syphilis.
2. Treponema-specific tests. Antibodies to the endoflagellae of
Treponema pallidum
are formed as syphilis progresses. As a result of antigen relatedness, these antibodies also react with the endoflagellae of other species of treponema. The endoflagellae of
Treponema phagedenis
(biotype Reiter) have thus also been used as an antigen for the diagnosis of syphilis (Flagellum ELISA, R. V. W. van Eijk et al.,
Genitourin. Med
. 62, 367-372 (1988)). In the flagellum ELISA, the cut off for a positive test result is a compromise between sensitivity and specificity, as a result of which 0.8% of results are false positives and 2.7% false negatives.
3
. Treponema pallidum
-specific tests. These tests detect antibodies which react with
Treponema pallidum
or antigen preparations from this pathogen. These test systems include the
Treponema pallidum
haemagglutination test (TPHA), the fluorescent
Treponema pallidum
antibody absorption test (FTA-ABS) and the Nelson test (
Treponema pallidum
immobilisation test, TPI) together with ELISA systems based on sonicate antigen. TPHA and FTA-ABS are generally used in diagnostics.
The Nelson test involves a microscopic assessment of the extent to which complement activating antibodies in the patient's serum inhibit the mobility of
Treponema pallidum
. Due to its high cost, the Nelson test is used virtually exclusively for scientific investigations.
In TPHA, erythrocytes to which the
Treponema pallidum
sonicate antigen is bound are agglutinated by serum antibodies from syphilis patients. At a false positive rate of 0.07% and false negative rate of 0.008%, TPHA is highly specific and sensitive with most false negative results occurring in the initial stage of syphilis. TPHA is positive starting from the 4
th
week of infection, exhibits sensitivity of 64% to 87% in primary syphilis with an initially low titre (80-320) which, on transition to secondary syphilis, may rise to above 5000 at a sensitivity of 100%. As the disease progresses further, TPHA remains positive when the titre has a tendency to fall during the latent stage.
In FTA-ABS, indirect immunofluorescence microscopy is used to detect the binding of specific antibodies in the serum under investigation onto
Treponema pallidum
attached to a solid support via a fluorescent-labelled secondary antibody. Sensitivity of detection is 86% to 100% in primary syphilis, reaches 100% in secondary syphilis and 96% to 100% in the late stages. According to various sources, sensitivity for all stages is between 83% and 95%. Specificity, at 83% to 89% (excluding “borderline” findings) is not very high. Isolated FTA-positive sera with an otherwise negative syphilis serology are caused, for example, by Lyme borreliosis. FTA-ABS is performed as a confirmatory test to validate positive TPHA findings. Veldkamp and Visser (
Brit. J. Vener. Dis
. 51, 227-231 (1975)) provide the first description of an ELISA (enzyme-linked immunosorbent assay) in which the sonicate antigen of
Treponema pallidum
, which is adsorptively bound to solid phases, reacts with specific antibodies from the material under investigation. The bound antibodies are detected by reaction with an enzyme-labelled, species-specific secondary antibody to class IgG or IgM immunoglobulins and a subsequent enzymatic colour reaction. The commercial indirect enzyme immunoassay Captia Syphilis G (Mercia Diagnostics, Guildford, England) is also based on this principle. This assay has been found to have a sensitivity of 98.4% and a specificity of 99.3% (Young et al.,
J. Clin. Pathol
. 45, 37-41, (1992),
Genitourin. Med
. 65, 72-78 (1989)). Captia Syphilis M detects a specific IgM which is formed right at the early stages of the disease (sensitivity in primary syphilis 82%). Since IgM antibodies cannot pass through the placenta, this assay is of significance in the diagnosis of congenital syphilis (sensitivity 100%, Ijsselmuiden et al.,
J. Clin. Microbiol
. 27, 152-157, (1989)). The assay is based on the principle of a solid phase bound capture antibody for IgM from the material under investigation, wherein specific IgM antibodies subsequently react with
Treponema pallidum
antigen. Detection is by means of an enzyme-labelled monoclonal antibody to endoflagellae and subsequent enzymatic colour reaction.
In the Syphilis BioEnza Bead Assay (Organon Teknika Corp., USA), the
Treponema pallidum
antigen is adsorptively bound to ferromagnetic particles. Binding of antibodies from the material under investigation is then detected by means of enzyme-labelled secondary antibodies and colour reaction. The sensitivity and specificity of this test are stated at 93% and 98.6% (Burdash et al.,
J. Clin. Microbiol
. 25, 808-811, (1987)).
The sonicate antigen mentioned above is obtained by mechanical processing of the pathogen with ultrasound.
The deficient in vitro culturability of
Treponema pallidum
, which has already been mentioned, naturally means that these pathogens can be made available only with great difficulty.
Two-stage diagnosis of syphilis is currently generally recognised. In the first stage, all the sera are investigated with a test (screening). In the second stage, positive sera are investigated with another test system (confirmatory test). Since, in many countries, the incidence of syphilis is low (<1%), 99% specificity of the screening test is not sufficient to ensure reliability of diagnosis. A positive predictive value of above 90% is only achieved with a confirmatory test based on a different detection principle.
Scr
Gerber Annegret
Krell Siegfried
Baskar Padma
Salter & Michaelson
Smith Lynette R. F.
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