Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-17
2004-03-02
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...
C435S007100, C435S007200, C435S007360, C435S007900, C435S007920, C435S007930, C435S007940, C435S007950, C435S071100, C424S263100, C424S185100, C424S184100, C424S139100, C424S130100, C530S300000, C530S350000
Reexamination Certificate
active
06699678
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns an improved method and kit for the diagnosis of
Chlamydia trachomatis
(
C. trachomatis
) in humans. More specifically, the present invention concerns a new mixture of peptides derived from the major outer membrane protein (MOMP), which together are capable of specifically reacting only with antibodies specific to any one of the serovars of
C. trachomatis
, and hence said mixture of peptides is particularly useful for identifying
C. trachomatis
infections in humans by binding specifically to antibodies, if present, in a body fluid sample obtained from an individual being tested.
BACKGROUND OF THE INVENTION
Chlamydia is a gram negative obligate intracellular bacteria that causes acute and chronic disease in mammalian and avian species. The genus Chlamydia is comprised of four species:
C. trachomatis, C. pneumonziae, C. precorum
and
C. psittaci.
The
C. trachornatis
species is divided into 15 serovars. Serovars A, B, Ba and C are agents of trachoma, a leading cause of preventable blindness, endemic in the Third World. Serovars L1-L3 are the agents of lymphogranuloma venereum. Serovars D-K are a common cause of sexually transmitted genital infection worldwide: cervitis, endometritis/salpingitis in females and uretritis in both males and females. Endometritis/salpingitis can lead to agglutination of salpinx, with a higher risk of extra-uterine pregnancy and infertility. The genital infection can cause acute infection and persistent infection occasionally without any clinical sign. Generally, these infections are treatable, once they are diagnosed; however, without any treatment, the infections can progress to severe chronic inflammation leading to infertility, ectopic pregnancy, induced abortion or pre-term child delivery. Moreover, the infants of infected mothers can themselves be infected during birth, leading to conjunctivitis or pneumonia.
Serological testing, i.e., the testing for the presence (or not) of anti-
C. trachomatis
antibodies in an individual, is now an established approach in many countries, and has been shown to provide a comprehensive answer for the detection of
C. trachomatis
infection. In suspected deep-seated infections, body fluid sampling reduces the necessity for invasive procedures which are required for direct antigen detection. In cases of lower urogenital infections, collection limitations such as effectiveness of scrape sampling procedure, specimen handling and transportation difficulties have to be weighed. Above all, there however still remains the problematic issue that most Chlamydial infections are asymptomatic. Therefore, an infection may persist for a long time, ascend the upper genital tract, causing deep and chronic infections, and increase the probability of false negative results in procedures designed for direct antigen detection, i.e., sampling of lower genital tract tissue with anti-
C. trachomatis
antibodies, or other suitable procedures, to directly detect the presence of the major
C. trachomatis
antigens such as the major outer membrane protein (MOMP) indicative of Chlamydial infection.
Serological testing for
Chlamydia trachomatis
, through the detection of various specific antibodies, is today an effective and highly accepted optional detection procedure. New and refined technologies apply the immuno markers IgM, IgA and IgG to characterize the presence and stage of infection.
The detection of specific IgM antibodies is indicative of acute Chlamydial infections. The absence does not, however, preclude the presence of on-going infection, however, especially in recurrent and chronic cases. The detection of specific IgA antibodies is now accepted as indicative of active Chlamydial infection and has been shown to be an important marker because of the shorter lifetime of IgA antibodies which persist only as long as antigenic stimulation exists. IgA antibody detection is, moreover, suitable for post-therapy follow-up. IgG antibody detection is a marker for Chlamydial-positive immune-response, either for current, chronic or past infections.
There exists a high level of serological cross-reactions between the three different species of Chlamydia. Most of the serological diagnostic assays for Chlamydia use either purified elementary bodies (microimmunofluorescence, MIF and ELISA tests), lipopolysaccharide, LPS or purified major outer membrane protein, MOMP (ELISA tests) as antigens. Genus specific epitopes are present in all the above antigens, therefore, low species specificity is observed. Moreover, a large proportion of the worldwide human population has been exposed to
C. pneumoniae
(with no clinical signs) and the prevalence of anti-Chlamydia antibodies is very high. Therefore, the differentiation between
C. pneumoniae
and
C. trachomatis
specific antibodies using conventional serological screening tests (MIF, ELISA, EIA, etc.) is not very effective.
THE PRIOR ART
A number of publications have been made in which there have been disclosed the isolation, purification and characterization of the major
C. trachomatis
outer membrane complex proteins, including the above-noted MOMP, and the use thereof for the purposes of generating anti-
C. trachomatis
antibodies and in immunoassays to detect the presence of anti-
C. trachomatis
antibodies in samples obtained from individuals suspected of being infected by
C. trachomatis
. For example, in U.S. Pat. No. 5,318,892 and its corresponding EP 0 456 524, there is disclosed the use of
C. trachomatis
outer membrane complex consisting of at least three polypeptides, including MOMP, for assaying anti-
C. trachomatis
antibodies. In U.S. Pat. No. 4,427,782, there is described the isolation and characterization of the
C. trachomatis
MOMP and its use in immunoassays. However, in the aforesaid published patents, there is not provided any nucleotide or amino acid sequences of the MOMP polypeptide, nor is there disclosed the possibility of using peptides derived from MOMP for the purposes of immunization against
C. trachomatis
disease or for use in immunoassays to detect anti-
C. trachonzatis
antibodies, indicative of infection by
C. trachoniatis
. It is well known in the art that large proteins such as MOMP are less effective than smaller antigenic peptides derived therefrom, both for the purposes of immunization against
C. trachomatis
infection and for use in immunoassays to detect anti-
C. trachomatis
antibodies. Further, the complete MOMP protein obtained from
C. trachomatis
also has a number of epitopes which are common to the MOMP protein obtained from the other above-noted species of Chlamydia, with the result that using the complete protein in immunoassays to specifically detect anti-
C. trachomatis
antibodies is not reliable because of the possibility of cross-reactivity between the various Chlamydia species, with the result that when positive results are obtained, it is not readily possible to determine whether the antibodies are specific to
C. trachomatis
or to the other Chlamydia species. Hence, in respect of
C. trachomatis
, false-positive results may be obtained and may even lead to an incorrect diagnosis of the agent causing the infection and subsequently to an incorrect mode of treatment of the infection. Moreover, large proteins like MOMP are often also difficult to produce and purify, especially when high levels of purity are imperative when such proteins are to be used in diagnostic kits, and such large proteins are often difficult to incorporate into a kit, for example, a kit based on ELISA in which the antigen is usually immobilized on a solid support.
In other publications, there has been described the cloning and sequencing of MOMP derived from
C. trachomatis
, the production of recombinant
C. trachomatis
MOMP polypeptide and its use for raising anti-MOMP antibodies and its use in immunoassays to detect anti-MOMP antibodies specific to
C. trachomatis
. The aforesaid cloning and sequencing of MOMP has been described, for example, in EP 0 192 033, in which publication there is also descr
Basker Padma
Lerner David Littenberg Krumholz & Mentlik LLP
Savyon Diagnostics Ltd.
Smith Lynette R. F.
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