Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-05-28
2001-12-11
Devi, S. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007920
Reexamination Certificate
active
06329157
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to combinations or mixtures of antigens which may be used in the detection of IgM or IgG antibodies to
Toxoplasma gondii
. Furthermore, the present invention also relates to methods of using these combinations of antigens, antibodies raised against these combinations of antigens or against the novel P29 antigen thereof, as well as kits and vaccine containing the antigens present in the combinations.
2. Background Information
Toxoplasma gondii
is an obligate intracellular parasite which is classified among the Coccidia. This parasite has relatively broad host range infecting both mammals and birds. The organism is ubiquitous in nature and exists in three forms: tachyzoite, cyst, and oocyst (Remington, J. S., McLeod, R., Desmonds, G., Infectious Diseases of the Fetus and Newborn Infant (J. S. Remington and J. O. Klein, Eds.), pp. 140-267, Saunders, Philadelphia (1995)). Tachyzoites, found during acute infection, are the invasive form capable of invading all nucleated mammalian cells. After the acute stage of infection, tissue cysts called bradyzoites are formed within host cells and persist within the host organism for the life of the host. Cysts are important in transmission of infection, especially in humans, as the ingestion of raw or undercooked meat can result in the ingestion of bradyzoites which can infect the individual resulting in an acute infection. Oocysts represent a stage of sexual reproduction which occurs only in the intestinal lining of the cat family from which they are excreted in the feces.
A
T. gondii
infection acquired through contaminated meat or cat feces in a healthy adult is often asymptomatic. In pregnant women and immunosuppressed patients, the clinical outcome can be very serious. An acute infection with
T. gondii
acquired during pregnancy, especially during the first trimester, can result in intrauterine transmission to the unborn fetus resulting in severe fetal and neonatal complications, including mental retardation and fetal death. Recrudesence of a previous
T. gondii
infection or an acute infection in an immunosuppressed individual can be pathogenic. Toxoplasmic encephalitis is a major cause of morbidity and mortality in AIDS patients. Toxoplasma infection has also been shown to be a significant cause of chorioretinitis in children and adults.
Diagnosis of infection with
T. gondii
may be established by the isolation of
T. gondii
from blood or body fluids, demonstration of the presence of the organism in the placenta or tissues of the fetus, demonstration of the presence of antigen by detection of specific nucleic acid sequences (e.g., DNA probes), or detection of
T. gondii
specific immunoglobulins synthesized by the host in response to infection using serologic tests.
The detection of
T. gondii
specific antibodies and determination of antibody titer are important tools used in the diagnosis of toxoplasmosis. The most widely used serologic tests for the diagnosis of toxoplasmosis are the Sabin-Feldman dye test (Sabin, A. B. and Feldman, H. A. (1948) Science 108, 660-663), the indirect hemagglutination (IHA) test (Jacobs, L. and Lunde, M. (1957) J. Parasitol. 43, 308-314), the IFA test (Walton, B. C. et al. (1966) Am. J. Trop. Med. Hyg. 15, 149-152), the agglutination test (Fondation Mérieux, Sérologie de I' Infection Toxoplasmique en Particulier à Son Début: Méthodes et Interprétation des Résultants, Lyon, 182 pp. (1975)) and the ELISA (Naot, Y. and Remington, J. S. (1980) J. Infect. Dis. 142, 757-766). The ELISA test is one the easiest tests to perform, and many automated serologic tests for the detection of Toxoplasma specific IgM and IgG are commercially available.
The current tests for the detection of IgM and IgG antibodies in infected individuals can vary widely in their ability to detect serum antibody. Hence, there is significant inter-assay variation seen among the commercially available kits. The differences observed between the different commercial kits are caused primarily by the preparation of the antigen used for the serologic test. Most kits use either whole or sonicated tachyzoites grown in tissue culture or in mice which contain a high proportion of extra-parasitic material, for example, mammalian cells, tissue culture components, etc. Due to the lack of a purified, standardized antigen or standard method for preparing the tachyzoite antigen, it is not surprising that inter-assay variability exists resulting in different assays having different performance characteristics in terms of assay sensitivity and specificity.
Given the limitations of serologic tests employing the tachyzoite antigen, purified recombinant antigens obtained by molecular biology are an attractive alternative in that they can be purified and standardized. In the literature, a number of Toxo genes have been cloned and expressed in a suitable host to produce immunoreactive, recombinant Toxo antigens. For example, the Toxo P22 (SAG2), P24 (GRA1), P25, P28 (GRA2), P30 (SAG1), P35, P41 (GRA4), P54 (ROP2), P66 (ROP1), and the Toxo P68 antigens have been described (Prince et al. (1990) Mol. Biochem. Parasitol 43, 97-106; Cesbron-Delauw et al. (1989) Proc. Nat. Acad. Sci. 86, 7537-7541; Johnson et al. (1991) Gene 99, 127-132; Prince et al. (1989) Mol. Biochem. Parasitol. 34, 3-13; Burg et al. (1988) J. Immunol. 141, 3584-3591; Knapp et al. (1989) EPA 431541A2; Mevelec et al. (1992) Mol. Biochem. Parasitol. 56, 227-238; Saavedra et al. (1991) J. Immunol. 147, 1975-1982).
It is plausible that no single Toxo antigen can replace the tachyzoite in an immunoassay for the detection of Toxo-specific immunoglobulins. This may be for several reasons. First, the antibodies produced during infection vary with the stage of infection, i.e., the antibodies produced by an infected individual vary over time reacting with different epitopes. Secondly, the epitopes present in a recombinant antigen may be different or less reactive than native antigen prepared from the tachyzoite depending on the host used for expression and the purification scheme employed. Thirdly, different recombinant antigens may be needed to detect the different classes of immunoglobulins produced in response to an infection, e.g., IgM, IgG, IgA, IgE.
In order to overcome the limitations of the tachyzoite antigen in terms of assay specificity and sensitivity, a search was begun for novel Toxo antigens which could be used in combination with known existing antigens in order to configure new assays for the detection of Toxo-specific immunoglobulins.
SUMMARY OF THE INVENTION
The present invention includes a composition comprising
Toxoplasma gondii
antigens P29, P30 and P35 as well as a composition comprising
Toxoplasma gondii
antigens P29, P35 and 66. These compositions may be used as diagnositic reagents, and the antigens within these compositions may be produced either recombinantly or synthetically.
Additionally, the present invention includes an isolated nucleic acid sequence represented by SEQ ID NO: 26 and a purified polypeptide having the amino acid sequence represented by SEQ ID NO: 27. The present invention also includes a polyclonal or monoclonal antibody directed against the purified polypeptide.
The present invention also encompasses a method for detecting the presence of IgM antibodies to
Toxoplasma gondii
in a test sample. This method comprises the steps of: a) contacting the test sample suspected of containing the IgM antibodies with a composition comprising P29, P35 and P66; and b) detecting the presence of the IgM antibodies.
Furthermore, the present invention includes an additional method for detecting the presence of IgM antibodies to
Toxoplasma gondii
in a test sample. This method comprises the steps of: a) contacting the test sample suspected of containing the IgM antibodies with a composition comprising antigen P29, P35 and P66 for a time and under conditions sufficient for the formation of IgM antibody/antigen complexes; b) adding a conjugate to the resulting IgM antibody/antigen complexes
Brojanac Susan
Chovan Linda E.
Howard Lawrence V.
Hunt Jeffrey C.
Maine Gregory T.
Abbott Laboratories
Becker Cheryl L.
Devi S.
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