Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-07-15
2001-05-08
Graser, Jennifer (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007100, C435S007900, C435S007920, C424S191100, C424S269100
Reexamination Certificate
active
06228601
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to polypeptides that are useful for diagnosing American trypanosomiasis, or Chagas disease, a disease caused by the infectious agent
Trypanosoma cruzi
. More particularly, the invention relates to recombinant
T. cruzi
polypeptides, synthesized using genetic engineering techniques, and to constructs and processes for producing the recombinant polypeptides, and to an assay for detecting
T. cruzi
infection which employs the polypeptides.
American trypanosomiasis, or Chagas disease, is an illness caused by the protozoan parasite,
T. cruzi
(1,2). This organism is transmitted by insects called reduviid bugs (3), by blood transfusion (4), and also from mother to fetus (5). Several years after acquiring
T. cruzi
infection, patients may develop the cardiac and gastrointestinal symptoms that are associated with chronic infection, which is life-long, but the majority of infected persons never develop clinical manifestations of Chagas disease and are unaware of being infected. The two drugs available for treating
T. cruzi
infections have low efficacy and often cause serious side effects. In practice, therefore, they have virtually no impact on the control of Chagas disease.
Chagas disease is a major cause of morbidity and death in Latin America, where an estimated 16-18 million people are chronically infected with
T. cruzi
(6). In recent years tens of thousands of
T. cruzi
-infected people have emigrated to the United States, especially from Central America, where the prevalence of
T. cruzi
infection is high, thus creating the risk of transfusion-associated transmission of the parasite here (7-9). Several such cases have been described (10-12).
Since clinical criteria cannot be depended upon for recognizing
T. cruzi
infection, blood tests are of paramount importance, both in patient care settings and in blood banks. Chronically infected persons uniformly have anti-
T. cruzi
antibodies. The diagnosis of
T. cruzi
infection is almost always made by detecting these antibodies in patients' blood, since parasitological approaches are laborious and lack sensitivity and, as noted, clinical evaluations lack specificity.
Immunological tests currently used to diagnose
T. cruzi
infection, such as complement fixation and indirect immunofluorescence tests, and enzyme-linked immunosorbent assays (ELISA), often produce inconsistent results and false-positive reactions (13). The occurrence of false-positive reactions can be a problem with specimens from patients with leishmaniasis, schistosomiasis, and other parasitic and infectious diseases, with samples from patients with autoimmune disorders and other illnesses, and with specimens from normal persons.
In large measure these problems with sensitivity and specificity occur because the assays are based on antigens extracted from parasites grown in the laboratory. The complexity and variability of mixtures of native antigens derived from cultured parasites, which persist even after fractionation and purification procedures are used, have been a major barrier to standardization of immunoassays. Because of the limitations of these immunoassays, experts generally agree that blood samples should be positive in three different assays, performed in parallel, before being accepted as positive.
An additional problem related to assays based on material derived from cultured parasites is that producing the antigens creates a serious biohazard for technical personnel, and laboratory-acquired cases of Chagas disease occur with disquieting frequency, both in the United States and abroad (14,15). Furthermore, some of the immunoassays currently available require sophisticated laboratory equipment and levels of technical expertise not generally available in the countries in which
T. cruzi
infection is endemic.
In response to the need for improved assays for detecting
T. cruzi
infection, considerable work has been invested in the development of new immunoassays. These efforts have accelerated in recent years as new technologies have become available that have the potential for serving as the basis of improved assays. Recombinant DNA technology has led to the molecular cloning of several antigenic
T. cruzi
proteins. Cloned segments of
T. cruzi
genes have been used to produce in bacteria portions of antigenic proteins (16-22). In research settings several of these, singly and in combination, have been used as target antigens in immunoassays. These assays have not been tested in field or blood bank trials, and none is available commercially.
U.S. Pat. No. 4,870,006 discloses the use of a recombinant protein in an assay for diagnosing
T. cruzi
infection. A 70-kilodalton heat shock protein constitutes the target antigen in this assay. No information regarding the sensitivity and specificity of the assay is provided in the patent.
In this context, therefore, a need exists for a highly sensitive and specific system for detecting
T. cruzi
infection that is safe, easy, and inexpensive to manufacture and perform.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a highly sensitive and specific assay for diagnosing infection with
T. cruzi.
It is a further object of the present invention to provide an assay for diagnosing
T. cruzi
infection that is safe, inexpensive to manufacture and easy to use.
In achieving these and other objects, there has been provided, according to one aspect of the present invention, a polypeptide having a sequence that corresponds to the amino acid sequence of at least one of the C-terminal and N-terminal nonrepetitive regions of the TCR27 protein. The inventive polypeptide additionally may comprise an amino acid sequence of one or more repeats from the central region of the TCR27 protein. In a preferred embodiment, the polypeptide corresponds to the N-terminal nonrepetitive region of the TCR27 protein and at least one repeat from the central region of the TCR27 protein, and does not correspond to the C-terminal nonrepetitive region. The polypeptides may further comprise a linker sequence at either the N-terminus or the C-terminus to facilitate attachment or conjugation to a carrier molecule in a liquid or solid support system. Isolated polynucleotides that encode the inventive polypeptides according to the present invention are also claimed, as are cells transformed with a recombinant plasmid that expresses a polypeptide according to the invention.
The present invention also provides a method for detecting the presence of antibodies to
T. cruzi
in an individual, comprising the steps of contacting a putative anti-
T. cruzi
antibody-containing sample from an individual with a polypeptide according to the invention that is attached or conjugated to a carrier molecule or attached or conjugated to a solid phase; allowing anti-
T. cruzi
antibodies in said sample to bind to said polypeptide; washing away unbound anti-
T. cruzi
antibodies; and adding a compound that enables detection of the anti-
T. cruzi
antibodies which are specifically bound to the polypeptide. The compound that enables detection of the anti-
T. cruzi
antibodies may be selected from the group consisting of a colorometric agent, a fluorescent agent, a chemiluminescent agent and a radionuclide.
Also provided in accordance with the present invention is a kit for diagnosing the presence of anti-
T. cruzi
antibodies in a sample, comprising a container in which a polypeptide having a sequence that corresponds to the amino acid sequence of at least one of the C-terminal and N-terminal nonrepetitive regions of the TCR27 protein is attached or conjugated to a carrier molecule or attached or conjugated to a solid phase; and directions for carrying out the method according to the invention. The kit additionally may comprise a container of a compound that binds to anti-
T. cruzi
antibodies and that renders said antibodies detectable.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It s
Kirchhoff Louis V.
Otsu Keiko
Foley & Lardner
Graser Jennifer
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