Methods and Materials for the Detection of Leishmania Infection

Abstract

The present invention provides rapid diagnostic assays for the detection of Leishmania which can readily be used in the field, leading to more rapid treatment. In certain embodiments, the inventive assays, including ELISA and lateral flow assays, employ antibodies that may be effectively employed to detect the Leishmania major antigen TSA, which is present in both promastigotes grown in culture and in amastigotes. Such assays may be employed to detect the presence of cutaneous leishmaniasis in a subject using scrapings, biopsies, and / or aspirates taken from cutaneous lesions.

Description

REFERENCE TO PRIORITY APPLICATION[0001]This application claims priority to U.S. provisional patent application No. 60 / 742,761, filed Dec. 6, 2005.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. W81XWH-05-C-0018 awarded by the Department of Defense.FIELD OF THE INVENTION[0003]The present invention relates generally to the diagnosis of Leishmania infection. More specifically, the invention relates to the immunological detection of cutaneous leishmaniasis.BACKGROUND OF THE INVENTION[0004]Leishmania organisms are intracellular protozoan parasites of macrophages that cause a wide range of clinical diseases in humans and domestic animals, primarily dogs. The life cycles of Leishmania involves a vertebrate host (e.g., a human) and a vector (a sand fly) that transmit...

AI Technical Summary

Benefits of technology

[0009]The present invention provides rapid diagnostic assays for the detection of Leishmania amastigotes, promastigotes and / or secreted proteins which can readily be used in the field, leading to more rapid treatment. In one embodiment, the present invention provides diagnostic tests, including ELISA and lateral flow assays, that may be effectively employed to detect a Leishmania major antigen that is present in both promastigotes grown in culture and, more importantly, in amastigotes—the form present in cutaneous lesions. Such assays may be employed to detect the presence of cutaneous leishmaniasis in a subject using scrapings, biopsies, and / or aspirates taken from cutaneous lesions. In another embodiment, diagnostic tests, including ELISA and lateral flow assays, are provided that may be effectively employed to detect a Leishmania major antigen that is present in secreted proteins and promastigotes but not in amastigotes. Such assays may be employed to detect the presence of cutaneous leishmaniasis in a subject using biological samples such as serum, and scrapings, biopsies, and / or aspirates taken from cutaneous lesions

[0010]In certain embodiments, the assays disclosed herein employ antibodies specific for the Thiol Specific Antigen (TSA also known as peroxidoxin; SEQ ID NO: 1 and 2). Preferably the antibodies employed in such assays bind to a conformational epitope of TSA that is present in amastigotes. In one embodiment, the assays employ a first TSA-specific antibody as a capture antibody, with a second TSA-specific antibody being employed as a detection antibody. Preferably, the detection antibody is labeled with a reporter group or agent. For example, a monoclonal antibody against TSA, such as the antibody C11C (which was initially raised against amastigotes), may be employed as the capture antibody, with a rabbit polyclonal antibody to recombinant TSA being employed as the detection antibody. In the case of an ELISA, further development of the assay may then be performed using a goat anti-rabbit IgG horseradish peroxidase label. For a lateral flow assay, the anti-TSA antibody is preferably labeled with a calorimetric or fluorescent indicator, such as colloidal gold or a fluorescent dye, thereby allowing a user to determine visually whether a test is positive or negative for cutaneous leishmaniasis.

Problems solved by technology

For example, leishmaniasis may be manifested as a cutaneous disease, which is a severe medical problem.

Current diagnostic procedures are not readily applicable to field situations and typically require centralized laboratory testing.

Serodiagnosis looking for specific antibodies in cutaneous leishmaniasis has been attempted with some success using secreted antigens but this test lacks sufficient sensitivity and specificity.

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