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.

1 . A method for detecting cutaneous leishmaniasis in a biological sample, comprising:
 (a) contacting the biological sample with an antibody specific for a polypeptide of SEQ ID NO: 1; and   (b) detecting in the biological sample the presence of antigens that bind to the antibody, thereby detecting cutaneous leishmaniasis in the biological sample.   
     
     
         2 - 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the antibody is a monoclonal antibody. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the presence of antigens that bind to the antibody is detected by means of ELISA. 
     
     
         8 . A method for detecting cutaneous leishmaniasis in a biological sample, comprising:
 (a) contacting the biological sample with a first antibody specific for a polypeptide of SEQ ID NO: 1 to form an antibody-polypeptide conjugate, wherein the first antibody is labeled with a reporter agent;   (b) contacting the antibody-polypeptide conjugate with a second antibody specific for a polypeptide of SEQ ID NO: 1 in order to capture the antibody-polypeptide conjugate; and   (c) detecting the presence of the captured antibody-polypeptide conjugate, thereby detecting cutaneous leishmaniasis in the biological sample.   
     
     
         9 - 13 . (canceled) 
     
     
         14 . The method of  claim 8 , wherein the first antibody is a polyclonal antibody. 
     
     
         15 . The method of  claim 8 , wherein the second antibody is a monoclonal antibody. 
     
     
         16 . The method of  claim 15 , wherein the second antibody is C11C. 
     
     
         17 . A dipstick for use in the method of  claim 8 . 
     
     
         18 . A dipstick for the detection of cutaneous leishmaniasis, comprising:
 (a) a lateral flow membrane;   (b) a first area positioned at a lower end of the lateral flow membrane for receiving a test sample, wherein the first area comprises a first antibody specific for a polypeptide of SEQ ID NO: 1, the antibody being labeled with a reporter agent;   (c) a second area positioned at an upper end of the lateral flow membrane comprising an immobilized control polypeptide; and   (d) a third area positioned between the first and second areas, wherein the third area comprises an immobilized second antibody specific for a polypeptide of SEQ ID NO: 1.   
     
     
         19 . The dipstick of  claim 18 , wherein the reporter agent is a colorimetric or fluorescent indicator. 
     
     
         20 . The dipstick of  claim 18 , wherein the reporter agent is colloidal gold or a fluorescent dye. 
     
     
         21 . The dipstick of  claim 18 , wherein the first and second antibodies are specific for a conformational epitope of the polypeptide of SEQ ID NO: 1 found in  L. major  promastigotes. 
     
     
         22 - 24 . (canceled) 
     
     
         25 . A diagnostic kit comprising a dipstick of  claim 17 . 
     
     
         26 . The diagnostic kit of  claim 25 , further comprising
 (a) a vessel containing lysing buffer;   (b) a rod for mixing a biological sample with the lysing buffer to provide a test solution; and   (c) a pipette for applying the test solution to the dipstick.   
     
     
         27 . (canceled) 
     
     
         28 . A method for detecting cutaneous leishmaniasis in a biological sample, comprising:
 (a) contacting the biological sample with the antibody WIC79.3; and   (b) detecting in the biological sample the presence of antigens that bind to the antibody, thereby detecting cutaneous leishmaniasis in the biological sample.   
     
     
         29 - 32 . (canceled) 
     
     
         33 . A method for detecting cutaneous leishmaniasis in a biological sample, comprising:
 (a) contacting the biological sample with the antibody WIC79.3 to form an antibody-polypeptide conjugate, wherein the WIC79.3 antibody is labeled with a reporter agent;   (b) contacting the antibody-polypeptide conjugate with WIC79.3 in order to capture the antibody-polypeptide conjugate; and   (c) detecting the presence of the captured antibody-polypeptide conjugate, thereby detecting cutaneous leishmaniasis in the biological sample.   
     
     
         34 - 38 . (canceled) 
     
     
         39 . A dipstick for use in the method of  claim 33 . 
     
     
         40 . A dipstick for the detection of cutaneous leishmaniasis, comprising:
 (a) a lateral flow membrane;   (b) a first area positioned at a lower end of the lateral flow membrane for receiving a test sample; wherein the first area comprises the antibody WIC79.3 labeled with a reporter agent;   (c) a second area positioned at an upper end of the lateral flow membrane comprising an immobilized control polypeptide; and   (d) a third area positioned between the first and second areas, wherein the third area comprises non-labeled antibody WIC 79.3.   
     
     
         41 - 42 . (canceled) 
     
     
         43 . A diagnostic kit comprising a dipstick of  claim 39 . 
     
     
         44 . The diagnostic kit of  claim 43 , further comprising:
 (a) a vessel containing lysing buffer;   (b) a rod for mixing a biological sample with the lysing buffer to provide a test solution; and   (c) a pipette for applying the test solution to the dipstick.   
     
     
         45 . (canceled)