Novel and practical serological assay for the clinical diagnosis of leishmaniasis

Abstract

Methods for the diagnosis of visceral, cutaneous and canine leishmaniasis in a subject suspected of being infected with the parasitic protozoa Leishmania is disclosed. Disclosed are antibody-capture enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to Leishmania parasite soluble antigens and antigen-capture ELISAs for the detection of Leishmania parasite soluble antigens in host samples. Also disclosed are immunodiagnostic kits for the detection of Leishmania parasite circulating antigens or IgM and IgG antibodies in a sample from subject having visceral, cutaneous or canine leishmaniasis. In these methods and kits, detection may be done photometrically or visually. The methods and kits also allow the visualization of Leishmania amastigotes or promastigotes in a sample.

1 . An immunoassay for detecting exposure to Leishmania parasites in a subject comprising the steps of: 
 contacting a sample from the subject suspected of having leishmaniasis with a soluble antigen prepared by utilizing a protein-free medium; and    detecting the presence or measuring the amount of an antibody or fragment thereof in the sample bound to the soluble antigen.    
     
     
         2 . The immunoassay of  claim 1  wherein the protein-free medium comprises D, xylose.  
     
     
         3 . The immunoassay of  claim 1  wherein the protein-free medium further comprises at least one of the following ingredients: Hepes buffer, L-glutamine and sodium bicarbonate without phenol red.  
     
     
         4 . The immunoassay of  claim 1  wherein the antibody is IgG or IgM and is specific for a Leishmania antigen.  
     
     
         5 . The immunoassay of  claim 1  wherein the sample is a serum sample.  
     
     
         6 . The immunoassay of  claim 5  wherein the serum sample is modified by diluting it 1:1000 in blocking buffer having 1.0% boiled casein.  
     
     
         7 . The immunoassay of  claim 1  wherein said immunoassay is capable of diagnosing visceral, cutaneous or canine leishmaniasis in a subject.  
     
     
         8 . The immunoassay of  claim 1  wherein the Leishmania soluble antigen preparation is prepared by using clones of  Leishmania donovani  or  Leishmania mexicana.    
     
     
         9 . An immunoassay for diagnosing leishmaniasis in a subject comprising the steps of: 
 contacting a sample from the subject with an antibody or fragment thereof that specifically binds the Leishmania exo-antigen; and    detecting the presence or measuring the amount of said antibody or fragment thereof bound to said Leishmania exo-antigen.    
     
     
         10 . The immunoassay of  claim 9  wherein the antibody or fragment thereof is adsorbed onto a substrate.  
     
     
         11 . A kit for the diagnosis of leishmaniasis in a subject comprising a substrate and a soluble antigen of either  L. donovani  or  L. mexicana  prepared by utilizing a protein-free medium packaged together for multiple or single use assays.  
     
     
         12 . The kit of  claim 11  wherein the substrate is coated with the soluble antigen.  
     
     
         13 . The kit of  claim 11  further comprising a positive control.  
     
     
         14 . The kit of  claim 11  further comprising a negative control.  
     
     
         15 . The kit of  claim 11  further comprising a diluent.  
     
     
         16 . The kit of  claim 11  further comprising an anti-human IgG conjugated to a label.  
     
     
         17 . The kit of  claim 11  further comprising a substrate chromogen.  
     
     
         18 . The kit of  claim 11  further comprising a substrate buffer.  
     
     
         19 . The kit of  claim 11  further comprising a blocking buffer.  
     
     
         20 . The kit of  claim 11  further comprising a stopping solution.  
     
     
         21 . A kit for the detection of an exo-antigen of a leishmania parasite in a sample comprising a fluorescein-labeled antibody that binds the exo-antigen and a counter-stain packaged together for multiple or single use assays.  
     
     
         22 . The kit of  claim 21  further comprising a protein stabilized buffer solution.  
     
     
         23 . The kit of  claim 21  further comprising sodium azide.  
     
     
         24 . The kit of  claim 22  wherein the fluorescein-labeled antibody is diluted in the protein stabilized buffer solution.  
     
     
         25 . The kit of  claim 21  wherein the counter-stain is Evans Blue.  
     
     
         26 . The kit of  claim 21  further comprising a solid substrate to which the sample to be tested is fixed.  
     
     
         27 . The kit of  claim 11  further comprising instructions.  
     
     
         28 . The kit of  claim 21  further comprising instructions.  
     
     
         29 . A diagnostic device comprising a Leishmania soluble antigen prepared by utilizing a protein-free medium and a means for detecting an antibody bound to the Leishmania soluble antigen.  
     
     
         30 . A diagnostic device comprising an antibody or fragment thereof that binds an exo-antigen found in a conditioned medium made by cultivating a Leishmania parasite in a protein-free medium.  
     
     
         31 . A method of preparing a diagnostic device comprising adsorbing to a substrate an antibody or fragment thereof which binds an exo-antigen found in conditioned medium made by cultivating a Leishmania parasite in a protein-free medium.  
     
     
         32 . A method of preparing a diagnostic device comprising adsorbing to a substrate a soluble antigen of a Leishmania parasite prepared by utilizing a protein-free medium.  
     
     
         33 . A method of detecting a Leishmania parasite in a sample comprising contacting the sample with an antibody specific for an exo-antigen found in a conditioned medium made by cultivating the Leishmania parasite in a protein-free medium; and 
 detecting the presence or measuring the amount of an antibody or fragment thereof in the sample bound to an antigen in the sample.    
     
     
         34 . A protein-free medium comprising an agent that balances the oncotic pressure across a semi-permeable cell membrane.  
     
     
         35 . The protein-free medium of  claim 34 , wherein the agent is D, xylose.  
     
     
         36 . The protein-free medium of  claim 34 , further comprising at least one of the following ingredients: Hepes buffer, L-glutamine and sodium bicarbonate without phenol red.  
     
     
         37 . A method for obtaining an exo-antigen from an organism comprising culturing the organism in a protein-free medium.  
     
     
         38 . The method of  claim 37 , wherein the organism is a leishmania parasite.  
     
     
         39 . The method of  claim 37 , wherein the protein-free medium is XOM.  
     
     
         40 . The exo-antigen obtained according to the method of claim  37 .