Method and device for identifying a mycobacterium species responsible for a mycobacterial infection

Abstract

The invention relates to a method for identifying a Mycobacterium species responsible for a mycobacterial infection in human or animal, comprising selecting a suitable mycobacterial species and strain; preparing at least one mycobacterial antigen, respectively antigen preparation; binding the antigen, respectively the antigen preparation to a suitable carrier; causing the binding antigen to react with antibodies from serum of an individual infected with a Mycobacterium species; making visible antigen-antibody reactions for a suitable antibody (sub-)class; and identifying the responsible Mycobacterium species on the basis of the reactions which are made visible. The invention further provides a diagnostic kit which takes the form of a dip-stick on which is arranged a carrier strip with mycobacterial antigens binding thereto, and means for visualizing antigen-antibody reactions occurring on the carrier after contact with the serum for testing. In another embodiment the diagnostic kit comprises a microtiter plate, in the wells of which a specified antibody is arranged, and means for making visible antigen-antibody reactions occurring in the wells after contact with the serum for testing. The third embodiment is an immunoblot with mycobacterial antigens separated by electrophoresis binding thereto, and means for visualizing antigen-antibody reactions occurring on the immunoblot after contact with the serum for testing.

What is claimed is:  
     
         1 . A method for detecting or identifying a Mycobacterium species in a biological sample, comprising: 
 (a) selecting a standard Mycobacterium;    (b) preparing an antigen preparation from a culture of the standard Mycobacterium, wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs);    (c) separating the ImCRACs of the antigen preparation according to molecular weight;    (d) binding the separated IMCRACS of the antigen preparation to a solid carrier;    (e) contacting the bound ImCRACs with the biological sample under conditions that permit antibodies in the biological sample to bind with the carrier-bound ImCRACs to provide a pattern of carrier-bound antibody-ImCRAC complexes;    (f) detecting the pattern of carrier-bound antibody-ImCRAC complexes; and    (g) comparing the pattern of carrier-bound antibody-ImCRAC complexes from the sample to a library of standard patterns of carrier-bound antibody-ImCRAC complexes characteristic of particular Mycobacterium species,    wherein matching of the pattern of carrier-bound antibody-ImCRAC complexes from the sample to one of the standard patterns of carrier-bound antibody-ImCRAC complexes from the library permits detection or identification of the Mycobacterium species in the sample.    
     
     
         2 . The method of  claim 1 , wherein the antigen preparation is separated by electrophoresis prior to step (d) and the carrier is a membrane to which the ImCRACS are bound by means of electroblotting.  
     
     
         3 . The method of  claim 2 , wherein the membrane is a nitrocellulose membrane.  
     
     
         4 . The method of  claim 1 , wherein the antigen preparation of the standard Mycobacterium is a total protein preparation.  
     
     
         5 . The method of  claim 4 , wherein the antigen preparation is a KP-100 or SP-100 fraction of the total protein preparation.  
     
     
         6 . The method of  claim 1 , wherein the carrier is a microtiter plate.  
     
     
         7 . The method of  claim 1 , wherein the pattern of the carrier-bound antibody-ImCRAC complexes is a banding pattern consisting of 29/33 KDa, 45/48 KDa, 64 KDa and combinations thereof.  
     
     
         8 . The method of  claim 1 , wherein the carrier-bound antibody-ImCRAC complexes are made visualizable by using at least one antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of IgG, IgM, IgA and combinations thereof.  
     
     
         9 . The method of  claim 8 , wherein the enzyme of the antibody-enzyme conjugate is peroxidase.  
     
     
         10 . The method of  claim 1 , wherein the carrier is a dip-stick.  
     
     
         11 . A method for detecting or identifying a Mycobacterium species in a biological sample, comprising: 
 (a) providing a test substrate having bound thereto an antigen preparation from a culture of the standard Mycobacterium, wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs) such that the ImCRACs of the antigen preparation separate according to molecular weight;    (b) contacting the bound ImCRACs of the test substrate with the biological sample under conditions that permit antibodies in the biological sample to bind with the substrate-bound ImCRACs to provide a pattern of substrate-bound antibody-ImCRAC complexes;    (c) detecting the pattern of substrate-bound antibody-ImCRAC complexes; and    (d) comparing the pattern of substrate-bound antibody-ImCRAC complexes from the sample to a library of standard patterns of substrate-bound antibody-ImCRAC complexes characteristic of particular Mycobacterium species,    wherein matching of the pattern of substrate-bound antibody-ImCRAC-complexes from the sample to one of the standard patterns of substrate-bound antibody-ImCRAC complexes from the library permits detection or identification of the Mycobacterium species in the sample.    
     
     
         12 . The method of  claim 11 , wherein the antigen preparation is separated by electrophoresis and the substrate is a membrane to which the IMCRACS are bound by means of electroblotting.  
     
     
         13 . The method of  claim 12 , wherein the membrane is a nitrocellulose membrane.  
     
     
         14 . The method of  claim 11 , wherein the antigen preparation of the standard Mycobacterium is a total protein preparation.  
     
     
         15 . The method of  claim 14 , wherein the antigen preparation is a KP-100 or SP-100 fraction of the total protein preparation.  
     
     
         16 . The method of  claim 11 , wherein the substrate is a microtiter plate.  
     
     
         17 . The method of  claim 11 , wherein the pattern of the substrate-bound antibody-ImCRAC complexes is a banding pattern consisting of 29/33 KDa, 45/48 KDa, 64 KDa and combinations thereof.  
     
     
         18 . The method of  claim 11 , wherein the substrate-bound antibody-ImCRAC complexes are made visualizable by using at least one antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of IgG, IgM, IgA and combinations thereof.  
     
     
         19 . The method of  claim 18 , wherein the enzyme of the antibody-enzyme conjugate is peroxidase.  
     
     
         20 . The method of  claim 11 , wherein the substrate is a dip-stick.  
     
     
         21 . Diagnostic kit comprising: 
 (1) a test substrate having bound thereto an antigen preparation from a culture of standard Mycobacterium, 
 wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs) that are separable according to molecular weight, and  
 wherein when the ImCRACs of the test substrate are contacted with a biological sample under conditions that permit antibodies in the biological sample to bind with the substrate-bound ImCRACs, a pattern of substrate-bound antibody-ImCRAC complexes is provided; and  
   (2) a means for visualizing the pattern substrate-bound antibody-ImCRAC complexes.    
     
     
         22 . The diagnostic kit of  claim 21 , wherein the test substrate is selected from the group consisting of a dip-stick, a microtiter plate, and an immunoblot.