Methods, immunoassays and devices for detection of anti-lipoidal antibodies

Abstract

Compositions, methods and devices for the detection of anti-lipoidal antibodies and the diagnosis of disease, for example, syphilis, are described. In particular, a method for immobilizing a lipoidal antigen, comprising cardiolipin, lecithin, and cholesterol, on a solid support (such as a nitrocellulose membrane) is described. The ability to immobilize a lipoidal antigen on a membrane satisfies a long-felt need for membrane-based assay for the detection of anti-lipoidal antibodies. Also described are immunoassay devices for concurrently performing treponemal and non-treponemal tests for syphilis.

1 . An immunoassay device comprising a microporous substrate having an anti-lipoidal antibody capture area, comprising:
 (a) an anchor antibody immobilized on the substrate; and   (b) a lipoidal antigen, comprising cardiolipin, lecithin, and cholesterol,   wherein the immobilized anchor antibody is specifically bound to one or more of the cardiolipin, lecithin or cholesterol components of the lipoidal antigen thereby forming an immobilized anchor antibody-lipoidal antigen complex.   
   
   
       2 . The immunoassay device of  claim 1 , wherein the substrate is a microporous membrane. 
   
   
       3 . The immunoassay device of  claim 1 , wherein the anchor antibody does not specifically bind an exogenous epitope in one or more of the cardiolipin, lecithin or cholesterol components of the lipoidal antigen. 
   
   
       4 . The immunoassay device of  claim 1 , wherein the lipoidal antigen comprises a micelle and not a liposome. 
   
   
       5 . The immunoassay device of  claim 1 , wherein the anchor antibody is specifically bound to the cardiolipin component of the lipoidal antigen. 
   
   
       6 . The immunoassay device of  claim 1  for determining at least one of the presence or amount of an anti-lipoidal antibody in a fluid sample, further comprising a sample application area and a flow path from the sample application area to the anti-lipoidal antibody capture area; wherein the at least one of the presence or amount of an anti-lipoidal antibody in a fluid sample can be detected by formation of a complex between an anti-lipoidal antibody in a fluid sample and the immobilized lipoidal antigen-anchor antibody complex. 
   
   
       7 . The immunoassay device of  claim 1 , further comprising a treponemal capture area comprising:
 (a) an immobilized treponemal antigen capable of being specifically bound by an anti- T. pallidum  antibody, or   (b) an immobilized anti- T. pallidum  antibody that specifically binds a mobile treponemal antigen.   
   
   
       8 . The immunoassay device of  claim 1 , wherein the substrate comprises nitrocellulose, nylon, polyvinylidene fluoride (PVDF), polyethersulfone, polycarbonate, polyester, cellulose acetate, mixed cellulose esters, or combinations thereof. 
   
   
       9 . The immunoassay device of  claim 1 , wherein the anchor antibody is a Fab fragment specific for cardiolipin. 
   
   
       10 . The immunoassay device of  claim 1 , wherein the anchor antibody is a plurality of Fab fragments produced from an immunoglobulin fraction from a subject infected with  T. pallidum.    
   
   
       11 . The immunoassay device of  claim 1 , wherein the lipoidal antigen is a USR antigen, a VDRL antigen, or a synthetic VDRL antigen. 
   
   
       12 . The immunoassay device of  claim 1 , wherein the anti-lipoidal antibody capture area comprises one or more lines. 
   
   
       13 . The immunoassay device of  claim 12 , wherein the one or more lines have a width from about 8 mm to about 15 mm. 
   
   
       14 . The immunoassay device of  claim 1 , wherein the device is a flow-through device. 
   
   
       15 . The immunoassay device of  claim 14 , further comprising an absorbent pad, which is in contact with the substrate. 
   
   
       16 . The immunoassay device of  claim 14 , wherein the substrate has a pore size from about 0.2 μm to about 8 μm. 
   
   
       17 . The immunoassay device of  claim 1 , wherein the device is a lateral flow device. 
   
   
       18 . The immunoassay device of  claim 17 , wherein the substrate has a pore size up to about 12 μm. 
   
   
       19 . The immunoassay device of  claim 17 , wherein the lateral flow device further comprises a conjugate pad located in the flow path, wherein the conjugate pad comprises a mobile or mobilizable detector reagent specific for the anti-lipoidal antibody. 
   
   
       20 . The immunoassay device of  claim 19 , wherein the detector reagent comprises gold-conjugated Protein A, gold-conjugated Fc-specific Protein G, or gold-conjugated anti-human antibody (Fc portion). 
   
   
       21 . The immunoassay device of  claim 19 , wherein the conjugate pad further comprises a mobile or mobilizable detector reagent specific for the anti- T. pallidum  antibody or the mobile treponemal antigen. 
   
   
       22 . The immunoassay device of  claim 21 , wherein the detector reagent specific for the anti- T. pallidum  antibody comprises gold-conjugated Protein A, gold-conjugated Fc-specific Protein G, or gold-conjugated anti-human antibody (Fc portion), or the detector reagent for the mobile treponemal antigen comprises gold-labeled anti-treponemal antigen antibody. 
   
   
       23 . The immunoassay device of  claim 1 , wherein the lipoidal antigen-anchor antibody complex is immobilized on the substrate by a method comprising:
 contacting the lipoidal antigen with one or more anchor antibodies specific for at least one of cardiolipin, lecithin, or cholesterol to form the lipoidal antigen-anchor antibody complex; and   applying the lipoidal antigen-anchor antibody complex to the substrate.   
   
   
       24 . The immunoassay device of  claim 1 , wherein the lipoidal antigen-anchor antibody complex is immobilized on the substrate by a method, comprising:
 immobilizing an anchor antibody specific for at least one of cardiolipin, lecithin, or cholesterol on the substrate;   blocking non-specific binding sites on the substrate;   contacting the immobilized anchor antibody with a lipoidal antigen to form a lipoidal antigen-anchor antibody complex; and   washing the substrate to remove any lipoidal antigen not specifically bound by the anchor antibody.   
   
   
       25 . A method for detecting anti-lipoidal antibodies in a subject, comprising:
 applying a biological sample from a subject to the immunoassay device of  claim 1 ; and   detecting the formation of a complex between an anti-lipoidal antibody present in the biological sample and the immobilized lipoidal antigen-anchor antibody complex, wherein detection of the formation of the complex detects the anti-lipoidal antibody in the subject.   
   
   
       26 . The method of  claim 25 , wherein detection of the anti-lipoidal antibody is used to diagnose syphilis in the subject. 
   
   
       27 . The method of  claim 25 , wherein the biological sample is blood, serum, skin ulcer exudate, urine, saliva, sputum, or cerebrospinal fluid. 
   
   
       28 . The method of  claim 25 , further comprising applying to the immunoassay device a detector reagent specific for the anti-lipoidal antibody. 
   
   
       29 . The method of  claim 25 , further comprising adding a detector reagent specific for the anti-lipoidal antibody to the biological sample prior to or concurrent with applying the sample to the immunoassay device. 
   
   
       30 . The method of  claim 27  wherein the detector reagent is labeled Protein A, Fc-specific Protein G, or anti-human antibody. 
   
   
       31 . The method of  claim 30 , wherein the label is an enzyme, colloidal gold particles, colored latex particles, a chemiluminescent agent, or a fluorescent agent. 
   
   
       32 . A method for diagnosing syphilis in a subject, comprising:
 applying a biological sample to the device of  claim 7 ;   detecting in the anti-lipoidal antibody capture area the formation of a first complex between an anti-lipoidal antibody present in the biological sample and the immobilized lipoidal antigen-anchor antibody complex; and   detecting in the treponemal capture area the formation of a second complex between:   (a) an anti- T. pallidum  antibody present in the biological sample and the immobilized treponemal antigen, or   (b) a treponemal antigen present in the biological sample and the immobilized anti- T. pallidum  antibody,   wherein detection of the formation of the first complex and the second complex is used to diagnose syphilis in the subject.   
   
   
       33 . The method of  claim 32 , wherein the biological sample is a human blood sample. 
   
   
       34 . The method of  claim 33 , wherein the human blood sample is whole blood or serum. 
   
   
       35 . An immunoassay device for determining at least one of presence or amount of an anti-lipoidal antibody in a fluid sample comprising:
 a microporous membrane;   a sample application area;   an anti-lipoidal antibody capture area comprising a lipoidal antigen-anchor antibody complex, which complex comprises an anchor antibody and a lipoidal antigen specifically bound by the anchor antibody, and which complex is immobilized on the membrane by the anchor antibody; wherein the lipoidal antigen comprises cardiolipin, lecithin and cholesterol; and   a flow path from the sample application area to the anti-lipoidal antibody capture area;   
     wherein the presence and/or amount of an anti-lipoidal antibody in a fluid sample applied to the sample application area can be detected by formation of a complex between the anti-lipoidal antibody and the immobilized lipoidal antigen-anchor antibody complex. 
   
   
       36 . A method for immobilizing immunoreactive cardiolipin on a substrate, the method comprising:
 contacting a lipoidal antigen, comprising immunoreactive cardiolipin, with one or more antibodies specific for at least one component of the lipoidal antigen to form a lipoidal antigen-antibody complex; and   applying the lipoidal antigen-antibody complex to a substrate,   wherein applying the lipoidal antigen-antibody complex to the substrate immobilizes the immunoreactive cardiolipin on the substrate.   
   
   
       37 . The method of  claim 36 , wherein the lipoidal antigen is a USR antigen, a VDRL antigen, or a synthetic VDRL antigen. 
   
   
       38 . The method of  claim 36 , wherein the antibody is an antibody fragment that will not substantially react with Protein A, Fc-specific Protein G, or anti-human antibody (Fc portion). 
   
   
       39 . The method of  claim 38 , wherein the antibody is a Fab fragment. 
   
   
       40 . The method of  claim 36 , wherein the antibody is isolated from serum of a  T. pallidum -infected or  T. pallidum -inoculated subject. 
   
   
       41 . The method of  claim 36 , wherein the substrate comprises nitrocellulose. 
   
   
       42 . The method of  claim 36 , comprising:
 contacting a lipoidal antigen, comprising immunoreactive cardiolipin, lecithin, and cholesterol, with an antibody fragment specific for cardiolipin, lecithin, or cholesterol to form a lipoidal antigen-antibody complex; wherein the antibody fragment does not substantially react with Protein A, Fc-specific Protein G, or anti-human antibody (Fc portion); and   applying the lipoidal antigen-antibody complex to a substrate, which immobilizes the immunoreactive cardiolipin on the substrate.   
   
   
       43 . The method of  claim 42 , comprising
 contacting a lipoidal antigen, comprising immunoreactive cardiolipin, with a Fab fragment specific for cardiolipin, lecithin, or cholesterol to form a lipoidal antigen-Fab complex; wherein the lipoidal antigen is a USR antigen, a VDRL antigen, or a synthetic VDRL antigen; and   applying the lipoidal antigen-Fab complex to nitrocellulose, which immobilizes the immunoreactive cardiolipin on the nitrocellulose.   
   
   
       44 . A method for immobilizing immunoreactive cardiolipin on a substrate, the method comprising:
 immobilizing one or more antibodies specific for at least one of cardiolipin, lecithin or cholesterol on a substrate;   blocking non-specific binding sites on the substrate;   applying a lipoidal antigen, comprising at least one immunoreactive cardiolipin, lecithin or cholesterol, to the substrate to form lipoidal antigen-immobilized antibody complexes; and   washing the substrate to remove any lipoidal antigen that is not specifically bound by the one or more immobilized antibodies.