Anti-T. Cruzi Antibodies and Methods of Use

Abstract

The present disclosure is directed to reagents and methods of using the reagents to detect epitopes of Trypanosoma cruzi.

1 . An immunodiagnostic reagent comprising one or more antibodies that specifically bind to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein said one or more antibodies are selected from the group consisting of an antibody specific for  T. cruzi  polypeptides comprised by FP3, Pep2, FP10 and FRA polypeptides. 
     
     
         2 . The immunodiagnostic reagent according to  claim 1 , wherein said immunodiagnostic reagent comprises two or more of said antibodies. 
     
     
         3 . The immunodiagnostic reagent according to  claim 1 , wherein said immunodiagnostic reagent comprises an antibody selected from the group consisting of:
 an antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein the  T. cruzi  polypeptide is FRA and further wherein said antibody has at last one binding constant selected from the group consisting of: an association rate constant (k a ) between about 7.0×10 5  M −1 s −1  to about 7.0×10 6  M −1 s −1 , an dissociation rate constant (k d ) between about 4.0×10 −3  s −1  to about 3.0×10 −1  s −1  and an equilibrium dissociation constant (K D ) between about 5.7×10 −10  M to about 4.3×10 −7  M;   (b) an antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein the  T. cruzi  polypeptide is Pep2 and further wherein said antibody has at least one binding constant selected from the group consisting of: an association rate constant (k a ) between about 1.0×10 6  M −1 s −1  to about 8.0×10 6  M −1 s −1 ; an dissociation rate constant (k d ) between about 6.0×10 −3  s −1  to about 4.0×10 −2  s −1  and an equilibrium dissociation constant (K D ) between about 7.5×10 −10  M to about 4.0×10 −8  M;   (c) an antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein the  T. cruzi  polypeptide is FP10 and further wherein said antibody has at least one binding constant selected from the group consisting of: (a) an association rate constant (k a ) between about 5.0×10 4  M −1 s −1  to about 3.0×10 5  M −1 s −1 : (b) an dissociation rate constant (k d ) between about 1.0×10 −4  s −1  to about 8.0×10 −4  s −1 ; and (c) an equilibrium dissociation constant (K D ) between about 3.3×10 −10  M to about 1.6×10 −8  M;   (d) an antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein the  T. cruzi  polypeptide is FP3 and further wherein said antibody has at least one binding constant selected from the group consisting of: an association rate constant (k a ) between about 2.0×10 5  M −1 s −1  to about 6.0×10 6  M −1 s −1 ; an dissociation rate constant (k d ) between about 2.0×10 −5  s −1  to about 8.0×10 −4  s −1 ; and an equilibrium dissociation constant (K D ) between about 3.3×10 −12  M to about 4.0×10 −9  M; and   (e) any combinations of (a)-(d).   
     
     
         4 . The immunodiagnostic reagent according to  claim 1 , wherein said antibody is a chimeric antibody. 
     
     
         5 . The immunodiagnostic reagent according to  claim 4 , wherein said immunodiagnostic reagent comprises a chimeric antibody selected from the group consisting of:
 (a) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:2;   (b) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:4;   (c) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:6; and   (d) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:8.   
     
     
         6 . The immunodiagnostic reagent according to  claim 4 , wherein said immunodiagnostic reagent comprises a chimeric antibody selected from the group consisting of:
 (a) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:2;   (b) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:4;   (c) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:6; and   (d) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:8.   
     
     
         7 . The immunodiagnostic reagent according to  claim 4 , wherein said immunodiagnostic reagent comprises a chimeric antibody selected from the group consisting of:
 (a) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 95% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:2;   (b) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 95% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:4;   (c) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 95% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:6; and   (d) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 95% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:8.   
     
     
         8 . The immunodiagnostic reagent according to  claim 4 , wherein said immunodiagnostic reagent comprises a chimeric antibody selected from the group consisting of:
 (a) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:2;   (b) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:4;   (c) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:6; and   (d) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:8.   
     
     
         9 . The immunodiagnostic reagent according to  claim 8 , wherein said chimeric antibody comprises a V H  region selected from the group consisting of:
 (a) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 10;   (b) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 14;   (c) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 18; and   (d) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:28.   
     
     
         10 . The immunodiagnostic reagent according to  claim 8 , wherein said chimeric antibody comprises a V L  region selected from the group consisting of:
 (a) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 12;   (b) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 16; and   (c) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:20; and   (d) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:26.   
     
     
         11 . The immunodiagnostic reagent according to  claim 8 , wherein said chimeric antibody comprises both a V H  region and V L  region selected from the group consisting of:
 (a) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 10 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:12;   (b) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:14 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:16;   (c) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 18 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:20; and   (d) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:28 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:26.   
     
     
         12 . The immunodiagnostic reagent of  claim 8 , wherein said chimeric antibody is substantially identical to the chimeric antibody expressed by a cell line deposited with the American Type Tissue Collection and selected from the group consisting of PTA-8136, PTA-8138, and PTA-8140. 
     
     
         13 . The immunodiagnostic reagent of  claim 1 , wherein said antibody is substantially identical to the antibody expressed by a cell line deposited with the American Type Tissue Collection and selected from the group consisting of PTA-8137, PTA-8139, PTA-8141 and PTA-8142. 
     
     
         14 . The immunodiagnostic reagent according to  claim 1 , wherein said immunodiagnostic reagent is a reagent selected from the group consisting of a detection reagent, a standardization reagent, and a positive control reagent. 
     
     
         15 . An antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide selected from the group consisting of  T. cruzi  polypeptides comprised by FP3, Pep2, FP10 and FRA polypeptides. 
     
     
         16 . The antibody according to  claim 15 , wherein said antibody is selected from the group consisting of:
 an antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein the  T. cruzi  polypeptide is FRA and further wherein said antibody has at last one binding constant selected from the group consisting of: an association rate constant (k a ) between about 7.0×10 5  M −1 s −1  to about 7.0×10 6  M −1 s −1 , an dissociation rate constant (k d ) between about 4.0×10 −3  s −1  to about 3.0×10 −1  s −1  and an equilibrium dissociation constant (K D ) between about 5.7×10 −10  M to about 4.3×10 −7  M;   (b) an antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein the  T. cruzi  polypeptide is Pep2 and further wherein said antibody has at least one binding constant selected from the group consisting of: an association rate constant (k a ) between about 1.0×10 6  M −1 s −1  to about 8.0×10 6  M −1 s −1 ; an dissociation rate constant (k d ) between about 6.0×10 −3  s −1  to about 4.0×10 −2  s −1  and an equilibrium dissociation constant (K D ) between about 7.5×10 −10  M to about 4.0×10 −8  M;   (c) an antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein the  T. cruzi  polypeptide is FP10 and further wherein said antibody has at least one binding constant selected from the group consisting of: (a) an association rate constant (k a ) between about 5.0×10 4  M −1 s −1  to about 3.0×10 5  M −1 s −1 : (b) an dissociation rate constant (k d ) between about 1.0×10 −4  s −1  to about 8.0×10 −4  s −1 ; and (c) an equilibrium dissociation constant (K D ) between about 3.3×10 −10  M to about 1.6×10 −8  M;   (d) an antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein the  T. cruzi  polypeptide is FP3 and further wherein said antibody has at least one binding constant selected from the group consisting of: an association rate constant (k a ) between about 2.0×10 5  M −1 s −1  to about 6.0×10 6  M −1 s −1 ; an dissociation rate constant (k d ) between about 2.0×10 −5  s −1  to about 8.0×10 −4  s −1 ; and an equilibrium dissociation constant (K D ) between about 3.3×10 −12  M to about 4.0×10 −9  M; and   (e) any combinations of (a)-(d).   
     
     
         17 . The antibody of  claim 15 , wherein the antibody is a chimeric antibody. 
     
     
         18 . The chimeric antibody according to  claim 17 , selected from the group consisting of:
 (a) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:2;   (b) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:4;   (c) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:6; and   (d) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:8.   
     
     
         19 . The chimeric antibody according to  claim 17 , selected from the group consisting of:
 (a) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:2;   (b) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:4;   (c) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:6; and   (d) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:8.   
     
     
         20 . The chimeric antibody according to  claim 17 , selected from the group consisting of:
 (a) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 95% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:2;   (b) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 95% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:4;   (c) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 95% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:6; and   (d) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence having at least 95% sequence identity with an amino acid sequence as set forth in SEQ ID NO.:8.   
     
     
         21 . The chimeric antibody of  claim 17 , wherein said chimeric antibody is one selected from the group consisting of a monoclonal antibody, a humanized antibody, a single-chain Fv antibody, an affinity maturated antibody, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′) fragment, a disulfide-linked Fv, and an anti-idiotypic antibody, or fragments thereof. 
     
     
         22 . The chimeric antibody of  claim 17 , wherein the antibody is monoclonal. 
     
     
         23 . The chimeric antibody of  claim 17 , wherein said chimeric antibody is substantially identical to the chimeric antibody expressed by a cell line deposited with the American Type Tissue Collection and selected from the group consisting of PTA-8136, PTA-8138, and PTA-8140. 
     
     
         24 . The antibody of  claim 15 , wherein said antibody is substantially identical to the antibody expressed by a cell line deposited with the American Type Tissue Collection and selected from the group consisting of PTA-8137, PTA-8139, PTA-8141 and PTA-8142. 
     
     
         25 . A cell line that expresses a chimeric antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide, wherein said cell line is selected from the group consisting of a cell line deposited with the American Type Tissue Collection and selected from the group consisting of PTA-8136, PTA-8138, and PTA-8140. 
     
     
         26 . A method of standardizing a  T. cruzi  detection assay comprising using the immunodiagnostic reagent according to  claim 1  as a sensitivity panel. 
     
     
         27 . A method for detecting a  T. cruzi  antigen comprising the steps of:
 (a) contacting a test sample suspected of containing a  T. cruzi  antigen with the immunodiagnostic reagent according to  claim 4  under conditions that allow formation of chimeric antibody:antigen complexes; and   (b) detecting any chimeric antibody:antigen complexes formed as indicating the presence of said  T. cruzi  antigen.   
     
     
         28 . The method of  claim 27 , wherein the chimeric antibody comprises a detectable label. 
     
     
         29 . The method of  claim 27 , wherein the antibody:antigen complexes is contacted with a secondary antibody or fragment thereof, wherein the secondary antibody or fragment thereof comprises a detectable label. 
     
     
         30 . A method for detecting a  T. cruzi  antibody comprising the steps of:
 (a) contacting a test sample suspected of containing a  T. cruzi  antibody with one or more  T. cruzi  antigens specific for the  T. cruzi  antibody under conditions that allow formation of antigen:antibody complexes; and   (b) detecting any antigen:antibody complexes formed as indicating the presence of said  T. cruzi  antigens,   wherein the immunodiagnostic reagent according to  claim 4  is used in said method as a positive control or standardization reagent.   
     
     
         31 . A diagnostic kit for the detection of  T. cruzi  comprising the immunodiagnostic reagent of  claim 1 . 
     
     
         32 . An isolated polypeptide comprising a portion of a chimeric antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide selected from the group consisting of  T. cruzi  polypeptides comprised by FP3, Pep2, FP10 and FRA polypeptides. 
     
     
         33 . The isolated polypeptide according to  claim 32 , wherein said chimeric antibody is selected from the group consisting of:
 (a) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:2;   (b) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:4;   (c) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:6; and   (d) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:8.   
     
     
         34 . The isolated polypeptide according to  claim 32 , wherein said polypeptide comprises a V H  region selected from the group consisting of:
 (a) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 10;   (b) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 14;   (c) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 18; and   (d) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:28.   
     
     
         35 . The isolated polypeptide according to  claim 32 , wherein said chimeric antibody comprises a V L  region selected from the group consisting of:
 (a) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 12;   (b) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 16;   (c) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:20; and   (d) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:26.   
     
     
         36 . The isolated polypeptide according to  claim 32 , wherein said chimeric antibody comprises both a V H  region and V L  region selected from the group consisting of:
 (a) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 10 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:12;   (b) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 14 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:16;   (c) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 18 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:20; and   (d) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:28 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:26.   
     
     
         37 . An isolated polynucleotide encoding a portion of a chimeric antibody that specifically binds to a diagnostically relevant region of a  T. cruzi  polypeptide selected from the group consisting of  T. cruzi  polypeptides comprised by FP3, Pep2, FP10 and FRA polypeptides. 
     
     
         38 . The isolated polynucleotide according to  claim 37 , wherein said chimeric antibody is selected from the group consisting of:
 (a) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:2;   (b) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:4;   (c) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:6; and   (d) a chimeric antibody that specifically binds to an epitope comprised by an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:8.   
     
     
         39 . The isolated polynucleotide according to  claim 37 , wherein said polypeptide comprises a V H  region selected from the group consisting of:
 (a) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 10;   (b) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 14;   (c) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 18; and   (d) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:28.   
     
     
         40 . The isolated polypeptide according to  claim 37 , wherein said chimeric antibody comprises a V L  region selected from the group consisting of:
 (a) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 12;   (b) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 16;   (c) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:20; and   (d) a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:26.   
     
     
         41 . The isolated polypeptide according to  claim 37 , wherein said chimeric antibody comprises both a V H  region and V L  region selected from the group consisting of:
 (a) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 10 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:12;   (b) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:14 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:16;   (c) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.: 18 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:20; and   (d) a V H  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:28 and a V L  region comprising an amino acid sequence substantially identical to the sequence as set forth in SEQ ID NO.:26;   
     
     
         42 . A method of purifying an antigen comprising a  T. cruzi  amino acid sequence having at least 70% sequence identity with a  T. cruzi  amino acid sequence comprised by the amino acid sequences as set forth in SEQ ID NOs.:1, 3, 5 or 7, comprising:
 (a) contacting a sample suspected of containing a  T. cruzi  polypeptide with the immunodiagnostic reagent according to  claim 1  under conditions that allow formation of chimeric antibody:antigen complexes;   (b) isolating the chimeric antibody:antigen complexes formed; and   (c) separating the antigen from the antibody.