Diagnostic assays for detection of cryptosporidium parvum

Abstract

This invention provides a novel Cryptosporidium parvum protein disulfide isomerase polypeptide, and nucleic acids that encode this polypeptide. The inventionalso provides methods, reagents, and kits that are useful for diagnosing infection by Cryptosporidium parvum . The methods are based on the discovery of binding agents, including recombinant polyclonal antibodies, that bind to the protein disulfide isomerase polypeptide of C. parvum.

What is claimed is:  
     
         1 . A method of diagnosing infection of a mammal by a Cryptosporidium species, the method comprising: 
 contacting a stool sample obtained from the mammal with a capture reagent which binds to Cryptosporidium protein disulfide isomerase, wherein the capture reagent forms a complex with the protein disulfide isomerase if the protein disulfide isomerase is present in the stool sample; and    detecting whether protein disulfide isomerase is bound to the capture reagent, wherein the presence of protein disulfide isomerase is indicative of Cryptosporidium infection of the mammal.    
     
     
         2 . The method of  claim 1 , wherein the protein disulfide isomerase comprises an amino acid sequence at least ten consecutive amino acids of which are substantially identical to a subsequence of an amino acid sequence AWFCGTNEDFA KYASNIRKVAADYR EKYAFVF (SEQ ID NO: 3).  
     
     
         3 . The method of  claim 2 , wherein the protein disulfide isomerase has an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO:2.  
     
     
         4 . The method of  claim 1 , wherein the capture reagent comprises an antibody which binds to protein disulfide isomerase.  
     
     
         5 . The method of  claim 4 , wherein the antibody is a recombinant antibody.  
     
     
         6 . The method of  claim 5 , wherein the antibody is a recombinant polyclonal antibody.  
     
     
         7 . The method of  claim 6 , wherein the recombinant polyclonal antibody is SCPc.4.PC.  
     
     
         8 . The method of  claim 1 , wherein the capture reagent is immobilized on a solid support.  
     
     
         9 . The method of  claim 8 , wherein the capture reagent is immobilized on the solid support prior to contacting the capture reagent with the test sample.  
     
     
         10 . The method of  claim 1 , wherein the detection of the protein disulfide isomerase is performed by contacting the protein disulfide isomerase with a detection reagent which binds to the protein disulfide isomerase.  
     
     
         11 . The method of  claim 10 , wherein the detection reagent comprises an antibody which binds to protein disulfide isomerase.  
     
     
         12 . The method of  claim 10 , wherein the detection reagent comprises a detectable label.  
     
     
         13 . The method of  claim 12 , wherein the detectable label is selected from the group consisting of a radioactive label, a fluorophore, a dye, an enzyme, and a chemiluminescent label.  
     
     
         14 . A kit for diagnosing infection of a mammal by an Cryptosporidium species, the kit comprising: 
 a solid support upon which is immobilized a capture reagent which binds to a protein disulfide isomerase of  Cryptosporidium parvum ; and    a detection reagent which binds to the protein disulfide isomerase.    
     
     
         15 . The kit according to  claim 14 , wherein the kit further comprises a positive control that comprises a protein disulfide isomerase.  
     
     
         16 . The kit according to  claim 15 , wherein the protein disulfide isomerase comprises an amino acid sequence of which at least ten consecutive amino acids are substantially identical to an amino acid sequence AWFCGTNEDFAKYASNIRKVAADYR EKYAFVF (SEQ ID NO: 3).  
     
     
         17 . A monoclonal antibody that specifically binds to a protein disulfide isomerase of  Cryptosporidium parvum , wherein the monoclonal antibody is CP.2.  
     
     
         18 . A recombinant polyclonal antibody preparation that specifically binds to protein disulfide isomerase of  Cryptosporidium parvum.    
     
     
         19 . The recombinant polyclonal antibody preparation of  claim 18 , wherein the protein disulfide isomerase comprises an amino acid sequence of which at least ten consecutive amino acids are substantially identical to an amino acid sequence AWFCGTNEDFAKYASNIRKVAADYREKYAFVF (SEQ ID NO: 3).  
     
     
         20 . The recombinant polyclonal antibody preparation of  claim 18 , wherein the antibody preparation is SCPc.4.PC.  
     
     
         21 . An isolated protein disulfide isomerase polypeptide which comprises an amino acid sequence of which at least ten consecutive amino acids are substantially identical to a subsequence of an amino acid sequence AWFCGTNEDFAKYASNIRKVAADYR EKYAFVF (SEQ ID NO: 3).  
     
     
         22 . The protein disulfide isomerase polypeptide of  claim 21 , wherein the polypeptide comprises an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 2.  
     
     
         23 . The protein disulfide isomerase polypeptide of  claim 21 , wherein the polypeptide comprises an amino acid sequence that is substantially identical to an amino acid sequence AWFCGTNEDFAKYASNIRKVAADYREKYAFVF (SEQ ID NO: 3).  
     
     
         24 . The protein disulfide isomerase polypeptide of  claim 23 , wherein the polypeptide comprises an amino acid sequence of SEQ ID NO: 3.  
     
     
         25 . The protein disulfide isomerase polypeptide of  claim 24 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.  
     
     
         26 . An isolated nucleic acid that comprises a polynucleotide sequence that encodes a polypeptide that comprises an amino acid sequence of which at least ten consecutive amino acids are substantially identical to a subsequence of an amino acid sequence AWFCGTNEDFAKYASNIRKVAADYREKYAFVF (SEQ ID NO: 3).  
     
     
         27 . The nucleic acid of  claim 26 , wherein the nucleic acid comprises a translation initiation codon that is in frame with codons that encode the amino acid sequence set forth in SEQ ID NO: 3.  
     
     
         28 . The nucleic acid of  claim 27 , wherein the nucleic acid comprises a polynucleotide sequence of SEQ ID NO: 1.  
     
     
         29 . The nucleic acid of  claim 26 , wherein the nucleic acid is operably linked to a promoter.  
     
     
         30 . The nucleic acid of  claim 29 , wherein the nucleic acid comprises an expression cassette.  
     
     
         31 . A recombinant cell that comprises an expression cassette of claim  30 .