Polyclonal-monoclonal elisa assay for detecting n-terminus pro-bnp

Abstract

A specific and sensitive in vitro ELISA assay and diagnostic test kit is disclosed for determining levels of NT-proBNP protein in a variety of bodily fluids, non-limiting examples of which are blood, serum, plasma, urine and the like. The NT-proBNP ELISA assay test employs the sandwich ELISA technique to measure circulating NT-proBNP in human plasma. In order to obtain antibodies with specific binding properties for targeted amino acid sequences within human proBNP, recombinant human proBNP (or rhproBNP) was expressed and purified for use as an immunogen. Polyclonal antibodies (PAb) to specific amino acid sequences were subsequently purified from goat serum by sequential affinity purification. Monoclonal antibodies were raised against specific polypeptides. Recombinant human NT-proBNP (or rhNT-proBNP) was expressed and purified in order to obtain material for use in calibration of a quantitative method for measurement of human NT-proBNP.

1 - 7 . (canceled) 
     
     
         8 . A kit for identifying a patient with congestive heart failure of New York Heart Association (NYHA) classes III or IV by determining concentration of N-terminal pro-brain natriuretic peptide (NT-probNP) in a bodily fluid sample, the kit comprising:
 isolated polyclonal antibodies specific for amino acid residues 26-51 of SEQ ID No: 1 attached to a solid support, wherein said polyclonal antibodies are capable of capturing immunogenic fragments of NT-proBNP in said sample.   
     
     
         9 . The kit of  claim 8 , further comprising:
 monoclonal antibodies produced by a hybridoma cell line designated as 6G11-F11-D12 and deposited with the American Type Culture Collection (ATCC) as Accession Number PTA-4844, wherein said monoclonal antibodies are specific for amino acid residues 1-25 of SEQ ID NO: 1, and wherein said monoclonal antibodies are labeled with a detector, wherein said monoclonal antibodies when added to said sample are capable of binding to said captured immunogenic fragments of NT-proBNP.   
     
     
         10 . The kit of  claim 9 , further comprising:
 a reporter that reacts with said detector to form a reaction product; wherein a measurement of said reaction product determines concentration of NT-proBNP in said sample, wherein determining whether said concentration of NT-proBNP in said sample is elevated above a selected cut-off concentration indicative of concentration of NT-proBNP in a sample of said bodily fluid from a normal human identifies that the patient has congestive heart failure of NYHA classes III or IV.   
     
     
         11 . The kit of  claim 10 , further comprising:
 a control sample.   
     
     
         12 . The kit of  claim 10 , further comprising:
 a set of NT-proBNP protein standards.   
     
     
         13 . The kit of  claim 8 , wherein said sample is plasma. 
     
     
         14 . The kit of  claim 9 , wherein the detector is biotin. 
     
     
         15 . The kit of  claim 10 , wherein the reporter is peroxidase-labeled streptavidin.