Polyclonal-monoclonal ELISA assay for detecting N-terminus proBNP
View Patent ↗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. A process 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, comprising:
(a) obtaining isolated polyclonal antibodies specific for amino acid residues 26-51 of SEQ ID No: 1 and attaching said isolated polyclonal antibodies to a solid support;
(b) obtaining a sample of a bodily fluid from said patient wherein said sample is suspected of containing immunogenic fragments of NT-proBNP;
(c) adding said sample to said isolated polyclonal antibodies of step (a) wherein said immunogenic fragments of NT-proBNP contained in said sample are captured by said isolated polyclonal antibodies;
(d) providing 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;
(e) labeling said monoclonal antibodies with a detector;
(f) adding said monoclonal antibodies of step (e) to said immunogenic fragments of NT-proBNP captured in step (c) wherein said monoclonal antibodies of step (e) bind to said immunogenic fragments of NT-proBNP captured in step (c);
(g) adding a reporter that reacts with said detector to form a reaction product; and
(h) measuring said reaction product to determine concentration of NT-proBNP in said sample; and
(i) determining if 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; wherein said elevated concentration identifies that the patient has congestive heart failure of NYHA classes III or IV.
2. The process of claim 1 wherein said bodily fluid is plasma.
3. The process of claim 1 wherein the detector is biotin.
4. The process of claim 3 wherein the reporter is peroxidase-labeled streptavidin.