Polyclonal-monoclonal ELISA assay for detecting N-terminus pro-BNP

1. A process for determining concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) in a bodily fluid sample, comprising:

(a) providing isolated polyclonal antibodies specific for the sequence of amino acid residues 26-51 of SEQ ID No: 1, wherein the isolated polyclonal antibodies are immobilized on a solid support;

(b) providing a bodily fluid sample from a patient, wherein said bodily fluid sample is suspected of containing antigenic fragments of NT-proBNP;

(c) contacting said bodily fluid sample to said isolated polyclonal antibodies immobilized on the solid support to form a first mixture wherein said antigenic fragments of NT-proBNP in said bodily fluid sample are captured by said isolated polyclonal antibodies;

(d) washing the first mixture, wherein the first mixture comprises said antigenic fragments of NT-proBNP captured by said isolated polyclonal antibodies immobilized on the solid phase, thereby forming a washed first mixture;

(e) 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 the sequence of amino acid residues 1-25 of SEQ ID NO:1;

(f) labeling said monoclonal antibodies with a detector, thereby forming labeled monoclonal antibodies;

(g) adding the labeled monoclonal antibodies of step (f) to the washed first mixture of step (d) wherein said monoclonal antibodies bind to said antigenic fragments of NT-proBNP captured by the isolated polyclonal antibodies immobilized on the solid phase in step (c), thereby forming a second mixture;

(h) washing the second mixture, wherein the second mixture comprises captured antigenic fragments of NT-proBNP bound by the labeled monoclonal antibodies;

(i) adding a reporter that reacts with said detector of the labeled monoclonal antibodies to form a reaction product; and

(j) measuring said reaction product to determine concentration of NT-proBNP in said sample.

2. The process of claim 1 , further comprising comparing the determined concentration of NT-proBNP in said sample with a selected cut-off concentration.

3. The process of claim 1 , wherein said bodily fluid sample is plasma.

4. The process of claim 1 , wherein the detector is biotin.

5. The process of claim 1 , wherein the reporter is peroxidase-labeled streptavidin.

6. A kit for determining a concentration of N-terminal pro-brain natriurectic peptide (NT-proBNP) in a bodily fluid sample, the kit comprising:

isolated polyclonal antibodies specific for the sequence of amino acid residues 26-51 of SEQ ID No: 1 that are attached to a solid support;

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 the sequence of amino acid residues 1-25 of SEQ ID NO:1, wherein the monoclonal antibodies are labeled with a detector; and

a reporter capable of reacting with said detector to form a reaction product.

7. The kit of claim 6 , wherein the solid support attached with isolated polyclonal antibodies is a microtitration strip, and wherein the polyclonal antibodies specific for the sequence of amino acid residues 26-51 of SEQ ID NO: 1 are goat antibodies.

8. The kit of claim 6 , wherein the detector is biotin.

9. The kit of claim 8 , wherein the reporter is peroxidase-labeled streptavidin.

10. The kit of claim 9 , wherein the peroxidase is a horseradish peroxidase.

11. The kit of claim 6 , further comprising a chromogen solution that comprises 3, 3′, 5, 5′- tetramethylbenzidine (TMB) substrate solution.

12. The kit of claim 6 , further comprising a wash concentrate that comprises phosphate buffered saline with nonionic detergent.

13. The kit of claim 6 , further comprising a stopping solution that comprises sulfuric acid.