Highly sensitive particle enhanced assay for the quantification of nt-probnp

Abstract

NT-proBNP can be determined in a biological sample using at least one antibody which recognizes an epitope of NT-proBNP in both a glycosylated and non-glycosylated form of NT-proBNP. Said antibody is preferably an isolated polyclonal antibody or a mixture of monoclonal antibodies coated onto a particle, preferably coated onto said particle in a coating ratio of 6-60%, forming a layer or multiple layers of antibodies on said particle. The assay, realized in the form of a nephelometric or turbidimetric assay, can be applied to a wide range of automated clinical analyzers.

1 . A method for determining the concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) in a sample, said method comprising the following steps:
 (i) contacting the sample with a mixture of monoclonal antibodies comprising at least a first monoclonal antibody which specifically binds to NT-proBNP within the amino acid sequence of SEQ. ID. NO. 2 and at least a second monoclonal antibody which specifically binds to NT-proBNP within the amino acid sequence of SEQ. ID. NO. 3, and conducting a step of calibration using a calibrator comprising a known concentration of a substance chosen from a peptide having the amino acid sequence of SEQ. ID. NO. 4, SEQ. ID. NO 5, SEQ. ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID. NO. 8, SEQ. ID. NO. 9, or SEQ. ID. NO. 10;   
       wherein said at least one monoclonal antibody is immobilized to a particle, and wherein the calibrator is non-glycosylated; and
 (ii) detecting a change in reflectance, scattering or transmittance of the sample wherein said change is indicative of the concentration of NT-proBNP in the sample. 
 
     
     
         2 . The method according to  claim 1 , wherein said at least one first monoclonal antibody specifically binds to NT-proBNP within the amino acid sequence of SEQ. ID. NO 4, SEQ. ID. NO 5, SEQ. ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID. NO. 8, SEQ. ID. NO. 9, or SEQ. ID. NO. 10. 
     
     
         3 . The method according to  claim 1 , wherein said at least one monoclonal antibody specifically binds to NT-proBNP within the amino acid sequence of SEQ. ID. NO 5, SEQ. ID. NO. 7, or SEQ. ID. NO. 8. 
     
     
         4 . The method according to  claim 3 , wherein said at least one monoclonal antibody specifically binds to the amino acids of SEQ. ID. NO. 8. 
     
     
         5 . The method according to  claim 1 , wherein said at least one monoclonal antibody is bound to a latex particle having an average size in the range of 150-300 nm. 
     
     
         6 . The method of  claim 1 , further comprising, on the basis of said concentration of NT-proBNP in the sample, diagnosing, monitoring, or stratifying patients with heart failure or subjects at risk of developing heart failure. 
     
     
         7 . The method according to  claim 1 , wherein said at least one monoclonal antibody binds specifically to NT-proBNP with a K D  to NT-proBNP of less than 10.0E-09 M. 
     
     
         8 . The method according to  claim 7 , wherein said at least one monoclonal antibody binds specifically to NT-proBNP with a K D  to NT-proBNP of less than 5.0E-09 M. 
     
     
         9 . The method according to  claim 8 , wherein said at least one monoclonal antibody binds specifically to NT-proBNP with a K D  to NT-proBNP of less than 2.0E-09 M. 
     
     
         10 . The method according to  claim 1 , wherein said at least one monoclonal antibody binds specifically to a peptide having the amino acid sequence of SEQ. ID. NO 5, SEQ. ID. NO. 7, or SEQ. ID. NO. 8 with a K D  of less than 5.0E-09 M. 
     
     
         11 . The method according to  claim 10 , wherein said at least one monoclonal antibody binds specifically to a peptide having the amino acid sequence of SEQ. ID. NO 5, SEQ. ID. NO. 7, or SEQ. ID. NO. 8 with a K D  of less than 2.0E-09 M.