Goodpasture Antigen Binding Protein and Its Detection

Abstract

The present invention provides native Goodpasture antigen binding protein isoforms, monoclonal antibodies directed against such proteins, and methods for their use.

We claim: 
     
         1 . A substantially purified recombinant polypeptide comprising the general formula X-SEQ ID NO:2, wherein X is a detectable polypeptide. 
     
     
         2 . A recombinant nucleic acid encoding a polypeptide of  claim 1 . 
     
     
         3 . A recombinant expression vector comprising the recombinant nucleic acid of  claim 2  operatively linked to a promoter sequence. 
     
     
         4 . A host cell transfected with the recombinant expression vector of  claim 3 . 
     
     
         5 . A substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:2 (91 kD GPBP) or SEQ ID NO:4 (77 kD GPBP), wherein the polypeptide comprises one or more post-translational modifications (PTMs) directly and/or indirectly involving amino acids 305-344 (GGPDYEEGPNSLINEEEFFDAVEAALDRQDKIE EQSQSEK) (SEQ ID NO: 10) and/or involving residues 371-396 PYSRSSSMSSIDLVSASDDVHRFSSQ (SEQ ID NO:9). 
     
     
         6 . The substantially purified polypeptide of  claim 5 , wherein the one or more PTMs are within residues 320-327 (EEFFDAVE, SEQ ID NO:5). 
     
     
         7 . The substantially purified polypeptide of  claim 5 , wherein the one or more PTMS are within residues 388-392 (DDVHR, SEQ ID NO:6). 
     
     
         8 . A substantially purified monoclonal antibody that selectively binds to the substantially purified polypeptide of  claim 5 . 
     
     
         9 . A substantially purified monoclonal antibody that specifically binds to the polypeptide of SEQ ID NO:2 and not to the polypeptide of SEQ ID NO:4. 
     
     
         10 . A method for detecting urinary Goodpasture antigen binding protein (GPBP), comprising
 (a) contacting a urine sample with a GPBP-binding molecule that binds to GPBP under conditions to promote selective binding of the GPBP-binding molecule to GPBP;   (b) removing unbound GPBP-binding molecules; and   (c) detecting complex formation between the GPBP-binding molecule and the GPBP in the urine sample.   
     
     
         11 . A method for isolating native GPBP isoforms, comprising a method selected from the group consisting of:
 (I): (a) subjecting a plasma sample to ammonium sulfate precipitation; (b) conducting ion-exchange chromatography (IEC) on the ammonium sulfate precipitated serum sample; (c) identifying IEC fractions containing native GPBP isoforms; (d) subjecting IEC fractions containing native GPBP isoforms to gel filtration chromatography (GFC); and (e) identifying GFC fractions containing native GPBP isoforms;   (II): (a) subjecting a urine sample to salt precipitation; (b) conducting double ion-exchange chromatography (IEC) on the salt precipitated protein sample; and (c) identifying IEC fractions containing native GPBP isoforms; and   (III): (a) passing a plasma sample or urine sample through an affinity column comprising a GPBP-binding molecule that selectively bind to native GPBP; (b) washing unbound protein from the plasma or urine sample from the affinity column; and (c) eluting native GPBP isoforms from the column.