Goodpasture Antigen Binding Protein and Its Detection
OwnerJuan Saus, Fernando Revertinventors
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Abstract
The present invention provides native Goodpasture antigen binding protein isoforms, monoclonal antibodies directed against such proteins, and methods for their use.
Claims11
A substantially purified recombinant polypeptide comprising the general formula X-SEQ ID NO:2, wherein X is a detectable polypeptide.
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).
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.
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.
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.
Dependencies are inferred from the claim text.