Objective: To develop a convenient, nonradiometric, and robust assay platform for the measurement of urinary protein biomarkers.
Performance Specification: Customer required the two combined ELISA results to correlate well to an FDA approved HPLC method.
Proposed Timeline: 24 weeks
Description: IBA was commissioned to develop an antibody based ELISA. Outlined below are the major steps IBA took to accomplish this task:
Purification of Urine Proteins
Liters of diabetic urine precipitated with ammonium sulfate
Precipitated proteins run over anti-HSA affinity chromatography column
Flowthrough run over a gel filtration column
MW range of 40-90kD collected
Characterization of the Urine Proteins
Non-reducing and reducing gels
N-terminal sequence
MS/MS identification
Generation of Anti-Urine Protein Antibodies
Polyclonal Ab’s produced
Characterization of the anti-protein antibodies by ELISA and Western blots
Development of an Assay
Proprietary polyclonal antibody utilized
Optimization of antibody and key reagent conecntrations
Optimization of standard curve
Sample analysis and correlation to client’s HPLC method
Result: Very sensitive assay with a good sample correlation to the clients HPLC method