Project Examples – Development of an Assay for Urinary Protein Biomarkers
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
Project Completion: 24 weeks
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