Project Examples – Development of an Assay for the Biomarker PIIINP
Objective: To develop a convenient, nonradiometric, and robust assay platform for the measurement of PIIINP.
Performance Specification: Development of two different sets of antibody pairs with appropiate sensitivity and specificity for human PIIINP.
Proposed Timeline: 24 weeks
Description: PIIINP is the N-terminal pro-peptide of collagen type III. PIIINP is useful as a biomarker of liver fibrosis. Due to increased levels in serum in response to growth hormone administration, our client required a test for its detection. Outlined below are the major steps IBA took to accomplish this task:
Purification of Bovine PIIINP
- 9 kg of fetal calf skin were homogenized, extracted with 20 L salt solution, precipitated with ethanol, digested with enzyme and purified by ion exchange chromatography
- Purification of ~400,000 fold
Characterization of the Bovine PIIINP
- Reducing SDS gels
- N-terminal sequence
- Bioequivalence to human PIINP was demonstrated using a commercial human PIIINP RIA
Generation of Anti-PIIINP Antibodies.
- Epitope selection
- Monoclonal and polyclonal Ab’s produced
- Characterization of the anti-PIIINP antibodies by ELISA and Westerns blots
- Antibodies were evaluated for their ability to bind human PIIINP by RIA
Development of an Assay
- Extensive pairing explored to obtain the best antibody pairs compared by sensitivity, selectivity etc.
- Optimiztion of selected antibody pairs (coating buffers, coating concentrations, biotinalyation of secondary antibodies, etc.)
- Optimization of standard curve
- Sample analysis and correlation to commercial ELISA
Results: Very sensitive assay with a good sample correlation to the commercial RIA
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CAPTURE
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DETECTION
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| Anti-N-Terminus MAb (18E5; lgG3, k) |
Anti-C-Terminus MAb (263; lgG2a, k) |
| Anti-Whole PIIINP MAb (297;lgG1, k) |
Anti-C-Terminus MAb (263; lgG2a, k) |
| Anti-N-Terminus MAb (6C12; lgG1, k) |
Anti-Whole-PIIINP MAb (297; lgG1a, k) |
| Anti-N-Terminus MAb (17G12; lgG1, k) |
Rabbit PAb Anti-PIIINP (5027) |
Project Completion: 24 weeks
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