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
Project Completion: 24 weeks