Purpose: Megakaryocyte Potentiating Factor (MPF), is cleaved from mesothelin precursor and is a potential tumor marker in the serum of patients with mesothelioma. Due to low level of MPF and mesothelin expression in normal tissues shedded MPF can be used as PD biomarker. It was shown that soluble mesothelin and MPF levels are elevated in mesothelioma patients and may serve as PD biomarker. The available commercial MPF immunoassays were found to be not sensitive enough and higher sensitivity assay development was requested.
Methods: MPF-specific hybridomas were screened both on Gyros platform as well as by surface plasmon resonance. The highest affinity clones were purified and their clonality and uniqueness was confirmed by HRMS. The pairing was performed on Gyros platform to select the lead pair and backups. MSD platform was selected for the assay and DOE was deployed for assay optimization.
Results: Total of 134 MPF specific hybridomas were generated, 11 best clones were purified and evaluated on both Gyros and SPR platforms. The lead pair had KD in subnanomolar range. Biotinylated capture and ruthenylated detector concentrations and other MSD assay parameters were optimized via DOE. 500 ng/mL was optimal for both capture and detector, and 10% cynomolgus monkey serum in 1% BSA/0.05% Tween20/PBS was selected as surrogate matrix. The optimized assay sensitivity was below 1 pg/mL, but for the analysis of clinical samples the assay range was raised 82 -50,000 pg/mL. The validated assay shows robust accuracy and single-digit %CV for all concentrations tested. QC performance was within acceptable limits, dilution linearity, specificity, stability and selectivity QC recovered within ± 20% of the expected concentrations.
Conclusion: An ultrasensitive MPF immunoassay was developed and validated on MSD platform and was used for the analysis of clinical samples.
Gerard Dalglish– Bristol-Myers Squibb Company
Frank Zambito– Bristol-Myers Squibb Company
Paul Mesko– Bristol-Myers Squibb Company
Christine Bee– Bristol-Myers Squibb Company
Rasa Santockyte– Bristol-Myers Squibb Company
Robert Neely– Associate Director - Pharmacodiagnostics, Bristol-Myers Squibb Company
Yan Zhang– Director, Analytical and Bioanalytical Operations, Bristol-Myers Squibb Company, Princeton, New Jersey
Renuka Pillutla– Executive Director, Bristol-Myers Squibb Company