Development of bead based multiplexed immunoassay for evaluation of midkine, syndecan-1, and ANGPTL4 in patient serum.

Autor: Tarhoni I; a Departments of Biochemistry , Rush University Medical Center , Chicago , IL, USA., Fhied CL; b Departments of Pathology , Rush University Medical Center , Chicago , IL, USA., Pool M; b Departments of Pathology , Rush University Medical Center , Chicago , IL, USA., Liptay MJ; c Departments of Cardiovascular and Thoracic Surgery , Rush University Medical Center , Chicago , IL, USA., Bonomi P; d Departments of Medical Oncology , Rush University Medical Center , Chicago , IL, USA., Seder CW; c Departments of Cardiovascular and Thoracic Surgery , Rush University Medical Center , Chicago , IL, USA., Borgia JA; a Departments of Biochemistry , Rush University Medical Center , Chicago , IL, USA.; b Departments of Pathology , Rush University Medical Center , Chicago , IL, USA.
Jazyk: angličtina
Zdroj: Journal of immunoassay & immunochemistry [J Immunoassay Immunochem] 2018; Vol. 39 (1), pp. 84-98. Date of Electronic Publication: 2018 Feb 02.
DOI: 10.1080/15321819.2017.1407338
Abstrakt: Background: Angiogenesis is associated with tumor progression in a range of malignancies. Herein, we develop custom immunobead assays for several mechanistically important targets and evaluated these against sera from cohorts of non-small cell lung cancer (NSCLC) patients.
Methods: Antigen "capture" antibodies for midkine, syndecan-1, and ANGPTL4 were independently conjugated to MagPlex® Microspheres using standard carbodiimide/NHS-based chemistry. These reagents served as the basis for quantitative sandwich assay assembly using biotinylated detection antibodies and R-phycoerythrin-conjugated streptavidin reporter system. Standard curves were created using dilution series of recombinant target proteins with assay performance characteristics calculated, accordingly. Finally, we evaluated a range of serum samples from NSCLC patients (n = 32) to verify assay performance.
Results: Multiplexed assays for midkine, syndecan-1, and ANGPTL4 were developed with three orders of magnitude in dynamic range, excellent intra- and inter-assay precision, and accuracy parameters (<10%, and <15% variability, respectively). Detection and quantifications limits were suitable for the three assays to efficiently evaluate sera across a range of disease stages with a four-fold dilution factor.
Conclusion: We successfully developed and analytically validated a 3-plex immunobead assay for quantifying midkine, syndecan-1, and ANGPTL4 in patient sera. This multiplexed assay will provide an important tool for future studies delineating the role of angiogenesis in lung cancer progression.
Databáze: MEDLINE
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