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

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

J Immunoassay Immunochem. 2018 Jan 08;:

Authors: Tarhoni I, Fhied CL, Pool M, Liptay MJ, Bonomi P, Seder CW, Borgia JA

Abstract
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 4-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.

PMID: 29309212 [PubMed - as supplied by publisher]

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