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

J Immunoassay Immunochem. 2018;39(1):84-98. doi: 10.1080/15321819.2017.1407338. Epub 2018 Feb 2.


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.

Keywords: ANGPTL4; Luminex; Midkine; angiogenesis; syndecan-1.

MeSH terms

  • Angiopoietin-Like Protein 4 / blood*
  • Carcinoma, Non-Small-Cell Lung / blood*
  • Humans
  • Immunoassay / methods*
  • Lung Neoplasms / blood*
  • Midkine
  • Nerve Growth Factors / blood*
  • Syndecan-1 / blood*


  • ANGPTL4 protein, human
  • Angiopoietin-Like Protein 4
  • MDK protein, human
  • Nerve Growth Factors
  • SDC1 protein, human
  • Syndecan-1
  • Midkine