Open AccessEstablishment of a bead‐based duplex assay for the simultaneous quantitative detection of Neuropilin‐1 and Neuropilin‐2 using xMAP technology and its clinical application
Author(s) -
Huang ZiLan,
Meng PeiPei,
Yang Yun,
Wang ShengYu,
Zhang Xiufang,
Luo FangHong,
Yan JiangHua,
Wu Ting
Publication year - 2019
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.22850
Subject(s) - neuropilin 1 , multiplex , chemistry , monoclonal antibody , antibody , bead , detection limit , chromatography , microbiology and biotechnology , biology , materials science , cancer research , immunology , bioinformatics , vegf receptors , composite material , vascular endothelial growth factor
Background Neuropilins (Nrps) are a new type of broad‐spectrum tumor marker. Currently, a method for accurate simultaneous quantification of Nrps is not available. We aimed to develop a bead‐based and duplexed flow cytometric assay that could be used for accurate and simultaneous quantification of Nrp1 and Nrp2 for scientific research or clinical diagnosis. Methods We coupled anti‐human Nrp1‐11# mAb and anti‐human Nrp2‐C3 mAb to magnetic beads 18# and 25#, respectively. Capturing antibodies and detecting antibodies were then combined to detect Nrps by a bead‐based Luminex assay, which was subsequently applied to quantify Nrps in clinical serum samples. Results The results showed that the detection value of Nrps ranged from 10 to 100 000 pg/mL for Nrp1 and from 25 to 100 000 pg/mL for Nrp2. The detection sensitivity reached 10 pg/mL for Nrp1 and 24.8 pg/mL for Nrp2. Intra‐assay variances ranged from 1.0% to 2.6% for Nrp1 and from 2.9% to 4.0% for Nrp2, and interassay variances ranged from 1.5% to 6.4% for Nrp1 and from 4.2% to 8.1% for Nrp2. The Nrp1 and Nrp2 recoveries were 96.6%‐103.6% and 95.6%‐102.3%, respectively. Irrelevant antigens had no interference in the paired‐detection system, and the mean fluorescence intensity (MFI) values were stable for months. Conclusion A bead‐based, duplexed flow cytometric assay (xMAP ® technology) was developed to detect Nrp1 and Nrp2. The assay provided rapid, high‐throughput results and was much more sensitive, specific, reproducible, and stable than existing assays. In addition, this assay could be applied in early‐stage cancer screening, tumor malignancy analysis, and prognosis assessment.