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. 2019 May;33(4):e22850.
doi: 10.1002/jcla.22850. Epub 2019 Feb 13.

Establishment of a bead-based duplex assay for the simultaneous quantitative detection of Neuropilin-1 and Neuropilin-2 using xMAP technology and its clinical application

Zi-Lan Huang  1 , Pei-Pei Meng  1 , Yun Yang  1 , Sheng-Yu Wang  1 , Xiu-Fang Zhang  2 , Fang-Hong Luo  1 , Jiang-Hua Yan  1 , Ting Wu  1
Affiliations

Establishment of a bead-based duplex assay for the simultaneous quantitative detection of Neuropilin-1 and Neuropilin-2 using xMAP technology and its clinical application

Zi-Lan Huang et al. J Clin Lab Anal. 2019 May.

Abstract

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.

Keywords: Neuropilins; bead-based immunoassay; duplex flow cytometry; xMAP technology.

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Conflict of interest statement

The authors have no financial interests to disclose.

Figures

Figure 1
Figure 1
A, Analysis of magnetic beads coupling mAb efficiency. B, Titer analysis of antibody biotinylation. C, Influence of storage time on the performance of detection system
Figure 2
Figure 2
Sandwich ELISA standard curves of detection for Nrp1 (A) and Nrp2 (B). Standard curves of Nrp1 and Nrp2 from the duplex assay (C). Each standard curve shows optical density (OD) values and median fluorescence intensities (MFIs), respectively, which are presented as means and standard deviations obtained from three independent assays
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