A sensitive and rapid chemiluminescence immunoassay for point-of-care testing (POCT) of copeptin in serum based on high-affinity monoclonal antibodies via cytokine-assisted immunization

Abstract

Purpose:

Antibodies are key reagents in the development of immunoassay. We attempted to develop high-performance CPP immunoassays using high-affinity monoclonal antibodies prepared via cytokine-assisted immunization.

Methods:

We used fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), and granulocyte-macrophage colony-stimulating factor (GM-CSF) to assist traditional subcutaneous immunization of preparing high-affinity monoclonal antibodies, and further to develop high-performance immunoassay methods for CPP.

Results:

This novel immune strategy significantly enhanced immune response against CPP. Six anti-CPP monoclonal antibodies (mAbs) with high affinity were successfully screened and selected for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect CPP in the range of 1.2–1250 pmol L^–1 with a detection limit of 6.25 pmol L^–1. Significantly, the whole incubation process can be completed in 30 min as compared to about 4.5 hr for the control ELISA kit. Furthermore, this assay exhibited high sensitivity and specificity, low intra-assay and inter-assay coefficients of variation (CVs < 15%). The developed assay was applied in the detection of CPP in 115 random serum samples and results showed a high correlation with data obtained using a commercially available ELISA kit (correlation coefficient, 0.9737).

Conclusion:

Our assay could be applied in the point-of-care testing of CPP in the serum samples, and also the method developed in this study could be adopted to explore the detection and diagnosis of other biomarkers for various diseases.

Keywords:

• copeptin
• cytokine-assisted immunization
• high-affinity antibodies
• chemiluminescence immunoassay
• point-of-care tests
• clinical application

Acknowledgments

This work is supported by the Program of Guangdong Provincial Science & Technology (2017A020208014) and the Program of Guangzhou Science & Technology (201802010030).

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

Table 1 The concentration of standard CPP solutions (pmol L⁻¹)

Table 2 Comparison of performance CPP detection methods

Table 3 Intra-assay and inter-assay tests

Table S1 The comparison of subcutaneous and cytokine-assisted immunization

Table S2 The affinity of the antibodies prepared via cytokine-assisted immunization

Table S3 Stability of the reagents at store (n=3)

Table S4 Cross-reactivity of CLIA to related compounds

Figure S1 Purification results of the mAb against CPP by SDS–PAGE.Notes: Lane 1: 6-20G5; lane 2: 6-8D2; lane 3: 6-18H3; lane 4: 6-5G8; lane 5: 6-2A9 ; lane 6: 6-3C10 ; lane 7: 6-18G10 ; lane 8: 6-11C3. There were only two straps after the mAb was purified, and these were heavy chain and light chain, respectively, CV. The results indicate that the purity of all mAbs was above 90% as analyzed on SDS-PAGE.Abbreviations: mAb, monoclonal antibody; CV, coefficient of variation

Figure S2 The titer of the mAbs against CPP.Abbreviation: mAbs, monoclonal antibodies