Finding a reliable assay for soluble neprilysin

Oi Wah Liew; Timothy C R Prickett; Elena Revuelta-López; Samantha S M Ling; Adriana Cserkóová; Josep Lupón; Jessica Y X Ng; Jenny P C Chong; Shera Lilyanna; Siew-Pang Chan; Qifeng Lin; Teck K Lim; Qingsong Lin; Jaume Barallat; Antoni Bayés-Genís; A Mark Richards

doi:10.1016/j.clinbiochem.2022.03.004

Finding a reliable assay for soluble neprilysin

Clin Biochem. 2022 Jun:104:51-58. doi: 10.1016/j.clinbiochem.2022.03.004. Epub 2022 Mar 21.

Authors

Oi Wah Liew¹, Timothy C R Prickett², Elena Revuelta-López³, Samantha S M Ling¹, Adriana Cserkóová⁴, Josep Lupón⁵, Jessica Y X Ng¹, Jenny P C Chong¹, Shera Lilyanna¹, Siew-Pang Chan¹, Qifeng Lin⁶, Teck K Lim⁶, Qingsong Lin⁶, Jaume Barallat⁷, Antoni Bayés-Genís⁵, A Mark Richards⁸

Affiliations

¹ Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Cardiovascular Research Institute, National University Health System, Singapore.
² Christchurch Heart Institute, University of Otago, New Zealand. Electronic address: tim.prickett@otago.ac.nz.
³ Heart Failure and Cardiac Regeneration (ICREC) Research Program, Health Science Research Institute Germans Trias i Pujol (IGTP), Badalona, Spain; Centro de Investigación Biomédica en Red Enfermedades Cardiovasculares (CIBERCV), Spain.
⁴ Heart Failure and Cardiac Regeneration (ICREC) Research Program, Health Science Research Institute Germans Trias i Pujol (IGTP), Badalona, Spain.
⁵ Centro de Investigación Biomédica en Red Enfermedades Cardiovasculares (CIBERCV), Spain; Heart Failure Clinic, Cardiology Service, Hospital Universitari Germans Trias i Pujol Badalona, Barcelona, Spain.
⁶ Department of Biological Sciences, National University of Singapore, Singapore.
⁷ Biochemistry Service, Hospital Universitari Germans Trias I Pujol, Badalona, Spain.
⁸ Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Christchurch Heart Institute, University of Otago, New Zealand; Cardiovascular Research Institute, National University Health System, Singapore.

PMID: 35331754
DOI: 10.1016/j.clinbiochem.2022.03.004

Abstract

Background: Lack of validation and standardization of research-use-only (RUO) immunoassays brings with it inherent threats to authenticity and functional quality. Poor correlation between different commercial neprilysin RUO immunoassays is concerning and discordant findings need to be resolved. We seek to identify and validate reliable neprilysin immunoassays to strengthen the scientific rigor and reproducibility of neprilysin-related investigation and of biomarker research in general.

Methods: Soluble neprilysin (sNEP) concentrations were determined in cohorts (n = 532) from Spain (Cohort 1), New Zealand (NZ, Cohort 2) and Singapore (Cohort 3), using commercial kits from six vendors. Apparent sNEP concentrations were correlated between different assays and with plasma neprilysin activity. Assay reliability was further validated by performance verification, MS analysis and cross-reactivity tests.

Results: sNEP in Cohorts 1 and 2 measured concurrently in Spain and NZ showed significant inter-laboratory correlation only for the Aviscera Bioscience sNEP ELISA SK00724-01. Neprilysin concentrations obtained with the R&D systems and SK00724-01 ELISAs correlated with each other but not with neprilysin activity. In Cohort 3, sNEP concentrations from the Perkin Elmer AlphaLISA and Biotechne ELLA assays agreed (r = 0.89) and both correlated with neprilysin activity (r = 0.87, 0.77 respectively). MS analysis detected authentic neprilysin in the AlphaLISA kit calibrator and in antibody pull-down material from human plasma. The AlphaLISA assay performed within acceptable limits (spike and recovery, dilutional linearity, inter- and intra-assay CV) and showed no cross-reactivity against neprilysin substrates and closely-related analogues.

Conclusion: AlphaLISA and ELLA assays provide reliable measures of sNEP concentrations. Reliability of other commercial neprilysin assays remains in question.

Keywords: Biomarker research; Commercial research-use-only (RUO) tests; Heart failure; Neprilysin; sNEP.

MeSH terms

Biomarkers
Enzyme-Linked Immunosorbent Assay
Heart Failure*
Humans
Neprilysin*
Reproducibility of Results
Spain

Substances

Biomarkers
Neprilysin