Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum

Anand-Ivell, Ravinder; Frederiksen, Hanne; Juul, Anders; Andersson, Anna-Maria; Albrethsen, Jacob; Nordkap, Loa; Kirstine Bang, Anne; Jørgensen, Niels

Abstract

BACKGROUND:
The circulating level of the peptide hormone insulin-like factor 3 (INSL3) is a promising diagnostic marker reflecting Leydig cell function in the male. Few commercial immunoassays of varying quality exist. Therefore, we decided to develop and validate a precise method for quantification of INSL3 by mass spectrometry.

METHODS:
We developed an assay in which the INSL3 A-chain is released from the INSL3 A-B heterodimer by chemical reduction and alkylation. The alkylated INSL3 A-chain is quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as substitute for serum INSL3. The method was compared to a validated and sensitive in-house serum INSL3 immunoassay using 97 serum samples from 12 healthy boys during pubertal transition. Adult levels were determined based on sera from 72 adult healthy males aged 18-40 years.

RESULTS:
An LC-MS/MS assay with limit of detection and limit of quantification (LOQ) of 0.06 and 0.15 ng/mL, respectively, and intra-assay CVs <9% in the relevant ranges was obtained. The LC-MS/MS compared well with the in-house immunoassay (Deming regression slope: 1.28; Pearson correlation: R=0.86). INSL3 concentrations increased with pubertal maturation in healthy boys. INSL3 concentrations were above the LOQ in all samples from the adult men. The mean (±2 SD range)for serum INSL3 concentrations in the adult men was 2.2 (0.5-3.9) ng/mL.

CONCLUSIONS:
We have developed a robust and sensitive method suitable for quantitation of serum INSL3 in a clinical setting using LC-MS/MS instrumentation available in modern clinical laboratories. The method paves the way for future studies into the clinical role of serum INSL3 measurements.

Citation

Anand-Ivell, R., Frederiksen, H., Juul, A., Andersson, A.-M., Albrethsen, J., Nordkap, L., Kirstine Bang, A., & Jørgensen, N. (2018). Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum. Clinical Chemistry and Laboratory Medicine, 56(11), https://doi.org/10.1515/cclm-2018-0171

• Journal Article Type: Article
• Acceptance Date: May 3, 2018
• Online Publication Date: May 30, 2018
• Publication Date: May 30, 2018
• Deposit Date: Jun 1, 2018
• Publicly Available Date: May 31, 2019
• Journal: Clinical Chemistry and Laboratory Medicine
• Print ISSN: 1434-6621
• Electronic ISSN: 1437-4331
• Publisher: De Gruyter
• Peer Reviewed: Peer Reviewed
• Volume: 56
• Issue: 11
• DOI: https://doi.org/10.1515/cclm-2018-0171
• Keywords: assay development; insulin-like factor 3 (INSL3); mass spectrometry
• Public URL: https://nottingham-repository.worktribe.com/output/934420
• Publisher URL: https://www.degruyter.com/view/j/cclm.2018.56.issue-11/cclm-2018-0171/cclm-2018-0171.xml
• Contract Date: Jun 1, 2018