Development and validation of an immunoassay for quantification of NCAM-1 in human plasma

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 197; p. 113981
• Main Authors: Guven, Arcan, Gray, Kayleigh, Peng, Kuan-Wei, Klotz, Allison, Kellogg, Mark D., Narain, Niven R., Kiebish, Michael A.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15-04-2021
• Subjects: Assay development; Assay validation; Biomarker; Biomarkers; Calibration; CD56 Antigen; Humans; Immunoassay; Ligand binding assay; NCAM-1; Neural Cell Adhesion Molecules; Quality Control; Immunoassay; Biomarker; Assay development; Ligand binding assay; Assay validation; NCAM-1
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2021.113981

•A ligand binding assay was developed for quantification of NCAM-1 in plasma.•Validated method met prespecified criteria in the range of 62.5–4000.0 ng/mL.•NCAM-1 assay has potential to be used as a biomarker assay.•Clinical testing of human plasma shows NCAM-1 assay is not influenced by age.
Neural Cell Adhesion Molecule 1 (NCAM-1), a multifunctional member of the immunoglobulin superfamily, is expressed on the surface of neurons, glia, skeletal muscle, and natural killer cells. NCAM-1 has been implicated as having a role in cell-cell adhesion, involved in development of the nervous system, and for cells involved in the expansion of T cells and dendritic cells which play an important role in immune surveillance. Sensitive and specific methods to quantify non-surface bound NCAM-1 are not available.
A sandwich ligand binding assay was developed for quantification of NCAM-1 in plasma and validated using an electro-chemiluminescent (ECL) technology.
The data presented here demonstrated that the validated method met all prespecified criteria for precision, linearity, and accuracy in the range of 62.5 ng/mL to 4000.0 ng/mL, the range believed to be most relevant for plasma. The bioanalytical validation of the assay established the inter-assay coefficient of variation <8 % for calibration points, <2 % for high quality control (HQC), <8 % for medium quality control (MQC) and <19 % for low quality control (LQC) samples. Purified NCAM-1 spike-recovery experiment in plasma was used to determine assay accuracy; nominal concentrations (%) of NCAM-1 ranged from 91 % to 112 % for high and low spike level, respectively. Assay performance was subsequently evaluated for parallelism, selectivity, interference, and stability.
NCAM-1 assay has been developed and validated in human plasma and met all assay validation parameters pre-determined during development. Clinical testing of human plasma samples indicated that NCAM-1 does not seem to be influenced by age and was slightly influenced by gender. NCAM-1 assay has potential to be used as a biomarker assay once the assay is subjected to appropriate clinical assessment and diagnostic thresholds are established.