开发和验证用于人血浆中 NCAM-1 定量的免疫分析方法。

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

机构信息

BERG, 500 Old Connecticut Path, Framingham, MA, 01701, USA.

出版信息

J Pharm Biomed Anal. 2021 Apr 15;197:113981. doi: 10.1016/j.jpba.2021.113981. Epub 2021 Feb 16.

DOI:10.1016/j.jpba.2021.113981

PMID:33657526

Abstract

BACKGROUND

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.

METHOD

A sandwich ligand binding assay was developed for quantification of NCAM-1 in plasma and validated using an electro-chemiluminescent (ECL) technology.

RESULTS

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.

CONCLUSION

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.

摘要

背景

神经细胞黏附分子 1（NCAM-1）是免疫球蛋白超家族的多功能成员，表达于神经元、神经胶质细胞、骨骼肌和自然杀伤细胞的表面。NCAM-1 被认为在细胞-细胞黏附中发挥作用，参与神经系统的发育，并参与 T 细胞和树突状细胞的扩增，这些细胞在免疫监视中发挥重要作用。目前尚无定量检测非表面结合的 NCAM-1 的敏感和特异方法。

方法

建立了用于定量检测血浆中 NCAM-1 的夹心配体结合测定法，并使用电化学发光（ECL）技术进行了验证。

结果

本文数据表明，该验证方法在 62.5ng/mL 至 4000.0ng/mL 的范围内满足精密度、线性和准确度的所有预设标准，该范围被认为与血浆最相关。该测定的生物分析验证确立了批内变异系数（CV）<8%，校准点<2%，高质量控（HQC）<8%，中质量控（MQC）<8%和低质量控（LQC）<19%。使用血浆中 NCAM-1 的纯化加标回收率实验来确定测定的准确度；高、低加标水平的 NCAM-1 名义浓度（%）分别为 91%至 112%。随后评估了测定的平行性、选择性、干扰和稳定性。