[1]王静静,邓新立,龚美亮,等.基于抗复合物抗体非竞争免疫夹心法检测血清25（OH）D水平的方法学评价[J].现代检验医学杂志,2025,40(06):190-195,200.[doi:10.3969/j.issn.1671-7414.2025.06.035]
　WANG Jingjing,DENG Xinli,GONG Meiliang,et al.Evaluation of A Non-competitive Immunosandwich Method for Detecting Serum 25(OH)D on the Basis of Anti-immunocomplex Antibodies[J].Journal of Modern Laboratory Medicine,2025,40(06):190-195,200.[doi:10.3969/j.issn.1671-7414.2025.06.035]
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基于抗复合物抗体非竞争免疫夹心法检测血清25（OH）D水平的方法学评价()

《现代检验医学杂志》[ISSN:/CN:]

卷:
第40卷
期数:
2025年06期
页码:
190-195,200
栏目:
论著
出版日期:
2025-11-15

文章信息/Info

Title:
Evaluation of A Non-competitive Immunosandwich Method for Detecting Serum 25(OH)D on the Basis of Anti-immunocomplex Antibodies
文章编号:
1671-7414（2025）06-190-06
作者:
王静静a，邓新立a，龚美亮a，邹 琳b
解放军总医院第二医学中心 a.检验科；b.保健一科，北京100853
Author(s):
WANG Jingjinga，DENG Xinlia，GONG Meilianga，ZOU Linb
a. Department of Clinical Laboratory；b. Department of Health Care，the Second Medical Center，Chinese PLA General Hospital，Beijing 100853，China
关键词:
25-羟基维生素D; 性能验证; 非竞争免疫夹心法; 液相色谱-串联质谱法
分类号:
R446.61
DOI:
10.3969/j.issn.1671-7414.2025.06.035
文献标志码:
A
摘要:
目的对基于抗复合物抗体的非竞争免疫夹心法（比对方法）检测25-羟基维生素D[25(OH)D]进行性能验证基础上，比较其与参比方法，即液相色谱-串联质谱法（LC-MS/MS）检测结果的一致性。方法依据中国合格评定国家认可委员会（CNAS）-GL037等文件对比对方法检测25(OH)D进行精密度、线性区间、定量下限、正确度验证。收集并检测2021年3～7月解放军总医院第二医学中心健康查体剩余血清样本1073份，评价两种方法检测25(OH)D的相关性（Passing-Bablok回归）、一致性相关系数（CCC）和偏差（Bland-Altman图），同时验证两种方法用于临床判定维生素D营养状况的符合程度（加权Kappa检验）。结果比对方法精密度：批内（重复性）CV<5%，实验室内CV<10%；在2.88～52.45ng/ml区间内线性关系良好；定量下限(LOQ)为2.26ng/ml。比对方法检测标准物质偏倚在±6%以内，检测维生素D外部质量评估计划（DEQAS）定值质控品偏倚在±15%以内，通过正确度验证标准。比对方法与参比方法的相关性和一致性均较好（r=0.965，CCC=0.946）；检测25(OH)D平均偏差为-1.54ng/ml(-7.42%)。两种方法判断维生素D营养状况的加权Kappa=0.801，比对方法判断维生素D正常、不足、缺乏与参比方法的总符合率为85.96%（918/1068）。结论两种方法检测25(OH)D的相关性和符合度好，比对方法检测性能满足临床需求。
Abstract:
Objective To verify the performance of an non competitive immune sandwich method (candidate method) in detecting 25(OH)D，and evaluate the measurement agreement of candidate method with liquid chromatography tandem mass spectrometry (LC-MS/MS, reference method). Methods According to the comparison of documents such as China National Accreditation Service for Conformity Assessment (CNAS)-GL037, the precision, linear interval, lower limit of quantitation (LOQ) and accuracy of the method for detecting 25(OH)D were verified. 1 073 residual serum samples were collected from March to July 2021 and 25(OH)D was detected by the two methods. Passing-bablok regression, concordance correlation coefficient (CCC) , Bland altman plots and Kappa test were used to analyze the consistency and bias. Weighted Kappa test was conducted to evaluate the consistency of two methods for clinical assessment of vitamin D nutritional status. Results The performance of candidate method：the within-run CV was lower than 5% and the in laboratory CV were lower than 10%；test results between 2.88～52.45 ng/ml showed good linear relationship；the lower limit of quantitation was 2.26 ng/ml，the bias of candidate method measuring standard reference materials were within ± 6% and within 15% measuring vitamin D external quality assessment scheme (DEQAS) samples. The comparison method and the reference method showed good correlation (r=0.965, CCC=0.946) with an average deviation of -1.54 ng/ml (-7.42%). The two methods coincidence rate in judging vitamin D status was 85.96% (918/1 068), and the weighted kappa value was 0.801. Conclusions The candidate method measurement performance met the clinical requirements, and showed good correlation and consistency with the reference method.

参考文献/References:

[1] LI J , ZHANG H Y, ZHU H D, et al. 25-hydroxyvitamin D concentration is positively associated with overall survival in advanced pancreatic cancer: A systematic review and me-ta-analysis [J]. Nutrition Research, 2023, 117: 73-82.
[2] 李杨亮,沈瑞,朱亮.老年急性心肌梗死患者血清载脂蛋白A、脂蛋白(a)、25(OH)D水平对主要不良心血管事件发生的预测价值[J].中国老年学杂志,2023,43(7):1549-1552. LI Y L, SHEN R , ZHU L . The predictive value of se-rum levels of apolipoprotein A, lipoprotein (a), and 25 (OH) D for major adverse cardiovascular events in el-derly patients with acute myocardial infarction[J]. Chi-nese Journal of Gerontology, 2023, 43(7): 1549-1552.
[3] 于跑,张沛,高春林,等.血清25-羟基维生素D3水平在IgA血管炎肾炎患儿中临床病理及预后意义[J].中国当代儿科杂志,2025,27(1):55-61. YU P , ZHANG P , GAO C L, et al. Clinicopathologi-cal significance and prognostic value of serum 25-hy-droxyvitamin D3 level in children with IgA vasculitis nephritis [J]. Chinese Journal of Contemporary Pediat-rics, 2025, 27(1): 55-61.
[4] 王坤英,张蓬杰,王剑茹,等.肾移植受者早期血清25-羟基维生素D水平对急性排斥反应的预测价值研究[J].现代检验医学杂志,2024,39(4):138-142. WANG K Y, ZHANG P J, WANG J R, et al. Study on the predictive value of serum 25-hydroxyvitamin D level in early renal transplantation for acute rejection[J]. Journal of Modern Laboratory Medicine, 2024, 39(4): 138-142.
[5] BAI Y C, WANG Y H, LI Q , et al. Binding affini-ty-guided design of a highly sensitive noncompetitive immunoassay for small molecule detection [J]. Food Chemistry, 2021, 351: 129270.
[6] 李海侠,龚美亮,邓新立,等.中国老年人维生素D水平多中心调查分析[J].中华检验医学杂志, 2021, 44(1): 39-44. LI H X, GONG M L, DENG X L, et al. Multicenter survey and analysis of vitamin D level in elderly people in China[J]. Chinese Journal of Laboratory Medicine, 2021, 44(1): 39-44.
[7] NIKOOYEH B, SAMIEE S M, FARZAMI M R, et al. Harmonization of serum 25-hydroxycalciferol assay re-sults from high-performance liquid chromatography, en-zyme immunoassay, radioimmunoassay, and immunoche-miluminescence systems: a multicenter study [J]. Journal of Clinical Laboratory Analysis, 2017, 31(6): e22117.
[8] 孔卓,李春艳.自动化磁珠法提取血清脂溶性维生素应用LC-MS/MS检测的性能评价[J].现代检验医学杂志,2024,39(1):131-135, 145. KONG Z , LI C Y. Performance evaluation of automated magnetic beads extraction method for extraction of serum fat-soluble vitamins by LC-MS/MS detection[J]. Journal of Modern Laboratory Medicine, 2024, 39(1): 131-135, 145.
[9] OMI K, ANDO T, SAKYU T, et al. Noncompetitive immuno-assay detection system for haptens on the basis of antimetatype antibodies[J]. Clinical Chemistry, 2015, 61(4): 627-635.
[10] WISE S A, CAMARA J E, BURDETTE C Q, et al. Interlabo-ratory comparison of 25-hydroxyvitamin D assays: vitamin D standardization program (VDSP) intercomparison study 2-Part 2 ligand binding assays -impact of 25-hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance[J]. Ana-lytical and Bioanalytical Chemistry, 2022, 414(1): 351-366.
[11] 中华人民共和国国家卫生和计划生育委员会. WS/T492-2016:临床检验定量测定项目精密度与正确度性能验证［EB/OL］.(2016-07-07)［2016-12-15］.https://www.nhc.gov. cn/wjw/s9492/201607/da37e5ec965a431d94f435b192dabe72/files/1739781647690_98720.pdf. National Health and Family Planning Commis-sion of the People’s Republic of China.WS/T492 2016:Performance verification of precision and correctness of quantitative determination items in clinical laboratory［EB/OL］.(2016 07-07)[2016 12 15].https://www.nhc.gov.cn/wjw/s9492/201607/da37e5ec965a431d94f435b192dabe72/files/1739781647690_98720.pdf.
[12] 中国合格评定国家认可委员会. CNAS-GL037:2019:临床化学定量检验程序性能验证指南[EB/OL].(2019-02-15)［2021-10-28］. https://www.cnas.org.cn/rkgf/sysrk/rkzn/art/2024/art_5e28b38d0c334c2a95429922ac5e87d0.html。China National Accreditation Service for Conformity Assessment . CNAS-GL037:2019:Guidance on the veri-fication of quantitative measurement procedures used in the clinical chemistry[EB/OL].(2019-02-15)[2021-10-28]. https://www.cnas.org.cn/rkgf/sysrk/rkzn/art/2024/art_5e28b38d0c334c2a95429922ac5e87d0.html.
[13] 中华人民共和国卫生部.WS/T 408-2012:临床化学设备线性评价指南[EB/OL].(2012-12-25)[2013-08-01]. https://www.nhc.gov.cn/wjw/s9492/201301/fb8630de9bf-04f389a1af97edb373a1b/files/1739781579701_72958.pdf. Ministry of Health of the People’s Republic of China.WS/T 408-2012:Guide to linearity evaluation of clinical chemistry analyzers[EB/OL].(2012-12-25)［2013-08-01］. https://www. nhc.gov.cn/wjw/s9492/201301/fb8630de9bf04f389a1af97ed-b373a1b/files/1739781579701_72958.pdf.
[14] 中华人民共和国国家卫生健康委员会.WS/T 677-2020:人群维生素D缺乏筛查方法[EB/OL].(2020-05-06)［2020-11-01］.https://www.nhc.gov.cn/wjw/ying-yang/202005/4edc890001cd4041bec5108bee468d66/files/1739783560802_56160.pdf. National Health Commission of the People’s Republic of China.WS/T 677 2020:Method for vitamin D deficiency screening[EB/OL].(2020-05-06)［2020-11-01］.https://www. nhc.gov.cn/wjw/yingyang/202005/4edc890001cd4041bec-5108bee468d66/files/1739783560802_56160.pdf.
[15] Clinical and and Laboratory Standards Institute.CLSI EP9-A2:Method comparison and bias estiomation using patients samples:Approved guideline:second Edition [S]. Wayne:PA, CLSI EP9-A2,2002.
[16] 宋旖川,徐安平,王默,等.三种方法检测糖化血红蛋白结果的一致性评价[J].中华检验医学杂志,2024,47(10):1197-1205. SONG Y C, XU A P, WANG M , et al. Comparison of different methods for HbA1c measurement[J]. Chinese Journal of Laboratory Medicine, 2024, 47(10): 1197-1205.
[17] ALTMAN D G.Practical statistics for medical research [M]. Virginia Beach: Chapman and Hall/CRC Press, 1990.
[18] GALIOR K, KETHA H, GREBE S, et al. 10 years of 25-hy-droxyvitamin-D testing by LC-MS/MS-trends in vitamin-D deficiency and sufficiency [J]. Bone Reports, 2018, 8: 268-273.
[19] 禹松林,方慧玲,程歆琦,等.五种自动化免疫学方法和液相色谱串联质谱方法测定25羟维生素 D 的比较[J].中华检验医学杂志,2015,38(7):475-479. YU S L, FANG H L, CHENG X Q, et al. Comparison of five automated 25OHD immunoassays and liquid chroma-tography tandem mass spectrometry method[J]. Chinese Journal of Laboratory Medicine, 2015, 38(7): 475-479.
[20] 张文杰,李鹏飞.用液相色谱-串联质谱法测定人血清中25-羟基维生素D的浓度及临床应用[J].中国临床药理学杂志,2023,39(12):1787-1791. ZHANG W J, LI P F. Determination of 25-hydroxyvitamin D in serum by liquid chromatography-tandem mass spectrom-etry and its clinical application[J]. The Chinese Journal of Clinical Pharmacology, 2023, 39(12): 1787-1791.
[21] 邹迎曙,王军,孙京昇.10种市售总25-羟基维生素D免疫检测系统测定偏差评价[J].中华检验医学杂志,2017,40(4):320-325. ZOU Y S, WANG J , SUN J S. Detection bias evaluation of ten commercial total 25-hydroxyvitamin D immunoassays[J]. Chi-nese Journal of Laboratory Medicine, 2017, 40(4): 320-325.
[22] LIU A P, ANFOSSI L , SHEN L ,et al. Non-competitive im-munoassay for low-molecular-weight contaminant detection in food, feed and agricultural products: A mini-review[J]. Trends in Food Science & Technology, 2017, 71:181-187.
[23] 龚美亮,王科宇,陈瑞,等.一种免疫分析系统与液相色谱-串联质谱检测25-羟维生素D的一致性评价[J].中华检验医学杂志,2021,44(7):621-626. GONG M L, WANG K Y, CHEN R , et al. Measure-ment agreement of 25-hydroxyvitamin D results de-rived from Roche immunoassay and liquid chromatog-raphy tandem mass spectrometry[J]. Chinese Journal of Laboratory Medicine, 2021, 44(7): 621-626.
[24] 龚美亮,李晓霞,陈瑞,等.免疫分析系统与液相色谱串联质谱检测25-羟基维生素D的一致性评价[J].中国医学装备,2021,18(5):30-33. GONG M L, LI X X, CHEN R , et al. Consistency evaluation between immunoassay system and LC-MS/MS in detecting 25-(OH)-D[J]. China Medical Equipment, 2021, 18(5): 30-33.
[25] LI L S, ZENG Q , YUAN J J, et al. Performance evaluation of two immunoassays for 25-hydroxyvitamin D[J]. Journal of Clinical Biochemistry and Nutrition, 2016, 58(3): 186-192.
[26] PUM J. A practical guide to validation and verification of analytical methods in the clinical laboratory [J]. Ad-vances in Clinical Chemistry, 2019: 90: 215-281.
[27] TRIPATHI A, ANSARI M, DANDEKAR P, et al. Analytical methods for 25-hydroxyvitamin D:advantages and limitations of the existing assays [J].The Journal of Nucritional Biochemistry,2022,109:109123.

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备注/Memo

备注/Memo:
基金项目：军委后勤保障部卫生局项目（编号：17BJZ45）。
作者简介：王静静（1992-），女，本科，技师，研究方向：临床检验医学，E-mail：axiaoyoua@163.com。
通讯作者：邹琳（1975-），女，博士，副主任医师，研究方向：老年医学，E-mail：meryl301@aliyun.com。
更新日期/Last Update: 2025-11-15