[1]谭玉华,曹春玲,张润锋,等.时间分辨荧光免疫法检测胎盘生长因子的方法建立及性能评价[J].现代检验医学杂志,2023,38(01):112-116+146.[doi:10.3969/j.issn.1671-7414.2023.01.021]
　TAN Yu-hua,CAO Chun-ling,ZHANG Run-feng,et al.Establishment and Performance Evaluation of A Time-resolved Fluorescence Immunoassay for the Detection of Placental Growth Factor[J].Journal of Modern Laboratory Medicine,2023,38(01):112-116+146.[doi:10.3969/j.issn.1671-7414.2023.01.021]
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时间分辨荧光免疫法检测胎盘生长因子的方法建立及性能评价()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年01期
页码:
112-116+146
栏目:
论著
出版日期:
2023-01-15

文章信息/Info

Title:
Establishment and Performance Evaluation of A Time-resolved Fluorescence Immunoassay for the Detection of Placental Growth Factor
文章编号:
1671-7414（2023）01-112-06
作者:
谭玉华1; 曹春玲2; 张润锋2; 潘晓芳2; 李高成1; 余海枷1; 梁天铖1; 冯健明1
（1. 广州市丰华生物工程有限公司体外诊断试剂研发中心，广州 510730；2. 广东省医疗器械质量监督检验所综合检验四室，广东中山 528437）
Author(s):
TAN Yu-hua1; CAO Chun-ling2; ZHANG Run-feng2; PAN Xiao-fang2; LI Gao-cheng1; YU Hai-jia1;  LIANG Tian-cheng1; FENG Jian-ming1
（1.R & D Center of IVD Reagents, Guangzhou Fenghua Bioengineering Co. Ltd, Guangzhou 510730, China； 2.Comprehensive Inspection Room 4, Guangdong Medical Device Quality Supervision and Inspection Institute, Guangdong Zhongshan 528437，China）
关键词:
时间分辨荧光免疫法; 胎盘生长因子; 性能评价
分类号:
R373.19；R446
DOI:
10.3969/j.issn.1671-7414.2023.01.021
文献标志码:
A
摘要:
目的 建立检测胎盘生长因子（placental growth factor, PLGF) 的时间分辨荧光免疫分析法（time-resolvedfluoroimmunoassay，TRFIA），并对其性能进行评价。方法 利用捕获 PLGF 单克隆抗体包被微孔板，铕标记检测 PLGF单克隆抗体，建立一种双抗体夹心TRFIA 定量测定孕妇血清中的 PLGF 浓度。对该方法的检测低限、生物检测限、功能灵敏度、精密度、线性、干扰试验、交叉反应试验和HOOK 效应等性能指标进行评价。选择125 例孕期在9 ～ 40周，无溶血、黄疸和脂血的孕妇血清剩余样本，用于方法学比对研究，比对试验结果的相关性采用线性回归分析。结果 该方法的捕获抗体包被浓度为7.0 μg/ml，铕标记物使用工作稀释度为1:500，样本最适反应时间为90 min，检测低限为1.00 pg/ml，生物检测限为8.00 pg/ml，功能灵敏度为10.00 pg/ml，批内CV 和批间CV 均在5.00% 以内，线性范围为9.00 ～ 10 500.00 pg/ml，分别在低浓度和高浓度质控品中添加有干扰物质的16 种干扰样本与基础样本检测结果的相对偏倚在-3.49% ～ 2.20% 内；检测5 000 pg/ml 糖基化 PLGF-1，5 000 pg/ml 糖基化 PLGF-2，5 000 pg/ml 未糖基化PLGF-3，10 000 pg/ml 未糖基化血管内皮生长因子(vascular endothelial growth factor，VEGF)/ PLGF-1 异二聚体，50 000pg/ml 糖基化VEGF165 和40 000 pg/ml 可溶性FMS 样酪氨酸激酶-1，交叉反应率分别为41.82%，27.86%，19.68%，0.042%，0.063% 和0.004 5%；检测样品 PLGF 浓度高达115 000 pg/ml 时仍未出现HOOK 效应；样本 PLGF 浓度在5.74 ～4 197.00 pg/ml 间，与电化学发光法（electrochemiluminescence，ECL）检测结果的线性回归方程为Y=1.070 9X-30.192，（r=0.980 6, tr=55.42，P ＜ 0.05）。结论 该方法灵敏度高、精密度好、线性范围宽、抗干扰能力强、特异度好、检测范围宽，与参比方法检测结果的相关性良好，可以满足临床需要。
Abstract:
Objective To establish a time-resolved fluoroimmunoassay (TRFIA) for the detection of placental growth factor (PLGF) and evaluate its performance. Methods A double-antibody sandwich TRFIA was established to quantitatively determine the concentration of PLGF in serum of pregnant women，which based on the microplate was coated with the monoclonal antibody to capture PLGF, and the detection of PLGF monoclonal antibody was labeled by europium. The performance indicators such as lower limit of detection, biological limit of detection, functional sensitivity, precision, linearity, interference test, cross-reaction test and HOOK effect of the method were evaluated. A total of 125 remaining serum samples from pregnant women with no hemolysis, jaundice and lipemia between 9 to 40 weeks of gestation were selected for methodological comparison study, and the correlation of the comparison test results was analyzed by linear regression. Results The concentration of capture antibody was 7.0 μg/ml .The working dilution of europium label was 1:500.The optimal reaction time of the sample was 90 min. The lower limit of detection was 1.00 pg/ml. The biological limit of detection was 8.00 pg/ml.The functional sensitivity was 10.00 pg/ml. The intra-assay CV and inter-assay CV were both within 5%. The linear range was 9.00 to 10 500.00 pg/ml. The relative bias of the detection results of the 16 interfering samples with interfering substances added to the low-concentration and high-concentration quality controls and the basic samples was within -3.49% to 2.20%. When detected 5 000 pg/ml glycosylated PLGF-1, 5 000 pg/ml glycosylated PLGF-2, 5 000 pg/ml unglycosylated PLGF-3, 10 000 pg/ml unglycosylated vascular endothelial growth factor (VEGF)/ PLGF-1 heterodimer, 50 000 pg/ml glycosylated VEGF165 and 40 000 pg/ml soluble FMS-like tyrosine Kinase-1 the cross-reaction rate(%) was 41.82%，27.86%，19.68%，0.042%，0.063% and 0.004 5%, respectively. The HOOK effect was not found in PLGF samples up to 115 000 pg/ml. When the concentration of PLGF in the sample was 5.74 to 4 197.00 pg/ml, the linear regression equation of TRFIA and electrochemiluminescence（ECL） detection results was Y=1.070 9X -30.192（r=0.980 6, tr=55.42，P ＜ 0.05）. Conclusion The quantitative detection of PLGF by TRFIA has high sensitivity, good precision, wide linear range, strong anti-interference ability, good specificity, wide detection range, and good correlation with the detection results of reference methods, which is valuable for clinical application.

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备注/Memo

备注/Memo:
作者简介： 谭玉华（1980-），男，医学硕士，临床医学检验技师，医疗器械工程师，二级企业培训师，三级健康管理师，研究方向：医疗器械（含体外诊断试剂）的研发、注册、应用与医学检验，E-mail: tanywhy@aliyun.com。
更新日期/Last Update: 2023-01-15