胰高血糖素样肽1定量ELISA方法的建立及初步临床应用

doi:10.3969/j.issn.1673-8640.2015.08.015

摘要/Abstract

摘要：

目的建立定量检测血清中胰高血糖素样肽1(GLP-1) 酶联免疫吸附试验(ELISA),并探讨其在2型糖尿病(T2DM)患者个体化治疗中的应用。方法以多克隆抗体作为捕获抗体,另一单克隆抗体作为标记抗体和系列稀释的标准液,建立能够定量检测血清中GLP-1总含量的ELISA,并初步确立生物参考区间。将研究对象根据糖代谢情况分为正常糖耐量(NGT)组(50例)、糖耐量受损(IGT)组(52例)和T2DM组(68例),分别测定空腹、服糖后1 h、服糖后2 h血清中GLP-1含量的动态变化,结合其它常规生化指标,评价GLP-1检测在T2DM患者治疗中的临床应用价值。结果所建立的方法在23.5~1 490 nmol/L范围内具有良好的定量关系(R²=0.999, P<0.05);初步建立成人生物参考区间为(79.5±15.6)nmol/L。T2DM组GLP-1浓度明显高于NGT组和IGT组(P<0.05)。动态监测研究对象糖耐量试验中血清GLP-1浓度变化,在IGT组和T2DM组出现服糖后GLP-1分泌异常不足的情况。结论成功建立了定量检测血清GLP-1的ELISA,可以动态监测IGT、T2MD患者体内GLP-1的分泌状况以指导临床个体化治疗。

关键词: 胰高血糖素样肽1, 酶联免疫吸附试验, 2型糖尿病, 糖耐量受损

Abstract:

Objective To establish an enzyme-linked immunosorbent assay (ELISA) for the quantitative measuring of glucagon-like peptide 1(GLP-1) in serum and to investigate the application in the individualized treatment of type 2 diabetes mellitus (T2DM) patients. Methods Using a polyclonal antibody as capture antibody, another monoclonal antibody as labeled antibody, serial dilutions as standard material, an ELISA was established for the quantitative measuring of GLP-1 in serum, and the biological reference interval was also established. All the subjects were classified into 3 groups, normal glucose tolerance (NGT) group (50 cases), impaired glucose tolerance (IGT) group (52 cases) and T2DM group (68 cases). The blood samples were collected at fasting time, 1 h after taking glucose and 2 h after taking glucose. The dynamic change of GLP-1 was evaluated with routine biological items, in order to evaluate the clinical application significance of GLP-1 for the treatment of T2DM. Results The established method had an good quantitative correlation in the range of 23.5-1 490 nmol/L(R²=0.999, P<0.05). The biological reference interval for adults was (79.5±15.6) nmol/L. The GLP-1 in T2DM group was higher than those in NGT and IGT groups (P<0.05). According to the dynamic change of GLP-1 concentration in the oral glucose tolerance test, the deficient secretion of GLP-1 after taking glucose among patients in IGT and T2DM groups could be concluded. Conclusions The ELISA for the quantitative measuring of GLP-1 in serum is successfully established. This method could be used to dynamically monitor the secretion of GLP-1 in IGT and T2DM patients and would be helpful for the individualized treatment of T2DM.

Key words: Glucagon-like peptide 1, Enzyme-linked immunosorbent assay, Type 2 diabetes mellitus, Impaired glucose tolerance

中图分类号:

R446.61

孙立山, 汪明东, 陆柳, 刘颖冰, 鹿麟, 郭金虎, 范列英. 胰高血糖素样肽1定量ELISA方法的建立及初步临床应用[J]. 检验医学, 2015, 30(8): 835-840.

SUN Lishan, WANG Mingdong, LU Liu, LIU Yingbing, LU Lin, GUO Jinhu, FAN Lieying. Establishment of an ELISA for the quantitative measuring of glucagon-like peptide 1 and the primary clinical application[J]. Laboratory Medicine, 2015, 30(8): 835-840.

图/表 6

表1 连续5 d标准液检测结果(A值)

项目	GLP-1标准液浓度(nmol/L)
项目	1 490	745	373	186	93	47	23.5	0
$x ?$ (n=10)	2.831	1.662	0.971	0.580	0.347	0.204	0.088	0.029
SD	0.124	0.104	0.078	0.045	0.027	0.022	0.015	0.009
99% 可信区间	2.459~3.023	1.351~1.973	0.736~1.206	0.444~0.710	0.264~0.429	0.136~0.271	0.043~0.133	0.002~0.056
CV(%)	4.38	6.24	8.07	7.76	7.81	11.02	17.00	31.50

表1 连续5 d标准液检测结果(A值)

项目	GLP-1标准液浓度(nmol/L)
项目	1 490	745	373	186	93	47	23.5	0
$x ?$ (n=10)	2.831	1.662	0.971	0.580	0.347	0.204	0.088	0.029
SD	0.124	0.104	0.078	0.045	0.027	0.022	0.015	0.009
99% 可信区间	2.459~3.023	1.351~1.973	0.736~1.206	0.444~0.710	0.264~0.429	0.136~0.271	0.043~0.133	0.002~0.056
CV(%)	4.38	6.24	8.07	7.76	7.81	11.02	17.00	31.50

图1 总GLP-1定量ELISA检测标准曲线

图2 120名健康人空腹血清GLP-1浓度分布

表2 血清总GLP-1 ELISA精密度试验结果

样本	检测次数	血清总GLP-1(nmol/L)		CV (%)
样本	检测次数	批内	批间	批内	批间
健康人	20	89.2±4.3	89.5±6.4	4.8	7.1
T2DM患者	20	201.0±5.5	201.5±13.7	3.7	6.8

表3 各组研究对象空腹状态下常规临床生化指标水平比较

组别	年龄(岁)	例数(男/女)	GLU(mmol/L)	IN (IU/mL)	GLP-1(nmol/L)	TG(mmol/L)
NGT组	45.3±14.9	50(28/22)	4.7±0.4	10.3±5.9	80.0±19.7	1.1±0.4
IGT组	53.7±13.2	52(29/23)	5.9±1.1	15.3±21.7	110.5±16.4	1.6±0.3
T2DM组	58.5±16.8	68(37/31)	8.2±2.5^*	26.7±14.3^*	145.1±24.5^*	1.4±0.2
组别	CHOL(mmol/L)	LDL-C(mmol/L)	HDL-C(mmol/L)	HbA_1c(%)	hs-CRP(mg/L)
NGT组	4.5±2.4	2.5±1.6	1.80±0.60	4.1±1.2	2.8±1.5
IGT组	4.6±0.3	3.2±0.3	1.21±0.14	6.2±0.2^*	13.7±3.2^*
T2DM组	4.2±0.3	2.8±0.2	1.14±0.07	7.0±0.4^*	13.6±3.4^*

表4 各组患者餐后血清GLP-1含量变化分析

组别	例数	GLP-1(nmol/L)
组别	例数	空腹	服糖后1 h	服糖后2 h
NGT组	50	80.0±19.7	150.0±67.9^#	70.4±39.8
IGT组	52	110.5±16.4	65.9±12.9^**##	69.5±27.1^##
T2DM组	68	145.1±61.3^**	104.2±42.1^*#	125.4±41.4^**

参考文献 12

[1]	CHIA CW, EGAN JM.Biology and therapeutic potential of GLP-1 in the treatment of diabetes[J].Drug Discov Today, 2005, 2(3): 295-301.
[2]	LUND A,VILSBØLL T, BAGGER JI, et al. The separate and combined impact of the intestinal hormones, GIP, GLP-1, and GLP-2, on glucagon secretion in type 2 diabetes[J].Am J Physiol Endocrinol Metab, 2011, 300(6): E1038-E1046.
[3]	BASU R, CHANDRAMOULI V, DICKE B, et al.Obesity and type 2 diabetes impair insulin-induced suppression of glycogenolysis as well as gluconeogenesis[J].Diabetes, 2005,54(7): 1942-1948.
[4]	ARONIS KN, TSOUKAS MA, MANTZOROS CS.Potential cardioprotective action of GLP-1: from bench to bedside[J].Metabolism, 2014, 63(8): 979-988.
[5]	HEIJBOER AC, FRANS A, LOMECKY M, et al.Analysis of glucagon-like peptide 1; what to measure[J]. Clin Chim Acta, 2011, 412(13-14): 1191-1194.
[6]	刘珊,谢波,郝素娟,等.胰高血糖素样肽-1及其类似物的生物学活性测定方法[J].中国生化药物杂志,2012, 33(5): 675-678.
[7]	CALANNA S, CHRISTENSEN M, HOLST JJ, et al.Secretion of glucagon-like peptide-1 in patients with type 2 diabetes mellitus: systematic review and meta-analyses of clinical studies[J].Diabetologia, 2013, 56(5): 965-972.
[8]	吴美芬, 武革, 蔡晓玲, 等. 初发2型糖尿病患者GLP-1水平变化对胰高血糖素及早相胰岛素分泌的影响[J].中国临床新医学, 2013, 22(8): 736-739.
[9]	NAUCK MA, FRIEDRICH N.Do GLP-1-based therapies increase cancer risk[J]. Diabetes Care, 2013, 36(Suppl 2): S245-S252.
[10]	TOFT-NIELSEN MB, DAMHOLT MB, MADSBAD S, et al.Determinants of the impaired secretion of glucagon-like peptide-1 in type 2 diabetic patients[J]. J Clin Endocrinol Metab, 2001, 86(8): 3717-3723.
[11]	NATHANSON D, ZETHELIUS B, BERNE C, et al.Plasma levels of glucagon like peptide-1 associate with diastolic function in elderly men[J]. Diabet Med, 2011, 28(3): 301-305.
[12]	SLEDDERING MA, BAKKER LE, JANSSEN LG, et al.Higher insulin and glucagon-like peptide-1 (GLP-1) levels in healthy, young South Asians as compared to Caucasians during an oral glucose tolerance test[J].Metabolism, 2014, 63(2): 226-232.

[1]	梁焯辉, 赵凡, 刘倩雯. 巨噬细胞移动抑制因子与老年2型糖尿病患者颈动脉血管钙化的关系[J]. 检验医学, 2023, 38(5): 460-465.
[2]	肖秀美, 梁超, 刘彩红, 李晓光. 新型冠状病毒中和抗体与特异性抗体检测结果一致性分析[J]. 检验医学, 2023, 38(4): 352-356.
[3]	邓旭, 庄文龙, 薛慧, 朱淑芹, 李颖. 中性粒细胞/淋巴细胞比值、纤维蛋白原与2型糖尿病患者颈动脉内中膜厚度的关系[J]. 检验医学, 2023, 38(4): 368-372.
[4]	游延军, 张欢, 向乾银, 曹长春, 王健, 曾红, 魏元敏, 吴斌, 黄敬双, 季纯宇, 代苗, 秦枫. 原发性膜性肾病标志物抗PLA2R抗体检测方法比较[J]. 检验医学, 2023, 38(12): 1153-1156.
[5]	吴颖, 张立红, 高吟. HDL-C对T2DM患者缺血性脑卒中发病风险的预测价值[J]. 检验医学, 2023, 38(11): 1052-1056.
[6]	黄山, 孙溪若, 吕松琴, 许宝妹, 聂磊, 王义娜, 李晓非. 干式荧光发光法在HIV感染初筛中的应用价值[J]. 检验医学, 2022, 37(2): 174-176.
[7]	区苑清, 赵凡, 叶爱莲, 李立峰, 赵海桃, 黎惠贞, 梁焯辉, 李粮. 血浆网膜素1水平与2型糖尿病患者代谢清除率的关系[J]. 检验医学, 2022, 37(1): 28-31.
[8]	许智越, 方勇, 卢亚运, 王修玉, 薛建华, 张伟英. 体检人群幽门螺杆菌感染与血糖异常的相关性研究[J]. 检验医学, 2021, 36(4): 412-416.
[9]	朱庆华, 王伟伟, 邹广慧, 董志武. 血清NGAL和Cys C联合检测在诊断2型糖尿病早期肾损伤中的价值[J]. 检验医学, 2021, 36(3): 281-284.
[10]	刘凤莲, 丁志芳. 抗突变型瓜氨酸波形蛋白抗体自动化检测的性能验证[J]. 检验医学, 2021, 36(12): 1264-1266.
[11]	张前进, 胡金娥, 胡一川, 丁颖, 沈艳秋, 陈慧芹. 血清降脂素及维生素D与2型糖尿病患者胰岛β细胞功能及胰岛素敏感性的相关性[J]. 检验医学, 2020, 35(9): 908-911.
[12]	周中卫, 居会祥, 金浩, 陈红梅, 孙明忠. 胎球蛋白A/脂联素比值在2型糖尿病合并代谢综合征中的价值[J]. 检验医学, 2020, 35(5): 414-419.
[13]	左雪灿, 楚艳玲, 李占平. T2DM合并微血管病变患者凝血四项与C肽、IR水平的关系[J]. 检验医学, 2020, 35(4): 327-328.
[14]	胡燕, 申鸽, 李琼, 曾紫嫣, 朱攀, 杨钢, 李萍, 陈煜枫, 张贞, 谢小兵. 2种SARS-CoV-2 抗体检测方法在COVID-19诊断中的价值[J]. 检验医学, 2020, 35(12): 1294-1297.
[15]	刘晓妮, 王颖, 李桃桃, 马小敏, 赵换牢. 2型糖尿病合并代谢综合征患者胰岛素抵抗指数与慢性炎症指标相关性研究[J]. 检验医学, 2019, 34(9): 826-830.