检测多天线AAG的Lectin-ELISA方法建立及初步应用评价

doi:10.3969/j.issn.1673-8640.2020.11.022

摘要/Abstract

摘要：

目的建立检测多天线α1-酸性糖蛋白（AAG）的凝集素-酶联免疫吸附试验（Lectin-ELISA）方法,探讨多天线AAG在原发性肝细胞肝癌（HCC）中的临床价值。方法根据曼陀罗凝集素（DSA）可特异性识别多天线结构AAG的原理构建检测DSA-AAG的Lectin-ELISA方法。采用该方法检测220例HCC患者（HCC组）、237例疾病对照者[疾病对照组,肝内胆管癌（ICC）74例（ICC组）、肝硬化（LC）120例（LC组）、慢性乙型肝炎（CHB）43例（CHB组）]和80名体检健康者（正常对照组）的血清DSA-AAG水平,同时收集HCC患者病理资料和实验室检测结果。采用Pearson相关分析评估各项目之间的相关性。采用Logistic回归方法建立多指标联合检测模型。采用受试者工作特征（ROC）曲线评估单项指标及联合检测模型诊断HCC的效能。结果 Lectin-ELISA检测血清DSA-AAG的线性回归系数r²=0.976,批间最大变异系数（CV）为10.51%,游离胆红素、结合胆红素、溶血和乳糜对Lectin-ELISA检测血清DSA-AAG无影响。HCC组血清DSA-AAG水平显著高于疾病对照组和正常对照组（P<0.001）,HCC组、疾病对照组血清AAG水平明显高于正常对照组（P<0.001）。癌症组（HCC组+ICC组）血清DSA-AAG和AAG水平均高于非癌症组（LC组+CHB组+正常对照组）（P<0.001）。HCC组血清DSA-AAG水平高于ICC组（P<0.001）,血清AAG水平低于ICC组（P<0.001）。HCC组DSA-AAG与甲胎蛋白（AFP）呈负相关（r=-0.029,P<0.05）。不同肿瘤大小和分期的HCC患者之间血清DSA-AAG水平差异均无统计学意义（P>0.05）。DSA-AAG、AAG及AFP单项和联合检测模型Logit_AAG1鉴别诊断HCC与非HCC的曲线下面积（AUC）分别为0.651、0.632、0.803和0.836,DSA-AAG、AAG单项和联合检测模型Logit_AAG2鉴别诊断AFP阴性HCC与非HCC的AUC分别为0.669、0.607、0.929。结论 DSA-AAG及相关联合检测模型对HCC及AFP阴性HCC的鉴别诊断可能有一定的作用。

关键词: α1-酸性糖蛋白, 糖基化, 凝集素, 原发性肝细胞癌

Abstract:

Objective To establish a lectin enzyme-linked immunosorbent assay（Lectin-ELISA） for the detection of multi-antennary alpha 1-acid glycoprotein（AAG）,and to investigate the clinical application role of multi-antennary AAG in hepatocellular carcinoma（HCC）. Methods A Lectin-ELISA for detecting datura stramonium agglutinin（DSA）-AAG was established based on the principle that DSA can specifically identify the AAG of multi-antennary structure. This method was used to detect serum DSA-AAG levels of 220 HCC patients（HCC group）,237 disease controls [disease control group,74 cases of intrahepatic cholangiocarcinoma（ICC）（ICC group）,120 cases of liver cirrhosis（LC）（LC group）,43 cases of chronic hepatitis B（CHB）（CHB group）] and 80 healthy subjects（healthy control group）. Meanwhile,the pathological data and laboratory determination results of HCC patients were collected. Pearson correlation analysis was performed. Logistic regression method was used to establish the multi-index joint determination model. Receiver operating characteristic（ROC） curves were used to evaluate the efficiency of single and combined determinations in the diagnosis of HCC. Results The linear regression coefficient（r²） of serum DSA-AAG determined by Lectin-ELISA was 0.976,and the maximum between-run coefficient of variation（CV） was 10.51%. Free bilirubin,binding bilirubin,hemolysis and chylous had no effect on serum DSA-AAG determined by Lectin-ELISA. Serum DSA-AAG level of HCC group was higher than those of disease control group and healthy control group（P<0.001）,and serum AAG levels of HCC group and disease control group were higher than that of healthy control group（P<0.001）. Serum levels of DSA-AAG and AAG in cancer group（HCC+ICC） were higher than those in non-cancer group（LC+CHB+healthy control）（P<0.001）. Serum DSA-AAG level of HCC group was higher than that of ICC group（P<0.001）,while serum AAG level was lower than that of ICC group（P<0.001）. There was a negative correlation between DSA-AAG and alpha-fetoprotein （AFP） in HCC group（r=-0.029,P<0.05）. There was no statistical significance in serum DSA-AAG level among HCC patients with different tumor sizes and stages（P>0.05）. The areas under curves（AUC） of DSA-AAG,AAG and AFP single and combined determination models Logit_AAG1 for differentiating HCC from non-HCC were 0.651,0.632,0.803 and 0.836,respectively. The AUC of DSA-AAG and AAG single and combined determination model Logit_AAG2 for the differential diagnosis of AFP negative HCC and non-HCC were 0.669,0.607 and 0.929,respectively. Conclusions DSA-AAG and combined determination model may play roles in the differential diagnosis of HCC and AFP negative HCC.

Key words: Alpha 1-acid glycoprotein, Glycosylation, Lectin, Hepatocellular carcinoma

中图分类号:

R446.1

关雯倩, 高致远, 冯惠娟, 洪松, 何羽童, 何璐, 高春芳. 检测多天线AAG的Lectin-ELISA方法建立及初步应用评价[J]. 检验医学, 2020, 35(11): 1177-1185.

GUAN Wenqian, GAO Zhiyuan, FENG Huijuan, HONG Song, HE Yutong, HE Lu, GAO Chunfang. Establishment of Lectin-ELISA for the detection of multi-antennary AAG and its preliminary application[J]. Laboratory Medicine, 2020, 35(11): 1177-1185.

图/表 12

参考文献 28

[1]	COHN E J,GURD F R N,SURGENOR D M,et al. A system for the separation of the components of human blood:quantitative procedures for the separation of the protein components of human plasma[J]. J Am Chem Soc,1950,72(1):465-474.
[2]	KAMIYAMA S,SCHMID K.Studies on the structure of alpha1-acid glycoprotein. Ⅱ. Preparation and characterization of a glycopeptide fraction[J]. Biochim Biophys Acta,1962,58:80-87.
[3]	ZHANG C,BI C,CLARKE W,et al.Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection[J]. J Chromatogr A,2017,1523:114-122.
[4]	FOURNIER T,MEDJOUBI-N N,PORQUET D.Alpha-1-acid glycoprotein[J]. Biochim Biophys Acta,2000,1482(1-2):157-171.
[5]	BERNTSSON J,ÖSTLING G,PERSSON M,et al.Orosomucoid,carotid plaque,and incidence of stroke[J]. Stroke,2016,47(7):1858-1863.
[6]	EL-BEBLAWY N M,ANDRAWES N G,ISMAIL E A,et al. Serum and urinary orosomucoid in young patients with type 1 diabetes:a link between inflammation,microvascular complications,and subclinical atherosclerosis[J]. Clin Appl Thromb Hemost,2016,22(8):718-726.
[7]	ANDERSEN P,EIKA C.Inhibition of thrombin-induced platelet aggregation by crude and highly purified alpha 1-acid glycoprotein[J]. Scand J Haematol,1980,25(3):202-204.
[8]	POS O,OOSTENDORP R A,VAN DER STELT M E,et al. Con A-nonreactive human alpha 1-acid glycoprotein(AGP) is more effective in modulation of lymphocyte proliferation than Con A-reactive AGP serum variants[J]. Inflammation,1990,14(2):133-141.
[9]	COSTELLO M,FIEDEL B A,GEWURZ H.Inhibition of platelet aggregation by native and desialised alpha-1 acid glycoprotein[J]. Nature,1979,281(5733):677-678.
[10]	RABEHI L,FERRIERE F,SAFFAR L,et al.alpha 1-acid glycoprotein binds human immunodeficiency virus type 1(HIV-1) envelope glycoprotein via N-linked glycans[J]. Glycoconj J,1995,12(1):7-16.
[11]	ORCZYK-PAWIĹOWICZ M,KTNIK-PRASTOWSKA I. Terminal monosaccharide expression on amniotic glycoproteins as biomarkers of fetus maturity[J]. Biochem Soc Trans,2011,39(1):344-348.
[12]	SAROHA A,BISWAS S,CHATTERJEE B P,et al.Altered glycosylation and expression of plasma alpha-1-acid glycoprotein and haptoglobin in rheumatoid arthritis[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2011,879(20):1839-1843.
[13]	TSUTSUMI M,TAKASE S.Usefulness of microheterogeneity of serum alhpa1-acidglycoprotein as a marker for alcohol abuse[J]. Alcohol,2001,25(3):181-184.
[14]	MONDAL G,CHATTERJEE U,DAS H R,et al.Enhanced expression of alpha1-acid glycoprotein and fucosylation in hepatitis B patients provides an insight into pathogenesis[J]. Glycoconj J,2009,26(9):1225-1234.
[15]	WANG M,ZHU J,LUBMAN D M,et al.Aberrant glycosylation and cancer biomarker discovery:a promising and thorny journey[J]. Clin Chem Lab Med,2019,57(4):407-416.
[16]	TANABE K,KITAGAWA K,KOJIMA N,et al.Multifucosylated alpha-1-acid glycoprotein as a novel marker for hepatocellular carcinoma[J]. J Proteome Res,2016,15(9):2935-2944.
[17]	TAKETA K,ICHIKAWA E,YAMAMOTO T,et al.Datura stramonium agglutinin-reactive alpha-fetoprotein isoforms in hepatocellular carcinoma and other tumors[J]. Tumour Biol,1990,11(4):220-228.
[18]	FANG M,ZHAO Y P,ZHOU F G,et al.N-glycan based models improve diagnostic efficacies in hepatitis B virus-related hepatocellular carcinoma[J]. Int J Cancer,2010,127(1):148-159.
[19]	OSNA N A,CARTER W G,GANESAN M,et al.Aberrant post-translational protein modifications in the pathogenesis of alcohol-induced liver injury[J]. World J Gastroenterol,2016,22(27):6192-6200.
[20]	OHTSUBO K,MARTH J D.Glycosylation in cellular mechanisms of health and disease[J]. Cell,2006,126(5):855-867.
[21]	BLOMME B,VAN STEENKISTE C,GRASSI P,et al.Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven[J]. Am J Physiol Gastrointest Liver Physiol,2011,300(5):G833-G842.
[22]	CLARK D,MAO L.Cancer biomarker discovery:lectin-based strategies targeting glycoproteins[J]. Dis Markers,2012,33(1):1-10.
[23]	中华人民共和国卫生和计划生育委员会. 原发性肝癌诊疗规范(2017年版)[J]. 传染病信息,2017,30(3):111-127.
[24]	WANG M,ZHU J,LUBMAN D M,et al.Aberrant glycosylation and cancer biomarker discovery:a promising and thorny journey[J]. Clin Chem Lab Med,2019,57(4):407-416.
[25]	FANG M,ZHAO Y P,ZHOU F G,et al.N-glycan based models improve diagnostic efficacies in hepatitis B virus-related hepatocellular carcinoma[J]. Int J Cancer,2010,127(1):148-159.
[26]	ZHANG D,HUANG J,LUO D,et al.Glycosylation change of alpha-1-acid glycoprotein as a serum biomarker for hepatocellular carcinoma and cirrhosis[J]. Biomark Med,2017,11(5):423-430.
[27]	MEHTA A,NORTON P,LIANG H Y,et al.Increased levels of tetra-antennary N-linked glycan but not core fucosylation are associated with hepatocellular carcinoma tissue[J]. Cancer Epidemiol Biomarkers Prev,2012,21(6):925-933.
[28]	IDE Y,MIYOSHI E,NAKAGAWA T,et al.Aberrant expression of N-acetylglucosaminyltransferase-Ⅳa and Ⅳb(GnT-Ⅳa and b) in pancreatic cancer[J]. Biochem Biophys Res Commun,2006,341(2):478-482.

组别	例数	AST/（U/L）		ALT/（U/L）		GGT/（U/L）		TB/（μmol/L）
HCC组	220	29.00（20.00~42.00）		29.00（20.00~42.00）		56.00（34.00~104.5）		14.00（10.90~18.38）
CHB组	43	223.00（88.00~519.00）		295.00（183.00~767.00）		114.00（63.00~165.00）		23.40（14.70~69.10）
LC组	120	32.00（24.25~44.75）		27.00（18.00~40.25）		46.50（28.25~79.75）		23.85（15.00~40.15）
ICC组	74	23.00（18.00~35.00）		24.50（16.00~34.25）		84.50（61.75~113.00）		14.35（10.70~18.43）
正常对照组	80			20.00（16.00~25.75）				14.65（12.00~16.63）
统计值		17.295		17.744		2.271		18.798
P值		<0.001		<0.001		0.132		<0.001
组别	DBil/（μmol/L）		Alb/（g/L）		TP/（g/L）		AFP/（μg/L）
HCC组	5.50（4.10~7.10）		42.01±3.74		69.62±8.28		61.00（4.70~1 210.00）
CHB组	9.60（5.20~47.00）		37.69±4.24		69.09±6.26		8.31（3.71~45.47）
LC组	10.20（6.00~20.00）		33.08±10.18		63.63±16.81		3.60（2.23~8.84）
ICC组	5.15（3.85~6.80）		42.26±3.46		68.67±5.12		2.90（2.00~4.70）
正常对照组			47.91±1.97		75.87±3.20		3.50（2.60~3.50）
统计值	22.056		46.845		161.700		82.406
P值	<0.001		<0.001		<0.001		<0.001

组别	例数	AST/（U/L）		ALT/（U/L）		GGT/（U/L）		TB/（μmol/L）
HCC组	220	29.00（20.00~42.00）		29.00（20.00~42.00）		56.00（34.00~104.5）		14.00（10.90~18.38）
CHB组	43	223.00（88.00~519.00）		295.00（183.00~767.00）		114.00（63.00~165.00）		23.40（14.70~69.10）
LC组	120	32.00（24.25~44.75）		27.00（18.00~40.25）		46.50（28.25~79.75）		23.85（15.00~40.15）
ICC组	74	23.00（18.00~35.00）		24.50（16.00~34.25）		84.50（61.75~113.00）		14.35（10.70~18.43）
正常对照组	80			20.00（16.00~25.75）				14.65（12.00~16.63）
统计值		17.295		17.744		2.271		18.798
P值		<0.001		<0.001		0.132		<0.001
组别	DBil/（μmol/L）		Alb/（g/L）		TP/（g/L）		AFP/（μg/L）
HCC组	5.50（4.10~7.10）		42.01±3.74		69.62±8.28		61.00（4.70~1 210.00）
CHB组	9.60（5.20~47.00）		37.69±4.24		69.09±6.26		8.31（3.71~45.47）
LC组	10.20（6.00~20.00）		33.08±10.18		63.63±16.81		3.60（2.23~8.84）
ICC组	5.15（3.85~6.80）		42.26±3.46		68.67±5.12		2.90（2.00~4.70）
正常对照组			47.91±1.97		75.87±3.20		3.50（2.60~3.50）
统计值	22.056		46.845		161.700		82.406
P值	<0.001		<0.001		<0.001		<0.001

A值	x	s	CV/%
高	0.867	0.08	9.23
中	0.730	0.07	9.59
低	0.666	0.07	10.51

A值	x	s	CV/%
高	0.867	0.08	9.23
中	0.730	0.07	9.59
低	0.666	0.07	10.51

组别	DSA-AAG（A值）	AAG/（g/L）
HCC组	0.47±0.07^*#	0.63±0.30^*
疾病对照组	0.44±0.09^*	0.66±0.36^*
正常对照组	0.39±0.10	0.45±0.15