纳米磁球法分离检测甲胎蛋白变异体方法的建立及应用
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摘要
目的:建立一种简便、快速和高敏感度检测甲胎蛋白(alpha-fetoprotein,AFP)变异体AFP-L3的新方法。方法:采用薄膜蒸发法制备扁豆凝集素(lens culinaris agglutinin,LCA)修饰的纳米功能磁球(LCA-纳米功能磁球),并通过磁性作用分离结合AFP-L3的LCA-纳米功能磁球,从而富集AFP-L3。结合人AFP定量检测试纸(时间分辨荧光法)检测AFP-L3的浓度,建立基于纳米功能磁球的AFP-L3检测方法。采用AFP-L3标准品比较LCA-纳米功能磁球法和已商品化的亲和离心层析柱法对AFP-L3的回收率,并应用上述2种方法分别检测了64例AFP阳性肝癌患者血清中AFP-L3的浓度并比较检测结果。选择830例血清样品,包括100例正常健康人群,300例慢性肝炎患者、228例肝硬化患者和202例肝癌患者,采用LCA-纳米功能磁球检测AFP-L3浓度,并比较各组的差异。结果:成功制备获得LCA偶联的纳米功能磁球,并建立了纳米磁球提取AFP-L3的方法。与亲和层析离心柱提取AFP-L3的方法进行比较,在AFP-L3浓度较高(4 000 ng/m L)时,LCA-纳米功能磁球法对AFP-L3的回收率显著优于亲和层析离心柱法(P<0.05),而在低浓度时两者的回收率相当。64例AFP阳性肝癌患者血清中AFP-L3的浓度检测结果显示,2种检测方法检测AFP-L3的阳性率均为37.5%,差异无统计学意义(P>0.05)。830例临床样本的检测结果显示,正常健康人群、慢性肝炎患者、肝硬化患者以及肝癌患者血清中AFP和AFP-L3的双阳性率分别为0.0%、3.3%、19.7%和60.9%,肝癌患者的阳性率明显高于良性肝病患者或正常人健康人群,和已报道结果一致。结论:采用LCA修饰的纳米功能磁球通过自动化提取AFP-L3的方法是一种准确、高效而简单的检测AFP-L3的方法。
Objective:To establish a new,simple,rapid and highly sensitive method of detecting alpha-fetoprotein(AFP) isoform(AFP-L3).Methods:Lentil(lens culinaris agglutinin,LCA)-modified lipid magnetic nano microsphere was prepared by using thin-film evaporation method,and LCA-modified magnetic nano microsphere was collected by using combination with AFP-L3 through magnetic property to enrich AFP-L3.AFP-L3 concentration was detected by using human AFP quantitative test(time-resolved fluorescence method) to establish the method for detecting AFP-L3 based on magnetic nano microsphere.The established method above was compared with the commercialized method of affinity entrifugation to observe the recovery of AFP-L3.The concentration of AFP-L3 in serum from 64 AFP-positive liver cancer patients was detected by using the above two methods.AFP-L3 concentration in serum samples from 830 individuals including 100 normal healthy people,300 cases of chronic hepatitis,228 cases of cirrhosis and 202 cases of liver cancer were detected to observe the difference by using magnetic nano microsphere test.Results:LCA-modified magnetic nano microsphere was successfully obtained,and the separating method of capturing AFP-L3 was established by using magnetic nano microsphere.The recovery of nano magnetic bead method was obviously superior to that of affinity centrifugal column method when AFP-L3(4000 ng/mL)concentration was high(P < 0.05);but the recovery was fairly the same when AFP-L3 concentration was low.Both positive rates of AFP-L3 detected by these two methods were 37.5%in 64 patients with AFP-positive liver cancer(P > 0.05).According to the results from 380 serum samples,the positive rates of AFP-L3 in normal controls and patients with chronic hepatitis,cirrhosis and liver cancer were 0.0%,3.3%,19.7%and 60.9%,respectively.The positive rate of liver cancer patients was significantly higher than that of the patients with benign liver diseases or the normal controls,which had no difference as compared with the reported results.Conclusion:The method of automatic AFP-L3 extraction by using LCA-modified magnetic nano microsphere is a kind of accurate,reliable,highly efficient and simple method for detecting AFP-L3.
Objective:To establish a new,simple,rapid and highly sensitive method of detecting alpha-fetoprotein(AFP) isoform(AFP-L3).Methods:Lentil(lens culinaris agglutinin,LCA)-modified lipid magnetic nano microsphere was prepared by using thin-film evaporation method,and LCA-modified magnetic nano microsphere was collected by using combination with AFP-L3 through magnetic property to enrich AFP-L3.AFP-L3 concentration was detected by using human AFP quantitative test(time-resolved fluorescence method) to establish the method for detecting AFP-L3 based on magnetic nano microsphere.The established method above was compared with the commercialized method of affinity entrifugation to observe the recovery of AFP-L3.The concentration of AFP-L3 in serum from 64 AFP-positive liver cancer patients was detected by using the above two methods.AFP-L3 concentration in serum samples from 830 individuals including 100 normal healthy people,300 cases of chronic hepatitis,228 cases of cirrhosis and 202 cases of liver cancer were detected to observe the difference by using magnetic nano microsphere test.Results:LCA-modified magnetic nano microsphere was successfully obtained,and the separating method of capturing AFP-L3 was established by using magnetic nano microsphere.The recovery of nano magnetic bead method was obviously superior to that of affinity centrifugal column method when AFP-L3(4000 ng/mL)concentration was high(P < 0.05);but the recovery was fairly the same when AFP-L3 concentration was low.Both positive rates of AFP-L3 detected by these two methods were 37.5%in 64 patients with AFP-positive liver cancer(P > 0.05).According to the results from 380 serum samples,the positive rates of AFP-L3 in normal controls and patients with chronic hepatitis,cirrhosis and liver cancer were 0.0%,3.3%,19.7%and 60.9%,respectively.The positive rate of liver cancer patients was significantly higher than that of the patients with benign liver diseases or the normal controls,which had no difference as compared with the reported results.Conclusion:The method of automatic AFP-L3 extraction by using LCA-modified magnetic nano microsphere is a kind of accurate,reliable,highly efficient and simple method for detecting AFP-L3.
引文
[1]Kim do Y,Han KH.Epidemiology and surveillance of hepatocellular carcinoma[J].Liver Cancer,2012,1(1):2-14.
[2]Zhou L,Liu J,Luo F.Serum tumor markers for detection of hepatocellular carcinoma[J].World J Gastroenterol,2006,12(8):1175-1181.
[3]孙桂珍,赵秀英,李俊红,等.凝集素微量离心柱法检测甲胎蛋白异质体用于肝癌的诊断及分析[J].中华医学杂志,2008,88(28):1986-1988.
[4]郑颖,单晓洁,任茗睿,等.微量离心柱法检测甲胎蛋白异质体对原发性肝癌诊断的意义[J].浙江实用医学,2009,14(1):3-4.
[5]Kim J,Lee JE,Lee J,et al.Magnetic fluorescent delivery vehicle using uniform mesoporous silica spheres embedded with monodisperse magnetic and semiconductor nanocrystals[J].J Am Chem Soc,2006,128(3):688-689.
[6]梁晓飞,王汉杰,田惠,等.载药磁性阳离子高聚物脂质体的制备及表征[J].高等学校化学学报,2008,29(4):858-861.
[7]FDA ClassⅡSpecial Controls Guideline Document:AFP-L3%Immunological Test Systems(FDA-2005-0030-0001)[EB/OL](2005-10-04)[2012-02-02].http://www.fda.gov/ohrms/dockets/98fr/o5d-0342-gd10001.pdf.
[8]汪子伟,卓广超,冷建杭,等.亲和吸附法在甲胎蛋白异质体检测中的应用[J].中华检验医学杂志,2005,28(2):203-205.
[9]Qian X,Peng XH,Ansari DO,et al.In vivo tumor targeting and spectroscopic detection with surfaceenhanced raman nanoparticle tags[J].Nat Biotechnol,2008,26(1):83-90.
[10]薛建祥,许凯黎,关赛芳,等.评多项肿瘤标志物检测对肝癌的诊断价值[J].肿瘤,1996,16(5):525-527.
[11]Stefaniuk P,Cianciara J,WiercinskaDrapalo A.Present and future possibilities for early diagnosis of hepatocellular carcinoma[J].World J Gastroenterol,2010,16(4):418-424.
[2]Zhou L,Liu J,Luo F.Serum tumor markers for detection of hepatocellular carcinoma[J].World J Gastroenterol,2006,12(8):1175-1181.
[3]孙桂珍,赵秀英,李俊红,等.凝集素微量离心柱法检测甲胎蛋白异质体用于肝癌的诊断及分析[J].中华医学杂志,2008,88(28):1986-1988.
[4]郑颖,单晓洁,任茗睿,等.微量离心柱法检测甲胎蛋白异质体对原发性肝癌诊断的意义[J].浙江实用医学,2009,14(1):3-4.
[5]Kim J,Lee JE,Lee J,et al.Magnetic fluorescent delivery vehicle using uniform mesoporous silica spheres embedded with monodisperse magnetic and semiconductor nanocrystals[J].J Am Chem Soc,2006,128(3):688-689.
[6]梁晓飞,王汉杰,田惠,等.载药磁性阳离子高聚物脂质体的制备及表征[J].高等学校化学学报,2008,29(4):858-861.
[7]FDA ClassⅡSpecial Controls Guideline Document:AFP-L3%Immunological Test Systems(FDA-2005-0030-0001)[EB/OL](2005-10-04)[2012-02-02].http://www.fda.gov/ohrms/dockets/98fr/o5d-0342-gd10001.pdf.
[8]汪子伟,卓广超,冷建杭,等.亲和吸附法在甲胎蛋白异质体检测中的应用[J].中华检验医学杂志,2005,28(2):203-205.
[9]Qian X,Peng XH,Ansari DO,et al.In vivo tumor targeting and spectroscopic detection with surfaceenhanced raman nanoparticle tags[J].Nat Biotechnol,2008,26(1):83-90.
[10]薛建祥,许凯黎,关赛芳,等.评多项肿瘤标志物检测对肝癌的诊断价值[J].肿瘤,1996,16(5):525-527.
[11]Stefaniuk P,Cianciara J,WiercinskaDrapalo A.Present and future possibilities for early diagnosis of hepatocellular carcinoma[J].World J Gastroenterol,2010,16(4):418-424.