基于量子点技术同步检测输血传染病病原体抗原的方法建立

1、基于量子点技术同步检测输血传染病病原体抗原的方法建立        【中文摘要】目的与意义在献血及输血治疗的过程中, HBV、HCV、HIV-1/2以及TP的检测是国家强制性检测项目,HBV与HCV是造成输血后肝炎的主要病原体。目前对上述多种病原微生物的实验室诊断方法较多,但各有不足。如ELISA无法同步检测多项指标、时间较长,金标法灵敏度较低,PCR和化学发光法需借助特殊设备,对实验室要求较高等。为解决输血性传染病的多种病原体的抗原同步检测,本研究拟以乙肝表面抗原和丙肝核心抗原的同步检测为例,建立一种高灵敏度和特

2、异性、稳定性好的抗原同步快速检测体系。将量子点技术与磁微粒技术相结合,以磁微粒富集以进步灵敏度,利用不同量子点在相同波长激发光下发射不同荧光的特性达到同步检测的目的。为建立针对多种输血传染病病原体抗原的同步检测液相芯片奠定基础。材料与方法1.磁微粒的活化及其与抗体的连接:选择粒径3m的羧基化磁微粒,采用碳二亚胺共价交联法,在活化剂EDC与NHS的不同浓度组合条件下活化磁微粒,选择最适的活化剂浓度组合。以不同浓度的鼠抗HBsAg-IgG、鼠抗HCcAg-IgG连接磁微粒,在不同反应时间、pH值的条件组合下考察抗体与磁微粒的连接效果,以Folin-酚法评价抗体连接效率。2.量子点的活化及其与抗体的

3、连接:采用碳二亚胺共价交联法以EDC及NHS活化发射波长为581nm、618nm的羧基化CdSe量子点,以不同浓度的兔抗HBsAg-IgG、兔抗HCcAg-IgG连接量子点并在不同反应时间、pH值的条件组合下考察抗体与磁微粒的连接效果,以Folin-酚法评价抗体连接效率。3.单指标检测的体系建立:以连接抗体的量子点作为荧光标记物,以连接抗体的磁微粒为载体作固-液分离,确定最适标本检测时间。作阴性血清检测以检测荧光强度并以2倍阴性对照均值+空缺对照均值作为单指标检测Cut off值判读结果。对不同浓度的HBsAg、HCcAg抗原标准品作单指标检测以确定检测范围。并作重复性实验和正确度评价。4.同

4、步检测HBsAg和HCcAg体系的建立:在单指标检测的基础上整合反应条件及最适标本检测时间,作阴性血清检测以检测荧光强度并以2倍阴性对照均值+空缺对照均值作为单指标检测Cut off值判读结果。混合激发光的记录以滤光片滤往一种颜色光线后记录一项结果,更换滤光片后记录另一结果。5.对不同浓度HBsAg、HCcAg抗原标准品混合物作同步检测以确定检测范围。6.对188例临床标本进行检测,并与ELISA试剂盒检测结果相比较。7.选取10ng/mL、100ng/mL、1g/mL三个浓度抗原标准品作同步检测的批内、天间重复性实验。8.以脂血、溶血、黄疸标本作干扰实验。以HAV、TP抗体阳性标本作特异性实

5、验。9.将预备好的试剂及反应后的复合物于4保存一定时间后观察保存时间对检测效果的影响。主要结果1.磁微粒的选择及连接抗体的条件选择:鼠抗HBsAg-IgG连接磁微粒的活化剂浓度为EDC 6mg/L、NHS 4mg/L,连接时间120min,最适pH值6.6,最适浓度20mg/mL,连接效率41%,并制备试剂1(R1HBV)。鼠抗HCcAg-IgG连接磁微粒的活化剂浓度为EDC 4mg/L、NHS4mg/L,连接时间120min,最适pH值6.6,最适浓度20mg/mL,连接效率44%,并制备试剂2(R2HBV)。上述R1HBV、R1HCV清洗后加进1%BSA封闭磁微粒表面剩余位点过夜。2.量子

6、点连接抗体的条件选择:兔抗HBsAg-IgG连接581nm量子点,连接时间120min,最适pH值6.2,最适浓度20mg/mL,连接效率45%,并制备试剂1 (R1HCV)。兔抗HBsAg-IgG连接581nm量子点,连接时间120min,最适pH值6.2,最适浓度10mg/mL,连接效率45%,并制备试剂2 (R2HCV)。3.量子点磁微粒单指标检测体系:于EP管内同时加进标本50L,R1各100L,R2各5L,反应体系pH值7.4,计算Cut off值为122和136(HBsAb与HCcAg),检测范围为2ng/mL-10g/mL、5ng/mL-5g/mL。检测时间分别缩短到30min、

7、40min。4.量子点磁微粒体系单项检测HBsAg和HCcAg标准品,批内重复性实验均匀CV值为9.7%、9.8%;天间重复性实验均匀CV值为10.3%、10.1%。5.量子点磁微粒同步检测体系:于EP管内同时加进标本5100L,R1各100L,R2各5L,反应体系pH值7.4,计算Cut off值为133/140,检测范围为2ng/mL-10g/mL,5ng/mL-5g/mL。检测时间从1h/3h同步缩短到40min。6.对188份临床标本的同步检测结果,HBsAg阳性:PCR方法36例,量子点-磁微粒法35例,ELISA方法34例。HCcAg阳性:PCR方法11例,量子点-磁微粒法11例,

8、 ELISA方法8例。对2例HBV与HCV混合感染的临床标本及5例标准品混合样本成功检出。HBsAg检测结果与PCR方法比较,符合率98.4%,灵敏度94.4%,特异度99.3%;HCcAg检测结果与ELISA方法比较符合率97.9%,灵敏度81.8%,特异度98.9%。7.同步检测HBsAg和HCcAg标准品,批内重复性实验均匀CV值为10.0%、10.1%;天间重复性实验均匀CV值为10.7%、10.6%。8.检测肉眼可见的溶血、黄疸、脂血标本,对结果无干扰。与其它常见致输血后感染的病原体(TP、HAV)无交叉反应。9.试剂R1、R2于4保存4周后使用,与新配置试剂检测标准品结果无明显差异

9、。结合目标抗原后的复合物标本,经4放置4周后荧光强度无明显衰减。最长放置8周后仍可检出荧光。结论1.本研究利用不同量子点在相同激发光照射下可发射不同颜色荧光的特性,选用输血传染病中造成输血后肝炎的两种主要病原体HBV和HCV的抗原检测为例,成功达到了同步检测不同抗原的目的,为建立针对多种输血传染病病原体抗原的检测体系建立奠定了基础。同时由于针对抗原作检测,相对抗体检测较大幅度提前了阳性检出时间,避免了因抗体形成的窗口期造成的漏检。2.以碳二亚胺法连接了不同源的抗体于羧基化磁微粒和量子点表面,以双抗体夹心法检测抗原,由于采用磁微粒作为固相载体,在不借助大型特殊设备的条件下获得了较高的灵敏度,对H

10、BsAg与HCcAg检测灵敏度分别为2ng/mL、5ng/mL。3.对临床标本的检测结果与ELISA试剂比较无明显性差异,但检测时间明显缩短,对HBsAg与HCcAg的检测时间分别从ELISA所需的1h、3h左右同步缩短到40min-50min。研究结果显示,抗原浓度与荧光强度呈正相关,通过图像软件计算可做到半定量分析。4.试剂制备所需较短,标本检测后的复合物易保存,荧光漂白作用小,利于结果的复查和动态监测。');【Abstract】 Objective and significanceDuring blood donation and transfusing, HBV, HCV, H

11、IV-1, HIV-2 and syphilis testing are compulsorily required in China nowadays. Hepatitis B virus and Hepatitis C virus mostly caused the PTH (post transfusion hepatitis). And the clinical laboratories usually use ELISA (Enzyme linked immunosorbent assay) to analyze those pathogenic microorganisms. Mo

12、st of these assays are for antibody test and the delay of antibody production (window phase) is inevitably can not be checked out. Morever, the different of testing kits use caused the different testing time.As the Quantum dots (QDs) with different size can be inspired different fluorescence by mono

13、chromatic light and the Magnetic Microspheres (MMS) can be enriched in a magnetic field, we may establish and apply testing system with high sensitivity, specificity, efficiency and speed for the simultaneously detection of antigens in liquid. Materials and methods1. The conjugation of MMS and Ab (r

14、eady for Reagent 1, R1): Chose the 3m diameter Carboxyl MMS and use the carbodiimide cross-link technique activate the MMS with EDC and NHS in different concentration, then linked mice anti-HBsAg-IgG and mice anti-HCcAg-IgG on them.To view the effect with different concentration of Ab, bounding time

15、 and pH value which act on the conjugation of MMS and Ab. Then examine the ratio of conjugation by folin-phenol method.2. The conjugation of QDs and Ab (ready for Reagent 2, R2): Activate QDs with emission wavelengths at 581nm and 618nm by EDC/NHS at the volume proportion of 1:50. To view the effect

16、 with different concentration of Ab, bounding time and pH value which act on the conjugation of QDs and Ab. Then examine the ratio of conjugation by folin-phenol method.3. The single test systems: Use the QD-Ab as the fluorescence labels and the MMS-Ab as carriers to solid-liquid separate. After obt

17、ained the optimal value of react time we analyzed the negative serums to get the average of double negative control adds average of blank as cut off values. Then we used R1, R2 to analyze mixed standard antigen at different concentrations of HBsAg and HCcAg to determine the detection limits.4. To es

18、tablish the simultaneously detection system: On the basis of single test systems we combined the conditions and choose the best react time.Then analyzed the negative serums to get the average of double negative adds average of blank as cut off values. Because of the overlapping of MMS, we use the fi

19、lters to obtain the different colors of fluorescence partly. Then we used R1, R2 to analyze mixed standard antigen at different concentrations of HBsAg and HCcAg to determine the detection limits.5. 188 unknown samples were analyzed by the simultaneously detection system and compared the results wit

20、h ELISA.6. We analyzed the individual and mixed standards at concentration of 10ng/mL, 100ng/mL, 1g /mL as the repetitive experiments.7. The samples with hemolysis, jaundice and lipemia were analyzed as the interference factors and the positive HAV, TP serums as the specificity experiments.8. The fl

21、uorescence of prepared reagents and the complexs after reaction were recorded in 8 weeks to investigate the stabilities.Results1. The condition for the conjugation of MMS and Ab: The concentration of activators EDC at 6mg/L and NHS at 4mg/L for mice anti-HBsAg-IgG, bonding time t=120min, pH value 6.

22、6 and the concentration of Ab was 20mg/mL. EDC at 4mg /L and NHS at 4mg /L for rabbit anti-HCcAg-IgG, bounding time at 120min, pH value 6.6 and the concentration of Ab was 20mg/mL. The efficiency ratios of conjugation were 41% and 44%. Add 1% BSA in R1 and store at room temperature for 4 hours and w

23、ashed with pH7.4 PBS for use.2. The condition for the conjugation of QDs and Ab: Bonding time t=120min, pH value 6.2 and the concentration of rabbit-anti-HBsAg-IgG was 20mg/mL to QD581. Bonding time t=120min, pH value 6.2 and the concentration of rabbit anti-HCcAg-IgG was 10mg/mL to QD618. The effic

24、iency ratios of conjugation were 45% and 45%.3. The single test systems: Serum 50L; R1, 100L; R2, 5L (after enrichment), add in EP tube totally then react under the condition of pH7.4, 37for 20min and 30min (HBV and HCV). The cut off value were 122,136. The detecting ranges were 2ng/mL10g/mL for HBs

25、Ab and 5ng/mL5g/mL for HCcAg. The time of detection were shortened from 1h and 3h to 30min and 40min.4. The parallel test system: Serum 100L; Each R1, 100L and each R2, 5L (after enrichment), add in EP tube totally then react at the condition of pH7.4, 37for 40min. The cut off value were 133,140 of

26、HBsAg and HCcAg. The detecting ranges were 2ng/mL10g/mL for HBsAb and 5ng/mL5g/mL for HCcAg.5. The results of 188 unknown samples analysis: Positive HBsAg serums were 36, 35, 34 and HCcAg were 11, 11, 8 by PCR, parallel test system and ELISA. The mixed 5 standards and 2 multiple infection serums wer

27、e detected correctly. The accordance rates, sensitivity, specificity of parallel test system were 98.4%, 94.4%, 99.3% for HBsAg and 97.9%, 81.8%, 98.9% for HCcAg between PCR. The accordance rates of parallel test system were 95.8%, 98.4% between ELISA for HBsAb and HCcAg.6. Standards analyze results

28、 of repetitive experiments show that the average coefficient variation (CV) of within-run and the inter-day-run of single test systems were 9.7%, 9.8% and 10.3%, 10.1% for HBsAb and HCcAg. The CV of within-run were 10.0%, 10.1% and of the inter-day-run were 10.7%, 10.6% for HBsAb, HCcAg of parallel

29、test system7. The analysis of samples with hemolysis, jaundice and lipemia show theses factors do not interfere the results and the test of positive HAV, TP serums show a good specificity.8. It show that the using effect of reagents and the intensity of fluorescence of the complexes after reacted wh

30、ich stored at 4did not change obviously after 4weeks.Conclusions1. In this study, by using the speciality of the different QDs can be inspired different fluorescence by monochromatic light, we took HBV and HCV which mostly caused PTH as examples and established the method that can detect different antigens in parallel.This research may helpful to the foundation of the antigens detect system in parallel and ensure the transfusion security. Because of the aiming at antigen detect