摘要
目的
弓形虫病是一种严重危害人类健康和影响优生优育的人畜共患疾病。ELISA法是目前诊断弓形虫感染的常规检测方法,主要是通过检测特异性IgG(免疫力水平指标)和IgM(近期感染指标)抗体的有无来判断机体的感染情况,但该方法存在不能同步检测IgG和IgM抗体,检测时间长,耗费试剂多,操作繁琐等缺点。
量子点是一种新型荧光标记材料,与传统荧光染料相比具有以下优点:激发波长范围较宽,同一波长的光能激发不同大小的量子点;发射波长窄而对称,同时使用具有不同光谱特征的量子点时,发射光谱间重叠较少;不同粒径和组成的量子点其发射波长不同。因此,应用不同发射波长的量子点对不同的待检分析物同时进行标记,可以达到多个指标同时检测的目的。
本研究将量子点标记技术与免疫磁珠技术相结合,研制了一种可同步检测抗弓形虫IgG和IgM抗体的检测体系,本方法不仅操作简便快速,还具有较高的特异性和敏感性,可用于弓形虫感染的筛查。
方法:
1.抗原固化磁性微球
采用碳二亚胺交联法将弓形虫重组抗原固化在羧基磁性微球表面。通过EDC和NHS溶液活化磁珠表面羧基后,活化的羧基再与抗原上的氨基进行反应,从而将弓形虫抗原固化在磁性微球表面,获得免疫磁珠。通过对磁性微球的粒径、EDC/NHS活化剂浓度等固化条件进行选择优化,建立稳定、高效的免疫磁性微球固化方法。
2.量子点标记二抗
采用碳二亚胺交联法将羧基化CdS/ZnS量子点与抗体进行标记.分别以发射波长为580nm、618nm的量子点分别标记羊抗人IgG和羊抗人IgM抗体,研究pH值,二抗加入浓度,反应时间对量子点发光的影响,制备适用于抗弓形虫IgG和IgM抗体同步检测体系的量子点标记二抗试剂。
3.分别建立抗弓形虫IgG、IgM抗体检测方法
以固化了弓形虫抗原的磁性微球作为固相载体,量子点标记二抗作为检测抗体,采用间接法原理,分别建立抗弓形虫IgG和IgM抗体的检测方法,并对检测条件进行优化。分别检测人抗弓形虫IgG、IgM标准品,观察本方法的线性范围、灵敏度、重复性和准确度。收集临床标本,分别用本方法与进口ELISA试剂盒进行检测并对结果进行比较,观察两种检测方法的结果有无统计学差异。
4.抗弓形虫IgG和IgM抗体同步检测体系的构建
在已建立的单个抗体检测方法的基础上,对关键检测条件进行统一和优化,构建抗弓形虫IgG和IgM抗体同步检测体系。同步检测人抗弓形虫IgG、IgM标准品,观察该体系的线性范围、灵敏度、重复性和准确度。用已构建的同步检测体系对280例临床标本进行同步检测,并将结果与进口ELISA试剂盒进行比较,探讨多指标同步检测的可行性。
主要结果
一、方法学建立
(1)抗原固化磁性微球
选择粒径为3μm的羧基磁性微球作为固相载体,加入EDC(6mg/ml)和NHS(4mg/ml)溶液进行活化,弓形虫抗原的最适固化浓度为50μg/mL,固化效率为54.2%。
(2)量子点标记二抗
实验结果表明,量子点标记二抗的最适反应pH值为6.0,羊抗人IgG、IgM抗体的最适标记浓度分别为40μg/mL、50μg/mL。
(3)抗弓形虫IgG、IgM抗体检测方法的建立
分别建立了抗弓形虫IgG和IgM抗体检测方法,经过实验优化,量子点羊抗人IgG、IgM的最适工作浓度分别为1:50、1:100,待测血清最适稀释度均为1:100。通过检测200例健康人血清样本确定了抗弓形虫IgG抗体和IgM抗体Cutoff值分别为: 0.45、0.44。
(4)同步检测抗弓形虫IgG和IgM抗体检测体系的建立
同步检测抗弓形虫IgG和IgM抗体,量子点标记羊抗人IgG、IgM的最适工作浓度分别为1:200、1:100,待测血清最适稀释度为1:100。检测200例健康人血清样本确定了抗弓形虫IgG抗体和IgM抗体Cutoff值分别为:0.55、0.51。同步检测抗弓形虫IgG和IgM抗体两个指标,仅需30min就可完成整个检测过程,而常规ELISA方法一次只能检测单个指标,完成两个指标的检测往往需要4h。
二、方法学评价
(1)抗弓形虫IgG、IgM抗体检测方法的方法学评价按已建立的方法分别检测人抗弓形虫IgG、IgM标准品,其最低检测限分别为:3.04IU/ml、3.11 IU/ml。批内重复性实验平均CV分别5.2%、5.4%,天间重复性实验平均CV分别为6.5%,6.6%。准确性实验偏倚系数分别为:6.0%,6.3%。20例其它TORCH系列阳性血清标本与本方法均无交叉反应。阻断实验结果显示,弓形虫IgG和IgM抗体的阻断率均在50%以上。
(2)同步检测抗弓形虫IgG、IgM抗体检测体系的方法学评价
同步检测人抗弓形虫IgG和IgM标准品,其最低检测限分别为:4.21IU/ml、4.32IU/ml;批内重复性实验平均CV分别为: 6.0%、6.2%;天间重复性实验平均CV为7.2%、7.4%;准确性实验偏倚系数分别为:7.6%,7.2%。
三、方法学比较
(1)抗弓形虫IgG、IgM抗体检测方法的方法学评价
收集临床标本,用已建立的抗弓形虫IgG、IgM抗体检测方法分别进行检测,并与进口ELISA试剂盒检测结果比较。抗弓形虫IgG和IgM抗体检测结果的符合率分别为: 96.5%、96.8%,采用SPSS10.0软件对数据进行配对χ2检验分析, P值均>0.05,认为两种方法分别检测抗弓形虫IgG和IgM抗体的测定结果无显著性差异。
(2)同步检测抗弓形虫IgG和IgM抗体检测体系的方法学评价
收集临床标本,用已构建的抗弓形虫IgG、IgM抗体同步检测体系进行同步检测,并与进口ELISA试剂盒检测结果比较。弓形虫IgG和IgM抗体检测结果的符合率分别为: 96.4%,97.1%,采用SPSS10.0软件对数据进行配对χ2检验分析, P值均>0.05,认为本方法同步检测抗弓形虫IgG和IgM抗体的测定结果与ELISA法无显著性差异。
结论
1.以磁性微球作为固相载体,在其表面成功固化了弓形虫重组抗原,固化效率最高可达54.2%。利用磁珠可快速富集的特性,在加快了检测速度同时,也极大的提高了检测灵敏度。本法同步检测抗弓形虫IgG和IgM抗体仅需30 min,较ELISA法的2.5h明显加快。
2.建立的抗弓形虫IgG、IgM抗体单指标检测方法,其灵敏度、检测范围、重复性、稳定性等达到临床初步应用的要求,为同步检测体系的建立奠定了基础。
3.选用不同发射波长的量子点分别标记不同二抗,经同一激发光激发,发射两种不同波长的光,实现了多指标的同步检测,为基于量子点标记同步检测更多抗体提供了技术支持。
4.构建的抗弓形虫IgG和IgM抗体同步检测体系,具有特异性较好、速度快、操作简便、灵敏度高等优点,为简化操作步骤、缩短报告时间创造了条件,也为进一步研制TORCH感染多种病原体的同步检测提供了一种新的技术手段。
Objective
Toxoplasmosis is an anthropozoonosis that can seriously harm human health and the promotion of good prenatal and postnatal care. ELISA is currently the conventional detection method to diagnose the infection of toxoplasma. It can determine the infection in the body mainly through the detection of specific IgG (index to indicate the immunity level) and IgM (index to indicate recent infection). However, the method can not detect IgG and IgM antibody simultaneously; furthermore, it requires a long period of detection and consumes a large amount of reagent with complicated operation procedures.
Quantum dot is a newly-developed material used in fluorescence labeling and it possesses the following advantages compared with traditional fluorescent dyestuff: it can arouse a wider range of wavelength, the light with the same wavelength can arouse different sizes of quantum dot; the emission wavelength is narrow and symmetrical, the overlapping part between emission spectrum is small when quantum dots with different spectral characteristics are used simultaneously; the quantum dots with different particle diameters and compositions have different emission wavelength. Therefore, the application of quantum dots with different emission wavelength in the labeling of material to be detected and analyzed can detect several indexes at the same time.
The quantum dot labeling technique and immune magnetic bead technique were combined together in this research. A detection system which can detect anti-toxoplasma IgG and IgM antibody simultaneously was developed. The method can be operated conveniently and rapidly with high level of specificity and sensitivity. It can be used in the screening of toxoplasma infection.
Method:
1. Antigen solidified magnetic microsphere
The carbodiimide crosslinking method was adopted to solidify the recombined toxoplasma antigen onto the surface of the carboxyl magnetic microsphere. The carboxyl on the surface of the magnetic microsphere was activated by the EDC and NHS solution. Subsequently, the activated carboxyl reacted with the amino-group on the antigen in order to solidify the toxoplasma antigen onto the surface of magnetic microsphere and in order to obtain the immune magnetic bead. A stable and highly-efficient solidification method for immune magnetic microsphere can be established through the optimization of solidification conditions, including the particle diameter of the magnetic microsphere and the concentration of activating agents such as EDC/NHS.
2. Labeling of second antibody using quantum dot
The carboxylated CdS/ZnS quantum dot and antibody were labeled using the carbodiimide crosslinking method. The quantum dots with the emission wavelength of 580nm and 618nm were used to label the sheep anti-human IgG antibody and the sheep anti-human IgM antibody. The effect of pH value, the application concentration of second antibody and the reaction time on the irradiancy of quantum dot was investigated. Quantum dot which was suitable for the synchronizing detection system for anti-toxoplasma IgG and IgM antibody was prepared in order to label the second antibody reagent.
3. Establishment of independent detection methods for anti-toxoplasma IgG and IgM antibody
The detection methods for anti-toxoplasma IgG and IgM antibody were established according to the theory of indirect method by taking the magnetic microsphere that had solidified the toxoplasma antigen as the solid phase carrier and by taking the second antibody that had been labeled by quantum dot as the detection antibody. Besides, the detection conditions were optimized. The standard samples of human anti-toxoplasma IgG and IgM were detected. The linear scope, sensitivity, repeatability and accuracy of this method were observed. Clinical samples were collected and were detected using this method and imported ELISA kit. The results were compared in order to determine if there was statistical difference between the results obtained using the two methods.
4. Establishment of the synchronizing detection system of anti-toxoplasma IgG and IgM antibody
Based on the established detection method used in single antibody, the key detection conditions were unified and optimized. The synchronizing detection system of anti-toxoplasma IgG and IgM antibody was established. The standard samples of human anti-toxoplasma IgG and IgM were detected simultaneously. The linear scope, sensitivity, repeatability and accuracy of this system were observed. 280 clinical samples were detected simultaneously using the established synchronizing detection system and the results were compared to the results obtained using the imported ELISA kit. The feasibility of the synchronizing detection of several indexes was discussed.
Main results
1. Establishment of methodology
(1) Solidification of magnetic microsphere by antigen The carboxyl magnetic microsphere with a particle diameter of 3μm was selected as the solid phase carrier. EDC (6mg/ml) and NHS (4mg/ml) were used for activation. The optimal solidification concentration for anti-toxoplasma was 50μg/mL with a solidification efficiency of 54.2%.
(2) Labeling of second antibody by quantum dot
The experimental results show that quantum dots marking second antibody the optimum reaction pH value of 6.0, sheep anti-human IgG, IgM concentrations were optimal labelling concentration 40μg/mL, 50μg/mL,respectively.
(3) Establishment of the detection method for anti-toxoplasma IgG and IgM antibody
The detection methods used for anti-toxoplasma IgG and IgM antibody were established separately. Based on the optimization of the experiment, the optimal operation concentration of quantum dot sheep anti-human IgG and IgM was 1:50 and 1:100, respectively. The optimal dilution rate for the serum to be tested was 1:100. Based on the study on the detection of the serum samples from 200 healthy people, the Cutoff value of anti-toxoplasma IgG and IgM antibody was 0.45 and 0.44, respectively.
(4) Establishment of the detection system for the synchronizing detection of anti-toxoplasma IgG and IgM antibody
Based on the synchronizing detection of anti-toxoplasma IgG and IgM antibody, the optimal operation concentration for the labeling of sheep anti-human IgG and IgM by quantum dot was 1:200 and 1:100, respectively. The optimal dilution rate for the serum to be tested was 1:100. Based on the study on the detection of the serum samples from 200 healthy people, the Cutoff value of anti-toxoplasma IgG and IgM antibody was 0.55 and 0.51, respectively. It took only 30min to detect the anti-toxoplasma IgG and IgM antibody while the conventional ELISA method can only detect one index each time. It usually took 4h to complete the two indexes.
2. Methodological evaluation
(1) Methodological evaluation on the detection method of anti-toxoplasma IgG and IgM antibody
Standard preparations of anti-Toxoplasma IgG and IgM antibody were taken to detect repeatedly for ten times with the coefficients of variation inter the batch were 5.2%, 5.4%, the minimum detection limit were: 3.04 IU/ml, 3.11 IU /ml. The standard preparations above were taken o detect once everyday , a total of ten times with the coefficients of variation between the batches were 6.5%, 6.6%. The accuracy of experimental coefficients of variation were: 6.0%, 6.3%. The 20 cases of other positive serum samples were no cross-reaction by these methods. Blocking experiments showed that IgG and IgM Toxoplasma gondii antibodies in the blocking rate of more than 50%.
(2) Methodological evaluation of the synchronizing detection method for anti- toxoplasma IgG and IgM antibody
Standard preparations of anti- toxoplasma IgG and IgM antibody were taken to synchronizing detect repeatedly for ten times at the same time with the coefficients of variation inter the batch were 6.0%, 6.2%, the minimum detection limit were: 4.21IU/ml, 4.32IU/ml. The standard preparations above were taken o detect once everyday ,a total of ten times with the coefficients of variation between the batches were 7.2%, 7.4%. The accuracy of experimental coefficients of variation were: 7.6%, 7.2%.
3. Methodological comparison
(1) Methodological comparison on the detection method of anti-toxoplasma IgG and IgM antibody
After the collection of clinical samples, the samples were detected using the established detection method for anti-toxoplasma IgG and IgM antibody and the results were compared to the results obtained using the imported ELISA kit. The coincidence rate for the detection results of anti-toxoplasma IgG and IgM antibody was 96.5% and 96.8%, respectively. The data were analyzed according to the pairedχ2 test using the SPSS10.0 software. All the P values were larger than 0.05. No significant difference was found between the detection results of anti-toxoplasma IgG and IgM antibody using the two methods.
(2) Methodological comparison of the synchronizing detection method for anti-toxoplasma IgG and IgM antibody
After the collection of clinical samples, the samples were detected using the established synchronizing detection system for anti-toxoplasma IgG and IgM antibody and the results were compared to the results obtained using the imported ELISA kit. The coincidence rate for the detection results of anti-toxoplasma IgG and IgM antibody was 96.4% and 97.1%, respectively. The data were analyzed according to the pairedχ2 test using the SPSS10.0 software. All the P values were larger than 0.05. No significant difference was found between the detection results of anti-toxoplasma IgG and IgM antibody using the synchronizing detection system.
Conclusion
1. If the magnetic microsphere was use as solid phase carrier, its surface successfully solidify the recombined toxoplasma antigen with the highest solidification efficiency of 54.2%. By taking advantage of the rapid enrichment of magnetic bead, not only the detection speed was accelerated but also the detection sensitivity was enhanced greatly. It only took 30min to detect anti-toxoplasma IgG and IgM antibody simultaneously using this method; it was much faster than that of ELISA which took 2.5h to complete the whole process.
2. The sensitivity, detection scope, repeatability and stability of the established detection method for single index of anti-toxoplasma IgG and IgM antibody had meet the requirements of preliminary clinical application. That had laid the foundation for the synchronizing detection system.
3. Different second antibodies were labeled using quantum dots with different emission wavelengths. The same exciting light was used in the arousing process and the light with two different wavelengths was emitted. The synchronizing detection of several indexes was realized and that provided technical support for the synchronizing detection of more antibodies based on the quantum dot labeling.
4. The established synchronizing detection system for anti-toxoplasma IgG and IgM antibody had excellent specificity, high speed, convenient operation and high level of sensitivity. It created the opportunities to simplify the operation procedure and to shorten the report time. It also provided a new technical methods to further develop the synchronizing detection for several pathogen infected by TORCH.
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