两种Bt毒素单链抗体制备与抗体检测技术研究
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摘要
苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)合成的杀虫晶体蛋白和用Bt基因改造的转基因植物对多种害虫具有特异性毒杀作用,是环境友好型生物农药,因此被广泛地用于防治农业和卫生害虫。杀虫晶体蛋白进入昆虫的中肠被消化酶转化为活性分子,与昆虫中肠细胞膜上的高亲和性特异受体结合后插入细胞膜造成细胞膜穿孔,破坏细胞内外的渗透平衡。大量水分子进入细胞,造成细胞肿胀、破裂,导致中肠溃烂,从而使害虫死亡；另一部分没有被毒素直接杀死的昆虫,一段时间后也会因细菌感染死亡。
Bt毒素在昆虫中肠pH=9.5的碱性环境下才能发挥作用,因此,该毒素被认为对哺乳动物基本无毒杀作用。随着大量转Bt基因植物直接或间接地进入人类的食物链后,针对Bt毒素的环境及食物安全性评价成为转基因领域中一个新的研究热点。围绕Bt毒素安全检测监控评价技术已经取得了显著的进展。生物测定法、免疫检测法(包括协同凝集反应、酶联免疫吸附、Western-blot、试纸条法、蛋白生物芯片等)、核酸检测法、仪器检测法(包括流式细胞仪、电泳、质谱等)均有研究报道,上述方法在毒素的毒力鉴定和安全检测中发挥了较好作用。但是,目前的方法中,有些方法的技术研发耗时长、费用高；有些与分子机制联系不紧、应用过程影响因素多；有些则存在仪器昂贵及对操作专业人员要求高等问题。抗体检测技术是近年来在医学以及农业技术领域快速发展的一种蛋白分析技术,具有快速、灵敏、方便、易普及的优点,在Bt毒素蛋白快速检测技术领域具有较好的应用前景。
基因工程抗体是指继多克隆抗体和单克隆抗体之后第三代通过基因工程技术制备出的多种新型抗体,其优点是制备快速、成本低、产量大,为抗体检测技术提供了高质量的抗体。单链抗体(single chain variable fragment, scFv)是一种小分子基因工程抗体,由于低或无免疫原性、分子量小、组织穿透力强、成本低、可大规模生产等特点被广泛应用于医学诊断、治疗的研究,而目前国内外利用针对Bt毒素scFv进行免疫检测的研究鲜见报道。在此背景下,本论文利用噬菌体抗体库技术对两种Bt毒素进行亲和淘选获得单链抗体,模拟其三维结构,并以Cry1Ac为对象建立了多种基于单链抗体的免疫检测方法,将基因工程抗体应用于转基因和生物农药检测及植物保护研究领域,拓宽了其应用范围,促进了免疫分析技术在食品安全领域的发展。同时单链抗体与CrylAc结合模型也为单链抗体亲和力成熟及Bt与受体的识别机制等研究提供了参考。本研究主要包括了以下几个方面：
1.构建了天然鼠源单链抗体噬菌体展示库
用Trizol试剂提取未经免疫的Balb/c小鼠脾脏总RNA。利用重链可变区和轻链可变区通用引物通过RT-PCR的方法扩增VH和VL基因,并通过SOE-PCR将两个可变区基因通过连接肽连接,将拼接成功的重组噬菌粒,电转化到感受态细胞中大量增殖,经辅助噬菌体超感染,构建了库容为2.04×107的天然噬菌体抗体库。利用该抗体库筛选到的抗-Bt单链抗体亲和力较低,因此确立Tomlinson.库为筛选库源。
2.建立了抗-Bt毒素单链抗体筛选及检验体系并获得了较高亲和力的单链抗体
以Cry1C和CrylAc为对象,建立了基于噬菌体抗体库淘选、展示及鉴定的技术体系。选用人源Tomlinson抗体库为筛选库,主要采用胰蛋白酶洗脱方式,经过四轮筛选和阳性克隆富集的过程,完成单克隆测序鉴定与ELISA检测验证。
抗-Cry1C的单链抗体筛选制备结果显示：竞争洗脱与胰蛋白酶处理联用的洗脱方法可以使富集率大幅提高。对表达稳定的1C6克隆进行可溶性表达,结果表明在30℃条件下添加1mM的IPTG诱导表达16h可以达到最大表达量。对1C6表达产物进行亲和纯化后用非竞争ELISA法测得亲和常数为(5.8+0.5)×106M-1。
抗-CrylAc的单链抗体筛选制备结果显示：经过四轮亲和淘选后,有一株阳性克隆大量富集,命名为1AC6,此单链抗体在诱导物IPTG浓度为0.8nM时,25℃诱导表达18h表达量最大。1AC6经亲和纯化后测得亲和常数为(3.33±0.05)×107M-1。利用计算机模拟技术,完成了1AC6和CrylAc的分子对接模型,结果显示重链的3个CDR区和轻链的CDR3区参与了与抗原的结合。1AC6的结合区域中有部分氨基酸与氨肽醇受体相似,该结果对进一步开展抗体亲和力成熟和受体结合机制等研究具有参考价值。
3.完成了Bt毒素抗体检测技术构建与验证测试
可溶性1C6单链抗体建立的间接ELISA检测方法结果显示：该方法的线性范围在0.023-4.35μg/mL之间(IC80-IC20)。米粉中Cry1C的添加回收实验结果表明其回收率在89.8%到97.2%之间,变异系数小于5.0%。优于国内报道的检测限为400ng/mL兔源多抗。因此该方法有望运用于检测食品和环境样品中的CrylC毒素。
抗-CrylAc多克隆抗体建立的时间分辨荧光免疫方法结果显示：该方法的最低检测限为0.3ng/mL,优于间接ELISA的检测方法,且检测时间比ELISA明显缩短。在实际样本测试中,黑土、黄壤、红壤、紫色土、潮土5种土壤中Cry1Ac毒素的平均回收率为75.03%,并且随着不同土壤中有机质含量的降低,Cry1Ac毒素蛋白的回收率逐渐升高,该现象对于土壤中Bt吸附的研究有一定的指导意义。
基于融合型1AC6建立的免疫-PCR结果显示：该技术的灵敏度优于单一使用ELISA法检测,辅助荧光检测法检测限可以达到0.2ng/mL,而间接ELISA为40ng/mL。引入噬菌体抗体较好地解决了传统免疫-PCR技术中报告DNA分子的选择、用量及标记方法等干扰实验结果的问题。
基于可溶性1AC6建立的双抗夹心ELISA结果显示：以1AC6为包被抗体,多克隆抗体为检测抗体,确定双抗夹心法工作浓度：1AC6浓度为2.5μg/mL,酶标抗体1600倍稀释时,对靶标抗原CrylAc的检测限为14ng/mL。单克隆抗体建立的此方法检测限优于单链抗体,因此后续需要利用体外亲和力成熟的方法对1AC6进行改造。
基于可溶性1AC6建立的胶体金免疫层析法结果显示：用1AC6制备金标抗体,其最适稳定标记量为20μg/mL。通过结果判定,该快速检测试纸条对CrylAc的检测限为58ng/mL,可以对转基因棉花种子进行定性检测。
利用1AC6单链抗体,建立的免疫-PCR、双抗夹心ELISA与胶体金免疫层析三种检测方法,在样本检测要求中均具有较好的实用价值。
As a Gram-positive, spore-forming bacterium, Bacillus thuringiensis(Bt) was initially characterized by its production of insecticidal parasporal crystals during sporulation, which has become a dominating bio-insecticide and genetic material of genetically modified (GM) crops worldwide. The Cry toxins are specifically toxic to the following insect:Lepidoptera, Coleoptera, Hymenoptera and Diptera. The toxinology study has conformed that the formation of ionic pores in the membrane of insect epithelial midgut cells after Bt toxin combining, leads to insect death. However, some reports showed that Bt toxins could infect human health and soil eco-system. For example, Bt transgenic rice could made a negative effect on bacteria and yeasts, while an active effect on actinomyces. In addition, crisis assessment need to be verified for GM crops, such as their potential allergenicity and environment residual persistence. Therefore, it is necessary to establish a convenient method for Cry toxins determination.
Currently, the detection of Bt-GM crops and Bt residue are mainly performed by instrument analysis, DNA-based and protein-based methods. The reversed phased high-performance liquid chromatography can distinguish the Bt-transgenic and non-transgenic corps. However because of its high cost and professional requirement for its operators, this method is not suited for most testing agency. The DNA-based method like polymerase chain reaction (PCR) focuses on the Bt DNA whether inserted into crops. Although PCR assay is highly sensitive, the complicated process, high cost and difficulty for a high throughput analysis restrict its application. Especially the technology can't evaluate the level of expression or residue. On the other hand, the immunoassay has been recognized as the most successful method, such as ELISA and colloidal gold strip. Nonetheless, it requires specific antibodies for binding targets.
Antibody phage display technology is an attractive alternative to hybridoma technology. It displays recombinant antibodies on the surface of phage particles that can be screened by enabling the phage to interact with immobilized ligands. It reduces batch to batch variation and the single chain variable fragments(scFvs) can be produced using prokaryotic or eukaryotic expression system.Under such background, this dissertation surrounding the topic of development of novel Bt toxin detection methods and has carried out the following research work.
1. Construction and application of a naive mouse scFv phage display library
The total RNA was extracted from spleen cells of non-immunized Balb/c mice. VH and VL were amplified using universal primers. To facilitate the display of antibody molecules in phages, the VH and VL fragments are assembled as single-chain molecules with a (Gly4Ser)3linker peptide resulting in the construction of scFv genes fused to the pⅢ gene in phagemid (pCANTAB5E) by digesting with sfi I and Not I, then transformed into competent E. coli cells via electroporation. The naive single chain antibody library contained2.04×107independent clones. Then the scFv phage library was used to pan scFv against Bt toxin. But the Kaff of functional scFv were too low to detect. So we choose the commercial human single fold scFv library for the next research.
2. Isolation of scFv specific for Bt toxin from human single fold scFv libraries
In this study, scFvs, which could specifically recognize and detect Cry1C, were isolated from naive phage displayed human antibody libraries (Tomlinson I+J) by iterative affinity selection procedure instead of immunization process. Following the competitive eluted phages were treated by trypsin, which disable the phages without scFvs. Meanwhile, with increasing selection pressure, after four rounds of panning,1C6individual scFv was obtained and sequenced. Thereafter, conformed novel anti-Cry1C scFv, was expressed in E. coli HB2151and purified by Ni metal ion affinity chromatography. The Kaff was (5.8±0.5)×106M-1.
Tomlinson J phage antibody library was panned for four rounds of "adhesion-elution-amplification". The specificity of the antigen binding activity of the selected clones was identified by ELISA and the variable genes were analyzed and identified. The1AC6positive clone was obtained, which could bind Cry1Ac specifically. The1AC6was expressed in HB2151and purified by Ni metal ion affinity chromatography. The experimental results showed that the best induced express condition is culturing overnight with0.8mM IPTG, at25℃. The immuno-activities of1AC6was confirmed by competitive inhibition ELISA. The Kaff was (3.33±0.05)×107M-1.
The3-D model of scFv-1AC6was homologically built with SWISS server. Then the interaction between scFv-lAC6and Cry1Ac was docked by GRAMM sever. The result showed that CDR involved in combination with antigen, which further implied that the1AC6could specificity bind with CrylAc.
3. Establish the novel immuno-detection methods for Cry toxin
Immunoassay is widely used in the area of rapid toxin detection. The research established several novel detection methods for Cry toxin based on polyclonal antibodies and scFv.
An indirect competitive ELISA assay of1C6was developed for the determination of CrylC toxin in the range from0.023to4.35μg/mL, and50%inhibition concentration (IC50) was0.39μg/mL. This approach showed ignorable cross-reactivity with toxin CrylAc and Cry1B. The ic-ELISA approach was exploited for the determination of CrylC in spiked ground rice samples with a mean recovery rate of92.5%and coefficient of variation less than5.0%. This study proves that phage display libraries provide a valuable system for the low-cost, rapid and continuous generation of specific antibody fragments directed against Cry1C toxin target and develop a simple detection method. The results show that1C6scFv could be a valuable tool for detection of CrylC in food and agricultural samples.
To establish a rapid detection of CrylAc residue in soil, the time-resolved fluorescence immunoassay (TRFIA) using goat anti-rabbit IgG conjugated with a Eu-N1as a tracer was developed to quantify CrylAc in five types of soil (black soil, yellow soil, red soil, purplish soil, fluvo-aquic soil). The results showed that the limit of detection of Cry1Ac achieved to0.3ng/mL by TRFIA, the detection range ranged from0.3to500.0ng/mL, and the coefficient of variations were both below10.0%. The mean recovery of CrylAc in soil was75.03%. The recovery of Cry1Ac in black soil with the highest organic matter content was the lowest, and the recoveries of CrylAc were reduction with the increase of the organic matter content in soil. The Cry1Ac residue detected by TRFIA and ELISA was a high correlation, and the coefficient of correlation was0.9967. Therefore, TRFIA was a simple, specific, highly sensitive, quantitative assay for Cry1Ac in soil.
Phage display is a very powerful technique since the selected phages maintain a physical link between the displayed scFv (phenotype) and the encoding gene (genotype). The novel immuno-PCR detection method was set up based on the character. The immuno-PCR assay showed high sensitivity with minimum detection limit of0.2ng/mL and found to be200times more sensitive than ic-ELISA. Under the optimized assay conditions, Cry1Ac protein can be determined in the concentration ranged from0.2to100ng/mL. As results showed this assay could be a powerful tool for the detection of trace amounts of Cry1Ac protein.
The double-antibody sandwich ELISA for detection of insecticidal protein Cry1Ac was developed. The1AC6was used as coating antibody and Cry1Ac-IgG as detecting antibody. The best working conditions were optimized for double-antibody sandwich ELISA. The results showed that the concentration of1AC6were2.5μg/mL and the dilution of IgG-HRP was1:1600are the best conditions. The sandwich ELISA has a detection limit of14ng/mL.
With the colloidal gold labeled1AC6, immnnochromatography assay for detection of insecticidal protein CrylAc were developed. IgG coated on the nitrocellulose membrane for the test line, and goat anti-rabbit antibody was blotted on the nitrocellulose membrane for the control line. The detection can be completed within10min and has a detection limit of58ng/mL for CrylAc. These results suggest the possibility for detecting transgenic Bt-crop with simply and reliably characters.
Bt毒素在昆虫中肠pH=9.5的碱性环境下才能发挥作用,因此,该毒素被认为对哺乳动物基本无毒杀作用。随着大量转Bt基因植物直接或间接地进入人类的食物链后,针对Bt毒素的环境及食物安全性评价成为转基因领域中一个新的研究热点。围绕Bt毒素安全检测监控评价技术已经取得了显著的进展。生物测定法、免疫检测法(包括协同凝集反应、酶联免疫吸附、Western-blot、试纸条法、蛋白生物芯片等)、核酸检测法、仪器检测法(包括流式细胞仪、电泳、质谱等)均有研究报道,上述方法在毒素的毒力鉴定和安全检测中发挥了较好作用。但是,目前的方法中,有些方法的技术研发耗时长、费用高；有些与分子机制联系不紧、应用过程影响因素多；有些则存在仪器昂贵及对操作专业人员要求高等问题。抗体检测技术是近年来在医学以及农业技术领域快速发展的一种蛋白分析技术,具有快速、灵敏、方便、易普及的优点,在Bt毒素蛋白快速检测技术领域具有较好的应用前景。
基因工程抗体是指继多克隆抗体和单克隆抗体之后第三代通过基因工程技术制备出的多种新型抗体,其优点是制备快速、成本低、产量大,为抗体检测技术提供了高质量的抗体。单链抗体(single chain variable fragment, scFv)是一种小分子基因工程抗体,由于低或无免疫原性、分子量小、组织穿透力强、成本低、可大规模生产等特点被广泛应用于医学诊断、治疗的研究,而目前国内外利用针对Bt毒素scFv进行免疫检测的研究鲜见报道。在此背景下,本论文利用噬菌体抗体库技术对两种Bt毒素进行亲和淘选获得单链抗体,模拟其三维结构,并以Cry1Ac为对象建立了多种基于单链抗体的免疫检测方法,将基因工程抗体应用于转基因和生物农药检测及植物保护研究领域,拓宽了其应用范围,促进了免疫分析技术在食品安全领域的发展。同时单链抗体与CrylAc结合模型也为单链抗体亲和力成熟及Bt与受体的识别机制等研究提供了参考。本研究主要包括了以下几个方面：
1.构建了天然鼠源单链抗体噬菌体展示库
用Trizol试剂提取未经免疫的Balb/c小鼠脾脏总RNA。利用重链可变区和轻链可变区通用引物通过RT-PCR的方法扩增VH和VL基因,并通过SOE-PCR将两个可变区基因通过连接肽连接,将拼接成功的重组噬菌粒,电转化到感受态细胞中大量增殖,经辅助噬菌体超感染,构建了库容为2.04×107的天然噬菌体抗体库。利用该抗体库筛选到的抗-Bt单链抗体亲和力较低,因此确立Tomlinson.库为筛选库源。
2.建立了抗-Bt毒素单链抗体筛选及检验体系并获得了较高亲和力的单链抗体
以Cry1C和CrylAc为对象,建立了基于噬菌体抗体库淘选、展示及鉴定的技术体系。选用人源Tomlinson抗体库为筛选库,主要采用胰蛋白酶洗脱方式,经过四轮筛选和阳性克隆富集的过程,完成单克隆测序鉴定与ELISA检测验证。
抗-Cry1C的单链抗体筛选制备结果显示：竞争洗脱与胰蛋白酶处理联用的洗脱方法可以使富集率大幅提高。对表达稳定的1C6克隆进行可溶性表达,结果表明在30℃条件下添加1mM的IPTG诱导表达16h可以达到最大表达量。对1C6表达产物进行亲和纯化后用非竞争ELISA法测得亲和常数为(5.8+0.5)×106M-1。
抗-CrylAc的单链抗体筛选制备结果显示：经过四轮亲和淘选后,有一株阳性克隆大量富集,命名为1AC6,此单链抗体在诱导物IPTG浓度为0.8nM时,25℃诱导表达18h表达量最大。1AC6经亲和纯化后测得亲和常数为(3.33±0.05)×107M-1。利用计算机模拟技术,完成了1AC6和CrylAc的分子对接模型,结果显示重链的3个CDR区和轻链的CDR3区参与了与抗原的结合。1AC6的结合区域中有部分氨基酸与氨肽醇受体相似,该结果对进一步开展抗体亲和力成熟和受体结合机制等研究具有参考价值。
3.完成了Bt毒素抗体检测技术构建与验证测试
可溶性1C6单链抗体建立的间接ELISA检测方法结果显示：该方法的线性范围在0.023-4.35μg/mL之间(IC80-IC20)。米粉中Cry1C的添加回收实验结果表明其回收率在89.8%到97.2%之间,变异系数小于5.0%。优于国内报道的检测限为400ng/mL兔源多抗。因此该方法有望运用于检测食品和环境样品中的CrylC毒素。
抗-CrylAc多克隆抗体建立的时间分辨荧光免疫方法结果显示：该方法的最低检测限为0.3ng/mL,优于间接ELISA的检测方法,且检测时间比ELISA明显缩短。在实际样本测试中,黑土、黄壤、红壤、紫色土、潮土5种土壤中Cry1Ac毒素的平均回收率为75.03%,并且随着不同土壤中有机质含量的降低,Cry1Ac毒素蛋白的回收率逐渐升高,该现象对于土壤中Bt吸附的研究有一定的指导意义。
基于融合型1AC6建立的免疫-PCR结果显示：该技术的灵敏度优于单一使用ELISA法检测,辅助荧光检测法检测限可以达到0.2ng/mL,而间接ELISA为40ng/mL。引入噬菌体抗体较好地解决了传统免疫-PCR技术中报告DNA分子的选择、用量及标记方法等干扰实验结果的问题。
基于可溶性1AC6建立的双抗夹心ELISA结果显示：以1AC6为包被抗体,多克隆抗体为检测抗体,确定双抗夹心法工作浓度：1AC6浓度为2.5μg/mL,酶标抗体1600倍稀释时,对靶标抗原CrylAc的检测限为14ng/mL。单克隆抗体建立的此方法检测限优于单链抗体,因此后续需要利用体外亲和力成熟的方法对1AC6进行改造。
基于可溶性1AC6建立的胶体金免疫层析法结果显示：用1AC6制备金标抗体,其最适稳定标记量为20μg/mL。通过结果判定,该快速检测试纸条对CrylAc的检测限为58ng/mL,可以对转基因棉花种子进行定性检测。
利用1AC6单链抗体,建立的免疫-PCR、双抗夹心ELISA与胶体金免疫层析三种检测方法,在样本检测要求中均具有较好的实用价值。
As a Gram-positive, spore-forming bacterium, Bacillus thuringiensis(Bt) was initially characterized by its production of insecticidal parasporal crystals during sporulation, which has become a dominating bio-insecticide and genetic material of genetically modified (GM) crops worldwide. The Cry toxins are specifically toxic to the following insect:Lepidoptera, Coleoptera, Hymenoptera and Diptera. The toxinology study has conformed that the formation of ionic pores in the membrane of insect epithelial midgut cells after Bt toxin combining, leads to insect death. However, some reports showed that Bt toxins could infect human health and soil eco-system. For example, Bt transgenic rice could made a negative effect on bacteria and yeasts, while an active effect on actinomyces. In addition, crisis assessment need to be verified for GM crops, such as their potential allergenicity and environment residual persistence. Therefore, it is necessary to establish a convenient method for Cry toxins determination.
Currently, the detection of Bt-GM crops and Bt residue are mainly performed by instrument analysis, DNA-based and protein-based methods. The reversed phased high-performance liquid chromatography can distinguish the Bt-transgenic and non-transgenic corps. However because of its high cost and professional requirement for its operators, this method is not suited for most testing agency. The DNA-based method like polymerase chain reaction (PCR) focuses on the Bt DNA whether inserted into crops. Although PCR assay is highly sensitive, the complicated process, high cost and difficulty for a high throughput analysis restrict its application. Especially the technology can't evaluate the level of expression or residue. On the other hand, the immunoassay has been recognized as the most successful method, such as ELISA and colloidal gold strip. Nonetheless, it requires specific antibodies for binding targets.
Antibody phage display technology is an attractive alternative to hybridoma technology. It displays recombinant antibodies on the surface of phage particles that can be screened by enabling the phage to interact with immobilized ligands. It reduces batch to batch variation and the single chain variable fragments(scFvs) can be produced using prokaryotic or eukaryotic expression system.Under such background, this dissertation surrounding the topic of development of novel Bt toxin detection methods and has carried out the following research work.
1. Construction and application of a naive mouse scFv phage display library
The total RNA was extracted from spleen cells of non-immunized Balb/c mice. VH and VL were amplified using universal primers. To facilitate the display of antibody molecules in phages, the VH and VL fragments are assembled as single-chain molecules with a (Gly4Ser)3linker peptide resulting in the construction of scFv genes fused to the pⅢ gene in phagemid (pCANTAB5E) by digesting with sfi I and Not I, then transformed into competent E. coli cells via electroporation. The naive single chain antibody library contained2.04×107independent clones. Then the scFv phage library was used to pan scFv against Bt toxin. But the Kaff of functional scFv were too low to detect. So we choose the commercial human single fold scFv library for the next research.
2. Isolation of scFv specific for Bt toxin from human single fold scFv libraries
In this study, scFvs, which could specifically recognize and detect Cry1C, were isolated from naive phage displayed human antibody libraries (Tomlinson I+J) by iterative affinity selection procedure instead of immunization process. Following the competitive eluted phages were treated by trypsin, which disable the phages without scFvs. Meanwhile, with increasing selection pressure, after four rounds of panning,1C6individual scFv was obtained and sequenced. Thereafter, conformed novel anti-Cry1C scFv, was expressed in E. coli HB2151and purified by Ni metal ion affinity chromatography. The Kaff was (5.8±0.5)×106M-1.
Tomlinson J phage antibody library was panned for four rounds of "adhesion-elution-amplification". The specificity of the antigen binding activity of the selected clones was identified by ELISA and the variable genes were analyzed and identified. The1AC6positive clone was obtained, which could bind Cry1Ac specifically. The1AC6was expressed in HB2151and purified by Ni metal ion affinity chromatography. The experimental results showed that the best induced express condition is culturing overnight with0.8mM IPTG, at25℃. The immuno-activities of1AC6was confirmed by competitive inhibition ELISA. The Kaff was (3.33±0.05)×107M-1.
The3-D model of scFv-1AC6was homologically built with SWISS server. Then the interaction between scFv-lAC6and Cry1Ac was docked by GRAMM sever. The result showed that CDR involved in combination with antigen, which further implied that the1AC6could specificity bind with CrylAc.
3. Establish the novel immuno-detection methods for Cry toxin
Immunoassay is widely used in the area of rapid toxin detection. The research established several novel detection methods for Cry toxin based on polyclonal antibodies and scFv.
An indirect competitive ELISA assay of1C6was developed for the determination of CrylC toxin in the range from0.023to4.35μg/mL, and50%inhibition concentration (IC50) was0.39μg/mL. This approach showed ignorable cross-reactivity with toxin CrylAc and Cry1B. The ic-ELISA approach was exploited for the determination of CrylC in spiked ground rice samples with a mean recovery rate of92.5%and coefficient of variation less than5.0%. This study proves that phage display libraries provide a valuable system for the low-cost, rapid and continuous generation of specific antibody fragments directed against Cry1C toxin target and develop a simple detection method. The results show that1C6scFv could be a valuable tool for detection of CrylC in food and agricultural samples.
To establish a rapid detection of CrylAc residue in soil, the time-resolved fluorescence immunoassay (TRFIA) using goat anti-rabbit IgG conjugated with a Eu-N1as a tracer was developed to quantify CrylAc in five types of soil (black soil, yellow soil, red soil, purplish soil, fluvo-aquic soil). The results showed that the limit of detection of Cry1Ac achieved to0.3ng/mL by TRFIA, the detection range ranged from0.3to500.0ng/mL, and the coefficient of variations were both below10.0%. The mean recovery of CrylAc in soil was75.03%. The recovery of Cry1Ac in black soil with the highest organic matter content was the lowest, and the recoveries of CrylAc were reduction with the increase of the organic matter content in soil. The Cry1Ac residue detected by TRFIA and ELISA was a high correlation, and the coefficient of correlation was0.9967. Therefore, TRFIA was a simple, specific, highly sensitive, quantitative assay for Cry1Ac in soil.
Phage display is a very powerful technique since the selected phages maintain a physical link between the displayed scFv (phenotype) and the encoding gene (genotype). The novel immuno-PCR detection method was set up based on the character. The immuno-PCR assay showed high sensitivity with minimum detection limit of0.2ng/mL and found to be200times more sensitive than ic-ELISA. Under the optimized assay conditions, Cry1Ac protein can be determined in the concentration ranged from0.2to100ng/mL. As results showed this assay could be a powerful tool for the detection of trace amounts of Cry1Ac protein.
The double-antibody sandwich ELISA for detection of insecticidal protein Cry1Ac was developed. The1AC6was used as coating antibody and Cry1Ac-IgG as detecting antibody. The best working conditions were optimized for double-antibody sandwich ELISA. The results showed that the concentration of1AC6were2.5μg/mL and the dilution of IgG-HRP was1:1600are the best conditions. The sandwich ELISA has a detection limit of14ng/mL.
With the colloidal gold labeled1AC6, immnnochromatography assay for detection of insecticidal protein CrylAc were developed. IgG coated on the nitrocellulose membrane for the test line, and goat anti-rabbit antibody was blotted on the nitrocellulose membrane for the control line. The detection can be completed within10min and has a detection limit of58ng/mL for CrylAc. These results suggest the possibility for detecting transgenic Bt-crop with simply and reliably characters.
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