免疫层析技术在口蹄疫POCT中的应用研究
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摘要
即时检验(point-of-care testing，POCT)是指接近病患进行的一种快速检测分析技术，它具有操作简便、结果判定快速，标本用量少、试剂稳定且便于保存和携带等优点。已经被广泛用于临床检测。口蹄疫是全球最重要的动物疫病之一，其大规模流行给畜牧业生产和进出口贸易造成巨大经济损失,准确的检测是防制口蹄疫的重要环节。口蹄疫的检测技术中病毒分离、ELISA和RT-PCR等方法要求有熟练的试验操作人员、特定的试验室和专用仪器设备，因而检测费用高。发展新型的POCT技术作为口蹄疫诊断方法的有益补充，已成为一个迫切需要解决的问题，也必将是未来诊断技术发展的方向。在现有的POCT技术中，以免疫层析技术为依托的快速诊断试纸条是最为快速简便、被大家认可和接受的方法。纳米技术和传统技术如免疫层析试纸条技术融合后,试纸条在POCT领域显示出巨大的优势。本研究利用胶体金、胶乳微球等标记物，分别开展针对口蹄疫病原、抗体、核酸诊断的新型免疫层析技术研究，开发相应的快速诊断试纸条的产品，对未来口蹄疫诊断在POCT领域的发展方向进行了探讨。
1．建立口蹄疫病毒O、A、Asia1型定型胶体金免疫层析方法
关于口蹄疫病原诊断的报道，大部分集中在口蹄疫与其临床症状相似的病原的鉴别诊断，对其血清型分型检测非常少。本研究利用柠檬酸三钠还原法制备胶体金颗粒，口蹄疫标准毒株O/AV99(L)、A/AV88(L)、Asia1/YN/BSh/58/B及流行株O/China99，A/AF72和Asia1/China05制备的多克隆抗体分别作为胶体金标记捕获抗体和硝酸纤维素膜上检测抗体，分别喷涂于玻璃纤维与硝酸纤维素膜，制备形成O、A、Asia1三种型别试纸条，三种试纸条组合形成定型诊断试剂盒。结果显示：试剂盒可检测到7.8×10~4LD50（1：128倍稀释乳鼠毒）的病毒量；对与口蹄疫临床症状相似疫病的病原，如猪水泡病（SVD）、水泡性口炎(VS)、猪水泡疹(VES)等抗原无交叉反应；对已知O、A、Asia1型和阴性样本的符合率分别为92.45%、91.66%、92.75%、100%定型准确率94.58%。这是国外及国内首个可同时鉴别口蹄疫三种血清型的试纸条，已获得国家发明专利。
2．建立口蹄疫病毒口蹄疫O、Asia1分型彩色胶乳免疫层析方法建立
为满足有限样品在一个反应中实现多重分析的要求，利用彩色胶乳微球做为标记物，结合免疫层析技术平台，研制口蹄疫O、Asia1分型彩色胶乳检测试纸条。选择两种不同颜色的单分散胶乳，用纯化的Asia1/XJ/AKT/03流行毒株兔抗体制备蓝色免疫胶乳；将纯化的O/GD/NX/92流行毒株兔抗体制备红色免疫胶乳。将两种免疫胶乳分别喷涂于不同玻璃纤维上，晾干叠加制成彩色胶乳反应垫。再分别将纯化的Asia1/YN/BSh和AV(99)L标准毒株的豚鼠抗体固定于同一硝酸纤维素膜的不同区域上作为两条检测带。最后组装成口蹄疫O、Asia1分型彩色胶乳试纸条。通过对阳性质控样品的检测，显示试纸条可检测到3.9×104LD50病毒含量；在检测与口蹄疫临床症状相似病原时无交叉反应，特异性好；不同批次间、同一批次内的试纸条，在检测阴、阳性参考样品时，结果完全一致；试纸条对检测已知血清型的74份田间样品的符合性实验中，O型、Asia1型样品的阳性符合率分别为95.24%，96.30%，阴性样品符合率为100%。本试验研发的口蹄疫O、Asia1型分型彩色胶乳试纸条具有很好的灵敏度、特异性、重复性和符合性，而且可以通过不同颜色进行定型检测，适合国内流行的口蹄疫病原型别的分型检测。
3．建立口蹄疫O型抗体检测胶体金免疫层析方法。
国内已报道的O型免疫抗体的试纸条，仅能进行猪血清中O型抗体免疫合格水平的定性检测，无法对牛、羊等其它免疫动物进行检测。本实验分别选用牛源和猪源两种O型疫苗株的146s抗原，利用双抗原夹心法原理结合免疫层析技术应用于胶体金垫和硝酸纤维膜上，可检测猪、牛、羊血清中的O型免疫抗体。同时研究通过试纸条显色强度建立了比色卡，可根据显色的强弱对应LPB-ELISA抗体效价，使试纸条对血清抗体水平评价从定性提高到半定量。敏感性试验中O型抗体试纸条对O型阳性血清检出率为95.3%；特异性试验中对于Asia1、A型阳性血清及阴性血清的特异性符合率分别为91.4%、93.3%和100%。通过与保护力试验的比较，待检血清在1:8稀释度时，试纸条显示阳性性结果,说明血清O型口蹄疫抗体达到免疫保护；出现阴性结果，说明该O型口蹄疫抗体未达免疫保护水平。该技术已申请发明专利，并进入复审。
4．建立适用于PCR产物检测的核酸免疫层析方法。
在POCT检测中,核酸检测有其不可替代的作用,因为其可以直观反映疾病感染状况及病原感染的严重程度，该试验的开展为胶体金快速试纸条应用于一个全新的领域进行了有益的尝试。试验利用PCR方法获得口蹄疫病毒3D核苷酸片段，在引物上标记了生物素和地高辛核酸探针，因此使扩增产物结合了生物素和地高辛。胶体金标记链霉亲和素可与PCR产物中的生物素结合。硝酸纤维素膜上端标记生物素化羊抗兔抗体作为质控条带，下端标记地高辛抗体以捕获PCR产物中的地高辛。组装形成试纸条后进行系列评价试验显示其可以准确检测口蹄疫病料样品为模板的PCR产物，可检测到0.4-1pg/mL DNA含量的模板，8.28μg/mL的PCR产物，敏感性高于核酸电泳，有较好的特异性，与其它病原扩增产物无交叉反应，对48份临床样品的符合率试验表明，其敏感性与特异性分别是90%和100%。
“Point-of-care testing”(POCT) refers to the use of the procedures oflaboratory medicine in the immediate vicinity of the patient The key advantages ofPOCT are that it investeigations can be performed rapidly and simply, the results areimmediately available at the patient’s bedside. reagent is stable and easy to preserveand carry.POCT is increasingly used in the clinical laboratories in recent years. footand mouth disease (FMD) is one of the most important known contagious animalvirual disease, Outbreaks can severely disrupt livestock production and result inembargoes by trade partners, and caused direct and indirect economic losses to benot uncommon Thereforce the accurate and prompt diagnosis and routinesurveillance become very important for the disease control and prevention of virusincursion. routine diagnostic methods include virus isolation from cell culture,ELISA, and reverse-transcriptase polymerase chain reaction (RT-PCR) whichrequirewell-trained laboratory personnel, special laboratory and expenseiv instruments inpractice.Thereby offering the possibility of implementing control procedures morerapidly and conveniently has become an urgent problem to be solved, and it will bepromsing for future diagnosis.Combination of nanotechnology and someconventional technologies such as lateral flow test makes immuno-chromatogaphicassay demonstrate enormous superiority in POCT. The aim of this study wasdeveloping and evaluating an immuno-chromatogaphic assay using a colloidal goldand latex particles as indicators for typing of FMDV antigen,quantification ofFMDV antibody and nucleic acids testing as well as The development prospects ofrapid diagnosis dipsticks in POCT were discussed.
1. Development of a rapid gold immunochromatographic strip test for thediagnosis of Foot-and-mouth Disease Virus O, A and Asia1Type
Most research focused on facilitating type-specific pen-side diagnosis todifferentiate FMDV and other vesicular viral pathogens by using FMDV serotype-specitic monoclonal antibodies, however,little report is on the developmentof serotyping diagnostic kits for FMDV. In this part, andtibodies obtained fromrabbits and guinea pigs immunized with cell-culture-adapted virus strains(O/CHA/99, A/GS/LX/66, Asia1/CHN/05) and suckling-mouse adapted virus strains(O/AV99(L), A/AV88(L), Asia1/YNBS/58) were used as capture antibodiesaccording to control of Characterization of the colloidal gold probe, optimizedantibody concentration and pH of conjugations colloidal gold,selected adsorptionprotein fixed and dissolved to nitrocellulose，it was development of a simple andrapid LFI for the detection of FMDV O, A and Asia1serotypes in samples fromanimal vesicular and epithelial fluids. The seriate results indicated that the sensitivityof the test kit can reach to7.8×104LD50.（128-diluted mouse tissue-derived virus）And it had the same results for positive and negative samples tested in triplicate. Nocross reaction was found with swine vesicular disease (SVD)、VS、VES antigen. bycross tests. In the clinical assay, the corresponding rate of goldimmunochromatographic strip test kit for FMDV O、A、Asia1type and negativesample was92.45%,91.66%,92.75%and100%respectively. The coincidence rate ofdetected FMDV serotype was94.58%. It is the fist report on the development of anLFI for typing FMDV serotypes O, A and Asia1which has been awarded the stateinvention patent.
2.Development of a rapid colour latex particles immunochromatographic striptest for the typing of Foot-and-Mouth Disease virus
To match a repuirement of multi-parameter detection for the typing of FMDVwith the limited samples.It was developed of colour latex particlesImmunochromatography assay for determination of foot-and-mouth diseasevirus(FMDV) O, Asia1type from the field samples. Monodispersed polystyreneMicrospheres with two distinct dyes and carboxyl group in the size rang of380nmwere prepared with seed polymerization and swelling. The purified anti-FMDVstrain Asia1/XJ/AKT/03, O/GD/NX/92rabbit antibodies were separately coupledwith blue and red latex particles. The purified anti-Asia1/YN/BSh and AV(99)L FMDV rabbit antibody were wrapped on to nitrocellulose membrane as two test line.The FMDV serotype diagnostic kit was then performed and conditions weredetermined. The results indicated that sensitivity of the test kit reached3.9×104LD50,and no cross reaction was found with SVD,VS,VES antigen by cross tests. In theclinical assay, a total74field samples were detected with the test strips. The result ofpositive samples for FMDV O, Asia1type and negative samples were95.24%,96.30%,100%respectively.
3. Development of a rapid gold immunochromatographic strip test detecting theantibodies against FMD type O
Although it has been repoted at2004that rapid chromatographic srip detedtedthe antibodies of serotype O.the assay could only detect the antibodies from swinesera and was not suitable for the other animals such as cattle and sheep.In the study,using inactivated146S antigen of two strains of serotype O FMDV which come fromswine and cattle cell bath respectively. One of antigen was labeled with colloidalgold, dispensed onto fiber glass. Another one was separately dispensed onto an NCmember to form a test line The diagnostic kit was employed to detect the antibodiesagainst FMD type O viruses from swine, cattle and sheep. According to the antibodytiters of liquid-phase blocking sandwich ELISA(LB-ELISA) and chromogenicsituation of gold Immunochromatography assay(GICA), the colourimetric card ofGICA was drawn. Which improved antibody titer evaluation from the qualitation tohalf quantitative. The sensitivity of the GICA for all samples was confirmed to be95.3%, and the The result of specificity for FMDV O, Asia1type and negativeserum was91.4%、93.3%and100%respectively. Using a1:8dilution of the sample,if the GICA result was positive, it meant the inoculated animal serum had serotype OFMDV immune protective antibodies. Howevre,if the result was negative, and theanti-FMDV serum was belonged to non-protection rang. This technology has alreadyapplied for patent invention, and is under the review procedure.
4.Development of oligonucleotide lateral-flow immunoassay for detection nucleicacid of PCR amplification
In POCT asssys, nucleic acid plays one of irreplaceable roles as it canreflectintuitively disease status and the severity of the virus infection.It will be adevelop a new application of immuno-chromatograpic strip. Nucleic acid lateral flowimmunoassay(NALFIA) that is a simple, sensitive and specific detection system. foramplified FMDV3D gene by PCR was described in the study. An ultrasensitivenucleic acid biosensor(NAB) based on streptavidin-labeled gold nano-particles duallabels and lateral flow strip biosensor(LFSB) was used in this system. The NALFIAwas evaluated using gel-electrophoresis analysis. The detection lower limit ofNALFIA was0.4-1pg/mL DNA in the template and8.28μg/mL product of PCR.Ithad an excellent consistency between gel-electrophoresis and NALFIA. Inconclusion, it was a good alternative to detect PCR product of FMDV3D gene.
1．建立口蹄疫病毒O、A、Asia1型定型胶体金免疫层析方法
关于口蹄疫病原诊断的报道，大部分集中在口蹄疫与其临床症状相似的病原的鉴别诊断，对其血清型分型检测非常少。本研究利用柠檬酸三钠还原法制备胶体金颗粒，口蹄疫标准毒株O/AV99(L)、A/AV88(L)、Asia1/YN/BSh/58/B及流行株O/China99，A/AF72和Asia1/China05制备的多克隆抗体分别作为胶体金标记捕获抗体和硝酸纤维素膜上检测抗体，分别喷涂于玻璃纤维与硝酸纤维素膜，制备形成O、A、Asia1三种型别试纸条，三种试纸条组合形成定型诊断试剂盒。结果显示：试剂盒可检测到7.8×10~4LD50（1：128倍稀释乳鼠毒）的病毒量；对与口蹄疫临床症状相似疫病的病原，如猪水泡病（SVD）、水泡性口炎(VS)、猪水泡疹(VES)等抗原无交叉反应；对已知O、A、Asia1型和阴性样本的符合率分别为92.45%、91.66%、92.75%、100%定型准确率94.58%。这是国外及国内首个可同时鉴别口蹄疫三种血清型的试纸条，已获得国家发明专利。
2．建立口蹄疫病毒口蹄疫O、Asia1分型彩色胶乳免疫层析方法建立
为满足有限样品在一个反应中实现多重分析的要求，利用彩色胶乳微球做为标记物，结合免疫层析技术平台，研制口蹄疫O、Asia1分型彩色胶乳检测试纸条。选择两种不同颜色的单分散胶乳，用纯化的Asia1/XJ/AKT/03流行毒株兔抗体制备蓝色免疫胶乳；将纯化的O/GD/NX/92流行毒株兔抗体制备红色免疫胶乳。将两种免疫胶乳分别喷涂于不同玻璃纤维上，晾干叠加制成彩色胶乳反应垫。再分别将纯化的Asia1/YN/BSh和AV(99)L标准毒株的豚鼠抗体固定于同一硝酸纤维素膜的不同区域上作为两条检测带。最后组装成口蹄疫O、Asia1分型彩色胶乳试纸条。通过对阳性质控样品的检测，显示试纸条可检测到3.9×104LD50病毒含量；在检测与口蹄疫临床症状相似病原时无交叉反应，特异性好；不同批次间、同一批次内的试纸条，在检测阴、阳性参考样品时，结果完全一致；试纸条对检测已知血清型的74份田间样品的符合性实验中，O型、Asia1型样品的阳性符合率分别为95.24%，96.30%，阴性样品符合率为100%。本试验研发的口蹄疫O、Asia1型分型彩色胶乳试纸条具有很好的灵敏度、特异性、重复性和符合性，而且可以通过不同颜色进行定型检测，适合国内流行的口蹄疫病原型别的分型检测。
3．建立口蹄疫O型抗体检测胶体金免疫层析方法。
国内已报道的O型免疫抗体的试纸条，仅能进行猪血清中O型抗体免疫合格水平的定性检测，无法对牛、羊等其它免疫动物进行检测。本实验分别选用牛源和猪源两种O型疫苗株的146s抗原，利用双抗原夹心法原理结合免疫层析技术应用于胶体金垫和硝酸纤维膜上，可检测猪、牛、羊血清中的O型免疫抗体。同时研究通过试纸条显色强度建立了比色卡，可根据显色的强弱对应LPB-ELISA抗体效价，使试纸条对血清抗体水平评价从定性提高到半定量。敏感性试验中O型抗体试纸条对O型阳性血清检出率为95.3%；特异性试验中对于Asia1、A型阳性血清及阴性血清的特异性符合率分别为91.4%、93.3%和100%。通过与保护力试验的比较，待检血清在1:8稀释度时，试纸条显示阳性性结果,说明血清O型口蹄疫抗体达到免疫保护；出现阴性结果，说明该O型口蹄疫抗体未达免疫保护水平。该技术已申请发明专利，并进入复审。
4．建立适用于PCR产物检测的核酸免疫层析方法。
在POCT检测中,核酸检测有其不可替代的作用,因为其可以直观反映疾病感染状况及病原感染的严重程度，该试验的开展为胶体金快速试纸条应用于一个全新的领域进行了有益的尝试。试验利用PCR方法获得口蹄疫病毒3D核苷酸片段，在引物上标记了生物素和地高辛核酸探针，因此使扩增产物结合了生物素和地高辛。胶体金标记链霉亲和素可与PCR产物中的生物素结合。硝酸纤维素膜上端标记生物素化羊抗兔抗体作为质控条带，下端标记地高辛抗体以捕获PCR产物中的地高辛。组装形成试纸条后进行系列评价试验显示其可以准确检测口蹄疫病料样品为模板的PCR产物，可检测到0.4-1pg/mL DNA含量的模板，8.28μg/mL的PCR产物，敏感性高于核酸电泳，有较好的特异性，与其它病原扩增产物无交叉反应，对48份临床样品的符合率试验表明，其敏感性与特异性分别是90%和100%。
“Point-of-care testing”(POCT) refers to the use of the procedures oflaboratory medicine in the immediate vicinity of the patient The key advantages ofPOCT are that it investeigations can be performed rapidly and simply, the results areimmediately available at the patient’s bedside. reagent is stable and easy to preserveand carry.POCT is increasingly used in the clinical laboratories in recent years. footand mouth disease (FMD) is one of the most important known contagious animalvirual disease, Outbreaks can severely disrupt livestock production and result inembargoes by trade partners, and caused direct and indirect economic losses to benot uncommon Thereforce the accurate and prompt diagnosis and routinesurveillance become very important for the disease control and prevention of virusincursion. routine diagnostic methods include virus isolation from cell culture,ELISA, and reverse-transcriptase polymerase chain reaction (RT-PCR) whichrequirewell-trained laboratory personnel, special laboratory and expenseiv instruments inpractice.Thereby offering the possibility of implementing control procedures morerapidly and conveniently has become an urgent problem to be solved, and it will bepromsing for future diagnosis.Combination of nanotechnology and someconventional technologies such as lateral flow test makes immuno-chromatogaphicassay demonstrate enormous superiority in POCT. The aim of this study wasdeveloping and evaluating an immuno-chromatogaphic assay using a colloidal goldand latex particles as indicators for typing of FMDV antigen,quantification ofFMDV antibody and nucleic acids testing as well as The development prospects ofrapid diagnosis dipsticks in POCT were discussed.
1. Development of a rapid gold immunochromatographic strip test for thediagnosis of Foot-and-mouth Disease Virus O, A and Asia1Type
Most research focused on facilitating type-specific pen-side diagnosis todifferentiate FMDV and other vesicular viral pathogens by using FMDV serotype-specitic monoclonal antibodies, however,little report is on the developmentof serotyping diagnostic kits for FMDV. In this part, andtibodies obtained fromrabbits and guinea pigs immunized with cell-culture-adapted virus strains(O/CHA/99, A/GS/LX/66, Asia1/CHN/05) and suckling-mouse adapted virus strains(O/AV99(L), A/AV88(L), Asia1/YNBS/58) were used as capture antibodiesaccording to control of Characterization of the colloidal gold probe, optimizedantibody concentration and pH of conjugations colloidal gold,selected adsorptionprotein fixed and dissolved to nitrocellulose，it was development of a simple andrapid LFI for the detection of FMDV O, A and Asia1serotypes in samples fromanimal vesicular and epithelial fluids. The seriate results indicated that the sensitivityof the test kit can reach to7.8×104LD50.（128-diluted mouse tissue-derived virus）And it had the same results for positive and negative samples tested in triplicate. Nocross reaction was found with swine vesicular disease (SVD)、VS、VES antigen. bycross tests. In the clinical assay, the corresponding rate of goldimmunochromatographic strip test kit for FMDV O、A、Asia1type and negativesample was92.45%,91.66%,92.75%and100%respectively. The coincidence rate ofdetected FMDV serotype was94.58%. It is the fist report on the development of anLFI for typing FMDV serotypes O, A and Asia1which has been awarded the stateinvention patent.
2.Development of a rapid colour latex particles immunochromatographic striptest for the typing of Foot-and-Mouth Disease virus
To match a repuirement of multi-parameter detection for the typing of FMDVwith the limited samples.It was developed of colour latex particlesImmunochromatography assay for determination of foot-and-mouth diseasevirus(FMDV) O, Asia1type from the field samples. Monodispersed polystyreneMicrospheres with two distinct dyes and carboxyl group in the size rang of380nmwere prepared with seed polymerization and swelling. The purified anti-FMDVstrain Asia1/XJ/AKT/03, O/GD/NX/92rabbit antibodies were separately coupledwith blue and red latex particles. The purified anti-Asia1/YN/BSh and AV(99)L FMDV rabbit antibody were wrapped on to nitrocellulose membrane as two test line.The FMDV serotype diagnostic kit was then performed and conditions weredetermined. The results indicated that sensitivity of the test kit reached3.9×104LD50,and no cross reaction was found with SVD,VS,VES antigen by cross tests. In theclinical assay, a total74field samples were detected with the test strips. The result ofpositive samples for FMDV O, Asia1type and negative samples were95.24%,96.30%,100%respectively.
3. Development of a rapid gold immunochromatographic strip test detecting theantibodies against FMD type O
Although it has been repoted at2004that rapid chromatographic srip detedtedthe antibodies of serotype O.the assay could only detect the antibodies from swinesera and was not suitable for the other animals such as cattle and sheep.In the study,using inactivated146S antigen of two strains of serotype O FMDV which come fromswine and cattle cell bath respectively. One of antigen was labeled with colloidalgold, dispensed onto fiber glass. Another one was separately dispensed onto an NCmember to form a test line The diagnostic kit was employed to detect the antibodiesagainst FMD type O viruses from swine, cattle and sheep. According to the antibodytiters of liquid-phase blocking sandwich ELISA(LB-ELISA) and chromogenicsituation of gold Immunochromatography assay(GICA), the colourimetric card ofGICA was drawn. Which improved antibody titer evaluation from the qualitation tohalf quantitative. The sensitivity of the GICA for all samples was confirmed to be95.3%, and the The result of specificity for FMDV O, Asia1type and negativeserum was91.4%、93.3%and100%respectively. Using a1:8dilution of the sample,if the GICA result was positive, it meant the inoculated animal serum had serotype OFMDV immune protective antibodies. Howevre,if the result was negative, and theanti-FMDV serum was belonged to non-protection rang. This technology has alreadyapplied for patent invention, and is under the review procedure.
4.Development of oligonucleotide lateral-flow immunoassay for detection nucleicacid of PCR amplification
In POCT asssys, nucleic acid plays one of irreplaceable roles as it canreflectintuitively disease status and the severity of the virus infection.It will be adevelop a new application of immuno-chromatograpic strip. Nucleic acid lateral flowimmunoassay(NALFIA) that is a simple, sensitive and specific detection system. foramplified FMDV3D gene by PCR was described in the study. An ultrasensitivenucleic acid biosensor(NAB) based on streptavidin-labeled gold nano-particles duallabels and lateral flow strip biosensor(LFSB) was used in this system. The NALFIAwas evaluated using gel-electrophoresis analysis. The detection lower limit ofNALFIA was0.4-1pg/mL DNA in the template and8.28μg/mL product of PCR.Ithad an excellent consistency between gel-electrophoresis and NALFIA. Inconclusion, it was a good alternative to detect PCR product of FMDV3D gene.
引文
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