兔出血症病毒单克隆抗体的制备及抗原捕获ELISA检测方法的建立
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摘要
兔出血症( Rabbit haemorrhagic disease,RHD )是由兔出血症病毒(Rabbit haemorrhagic disease virus,RHDV)引起的兔的一种急性、败血性、高度致死性传染病。该病于1984年在中国首次发现,其后在世界其它地区相继发生,给养兔业造成了巨大的经济损失。
鉴于RHD病程短、致死率高等特点,临床检测和早期诊断中对检测方法除要求敏感、准确外,还要求其快速化、简单化。现有的血清学检测方法因为商业养殖中对兔的严格免疫导致其无法判定是病毒感染还是疫苗免疫造成了抗体水平的升高而失去了其诊断意义。因此,在RHD的诊断中病原学检测发挥着主导作用。目前,已建立了多种实验室检测方法,其中琼脂扩散、血凝/抑制试验应用较广但灵敏性较低;反转录-聚合酶链式反应(RT-PCR)因仪器、试剂和操作技术要求更适合于实验室操作,相比之下酶标抗体技术操作相对简单又无需太多实验设备,比较适合在临床和检测中推广。然而迄今为止,只有国外少数公司研制成功了抗原检测ELISA试剂盒,其昂贵的成本使其在进行大规模临床检测时难以广泛应用。因此,研制灵敏、快速、操作简便的抗原检测ELISA试剂盒对于RHD的诊断和疫情防治具有重要意义。
本试验以分离纯化的地方毒株RHDV-TP株免疫BALB/c小鼠,制备了多株针对RHDV的单克隆抗体,对其生物学特性进行分析鉴定后,筛选出一株效价高、亲和力强的单克隆抗体DE_2株,并将其作为诊断试剂,建立了RHDV抗原捕获ELISA检测方法。
由于RHDV缺乏体外繁殖系统,本试验将RHDV-TP毒株攻击健康大白兔增殖病毒,发病死亡后采集其肝脏组织匀浆处理,以蔗糖密度梯度离心法纯化RHDV全病毒粒子,并将其作为免疫原免疫BALB/c小鼠。以真核表达系统表达的RHDV结构蛋白-VP6O作为基础建立间接ELISA检测方法进行单抗筛选。通过融合与克隆过程,筛选出6株针对RHDV的单克隆抗体AD4、AG10、BC9、BE8、BH3、DE_2;其腹水效价介于1:4000~1:3×104之间。竞争相加ELISA及Western-blot试验结果表明,6株单抗分别针对5个RHDV抗原位点,其中DE_2株针对线性抗原位点,DE_2株效价较高、亲和力强,有作为诊断试剂进行开发利用的前景。
将DE_2株单抗腹水纯化后作为捕获抗体包被酶标板,建立了抗原捕获ELISA诊断方法检测RHDV,并对各工作条件进行了优化,筛选出最佳工作体系,然后以对已知阳性抗原样品和阴性抗原样品及临床可疑样品进行检测,并与常规血凝/血凝抑制试验检测结果进行比对。结果显示对于已知阳性样品,捕获ELISA阳性检出率为100%;对于临床可疑样品,捕获ELISA阳性检出率为62.7%,常规血凝试验为55.2%,检出率比HA高7.5%,灵敏度为HA试验的3～13倍。最低病毒检出量测定试验表明,抗原捕获ELISA诊断方法对全病毒的最低检出浓度为26ng/mL,具有很高的灵敏度。
上述结果表明,基于DE_2株单克隆抗体的抗原捕获ELISA诊断方法可特异性地检测出发病兔肝脏组织中的RHDV,该方法快速、灵敏、准确,能够同时进行大规模的样品检测,省时省力且成本较低,是一种理想的RHDV抗原检测方法。另外,本试验制备的其他单克隆抗体分别针对不同的RHDV抗原表位,今后将对RHDV抗原表位筛选、RHDV的鉴别诊断等相关领域的研究中发挥重要作用。
Rabbit haemorrhagic disease (RHD) is an acute fatal disease of rabbits, which was first described in China in 1984. It has been spread all over the world till now. It can cause high economic losses in domestic and commercial rabbits as well as high mortality in wild rabbits.
Because RHD is a disease with short course and high mortality, a rapid and simple methods is necessary to clinical diagnosis and ealier period detection.There is no mean to detect antibodies because serological tests couldn’t differentiate that the antibody is produced by infection or immunifaction,so the pathogenic detecting are playing important roles.But these pathogenic detecting methods ,including HA/HI, AGP, RT-PCR,are not fit for the field test for their shortages such as low sensitivity or high requirements of technique and equipments.Compared to all methods above,ELISA kit is sensitive,rapid and convenient to operate.So ,developing an ELISA kit detecting RHDV antigen means great significance for the diagnosis and control of RHD.
In this test, BALB/c mices were immunized with RHDV local isolated strain named RHDV-TP. Six strains of hybridoma were obtained, which could secret monoclonal antibodies against RHDV.After the identification of these monoclonal antibodies, a McAb named DE_2 with high titre and affinity was selected to be capture antibody to establish a antigen capture ELISA.
As the rabbit hemorrhagic disease virus has not been grown in any cell cultures, viruses can be concentrated from the liver.Firstly,big white rabbits were challenged with RHDV-TP strain.Livers from the dead rabbits were collected and homogenated. Then the virus was purified with differential centrifugation and sucrose density gradient centrifugation.After fusions between hybridoma SP2/0 cells and spleen cells from the immunized mice, antibodies in supernatant were detected by indirect ELISA which was established with the only capsid protein–VP60 expressed in insect cells. Six strains hybridoma were prepared and named as AD4、AG10、BC9、BE8、BH3、DE_2.Their antibody titres in ascites ranged from 1:4000 to 1:3×104 in ELISA tests.It was proved in additivity ELISA and Western-blot assays that these six McAbs could specificly identify five different epitopes of RHDV.One McAb named DE_2 with high titre and affinity could cohere with a linear epidope stably.These characters showed that the DE_2 McAb was capable to be a diagnostic reagent.
An antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA) for detecting rabbit hemorrhagic disease (RHD)virus was developed with a monoclonal antibody capturing virus ,while the rabbit anti-RHDV serum was used as the second antibody to identify virus. Working conditions of the Ag-ELISA were optimized and its capabilities were evaluated then. In the present study, the optimum working concentration of McAb was 1μg/mL and that of rabbit anti-RHDV serum was 4μg/mL. Using this test , several positive and negtive samples and 67 liver tissue samples from suspectable infected rabbits in local farms were detected.All the known positive samples were detected with positive reslults.62.7% of 67 liver tissue samples had positive results in contrast to 55.2% by HA test. Detection of five positive RHDV samples proved the highest dilution titer by ELISA was 3~13 times higher than that detected by HA test.The detection limit of this assay is 26ng/mL to purified RHDV.
These results showed that the Ag-ELISA based on the McAb-DE_2 could detect out RHDV from liver samples of the infected rabbits correctly. The Ag-ELISA displayed excellent specificity , sensitivity and repeatability.Also it’s easy to operate and could save time and labour. The antigen-capture enzyme-linked immunosorbent assay was confirmed an excellent method for rapid diagnosis of RHD. In addition, 6 strains hybridoma cell secreting MAbs of RHDV stably obtained in this test would be used in the related study of RHDV ,such as screening the epitopes and discriminate detection ,in future.
鉴于RHD病程短、致死率高等特点,临床检测和早期诊断中对检测方法除要求敏感、准确外,还要求其快速化、简单化。现有的血清学检测方法因为商业养殖中对兔的严格免疫导致其无法判定是病毒感染还是疫苗免疫造成了抗体水平的升高而失去了其诊断意义。因此,在RHD的诊断中病原学检测发挥着主导作用。目前,已建立了多种实验室检测方法,其中琼脂扩散、血凝/抑制试验应用较广但灵敏性较低;反转录-聚合酶链式反应(RT-PCR)因仪器、试剂和操作技术要求更适合于实验室操作,相比之下酶标抗体技术操作相对简单又无需太多实验设备,比较适合在临床和检测中推广。然而迄今为止,只有国外少数公司研制成功了抗原检测ELISA试剂盒,其昂贵的成本使其在进行大规模临床检测时难以广泛应用。因此,研制灵敏、快速、操作简便的抗原检测ELISA试剂盒对于RHD的诊断和疫情防治具有重要意义。
本试验以分离纯化的地方毒株RHDV-TP株免疫BALB/c小鼠,制备了多株针对RHDV的单克隆抗体,对其生物学特性进行分析鉴定后,筛选出一株效价高、亲和力强的单克隆抗体DE_2株,并将其作为诊断试剂,建立了RHDV抗原捕获ELISA检测方法。
由于RHDV缺乏体外繁殖系统,本试验将RHDV-TP毒株攻击健康大白兔增殖病毒,发病死亡后采集其肝脏组织匀浆处理,以蔗糖密度梯度离心法纯化RHDV全病毒粒子,并将其作为免疫原免疫BALB/c小鼠。以真核表达系统表达的RHDV结构蛋白-VP6O作为基础建立间接ELISA检测方法进行单抗筛选。通过融合与克隆过程,筛选出6株针对RHDV的单克隆抗体AD4、AG10、BC9、BE8、BH3、DE_2;其腹水效价介于1:4000~1:3×104之间。竞争相加ELISA及Western-blot试验结果表明,6株单抗分别针对5个RHDV抗原位点,其中DE_2株针对线性抗原位点,DE_2株效价较高、亲和力强,有作为诊断试剂进行开发利用的前景。
将DE_2株单抗腹水纯化后作为捕获抗体包被酶标板,建立了抗原捕获ELISA诊断方法检测RHDV,并对各工作条件进行了优化,筛选出最佳工作体系,然后以对已知阳性抗原样品和阴性抗原样品及临床可疑样品进行检测,并与常规血凝/血凝抑制试验检测结果进行比对。结果显示对于已知阳性样品,捕获ELISA阳性检出率为100%;对于临床可疑样品,捕获ELISA阳性检出率为62.7%,常规血凝试验为55.2%,检出率比HA高7.5%,灵敏度为HA试验的3～13倍。最低病毒检出量测定试验表明,抗原捕获ELISA诊断方法对全病毒的最低检出浓度为26ng/mL,具有很高的灵敏度。
上述结果表明,基于DE_2株单克隆抗体的抗原捕获ELISA诊断方法可特异性地检测出发病兔肝脏组织中的RHDV,该方法快速、灵敏、准确,能够同时进行大规模的样品检测,省时省力且成本较低,是一种理想的RHDV抗原检测方法。另外,本试验制备的其他单克隆抗体分别针对不同的RHDV抗原表位,今后将对RHDV抗原表位筛选、RHDV的鉴别诊断等相关领域的研究中发挥重要作用。
Rabbit haemorrhagic disease (RHD) is an acute fatal disease of rabbits, which was first described in China in 1984. It has been spread all over the world till now. It can cause high economic losses in domestic and commercial rabbits as well as high mortality in wild rabbits.
Because RHD is a disease with short course and high mortality, a rapid and simple methods is necessary to clinical diagnosis and ealier period detection.There is no mean to detect antibodies because serological tests couldn’t differentiate that the antibody is produced by infection or immunifaction,so the pathogenic detecting are playing important roles.But these pathogenic detecting methods ,including HA/HI, AGP, RT-PCR,are not fit for the field test for their shortages such as low sensitivity or high requirements of technique and equipments.Compared to all methods above,ELISA kit is sensitive,rapid and convenient to operate.So ,developing an ELISA kit detecting RHDV antigen means great significance for the diagnosis and control of RHD.
In this test, BALB/c mices were immunized with RHDV local isolated strain named RHDV-TP. Six strains of hybridoma were obtained, which could secret monoclonal antibodies against RHDV.After the identification of these monoclonal antibodies, a McAb named DE_2 with high titre and affinity was selected to be capture antibody to establish a antigen capture ELISA.
As the rabbit hemorrhagic disease virus has not been grown in any cell cultures, viruses can be concentrated from the liver.Firstly,big white rabbits were challenged with RHDV-TP strain.Livers from the dead rabbits were collected and homogenated. Then the virus was purified with differential centrifugation and sucrose density gradient centrifugation.After fusions between hybridoma SP2/0 cells and spleen cells from the immunized mice, antibodies in supernatant were detected by indirect ELISA which was established with the only capsid protein–VP60 expressed in insect cells. Six strains hybridoma were prepared and named as AD4、AG10、BC9、BE8、BH3、DE_2.Their antibody titres in ascites ranged from 1:4000 to 1:3×104 in ELISA tests.It was proved in additivity ELISA and Western-blot assays that these six McAbs could specificly identify five different epitopes of RHDV.One McAb named DE_2 with high titre and affinity could cohere with a linear epidope stably.These characters showed that the DE_2 McAb was capable to be a diagnostic reagent.
An antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA) for detecting rabbit hemorrhagic disease (RHD)virus was developed with a monoclonal antibody capturing virus ,while the rabbit anti-RHDV serum was used as the second antibody to identify virus. Working conditions of the Ag-ELISA were optimized and its capabilities were evaluated then. In the present study, the optimum working concentration of McAb was 1μg/mL and that of rabbit anti-RHDV serum was 4μg/mL. Using this test , several positive and negtive samples and 67 liver tissue samples from suspectable infected rabbits in local farms were detected.All the known positive samples were detected with positive reslults.62.7% of 67 liver tissue samples had positive results in contrast to 55.2% by HA test. Detection of five positive RHDV samples proved the highest dilution titer by ELISA was 3~13 times higher than that detected by HA test.The detection limit of this assay is 26ng/mL to purified RHDV.
These results showed that the Ag-ELISA based on the McAb-DE_2 could detect out RHDV from liver samples of the infected rabbits correctly. The Ag-ELISA displayed excellent specificity , sensitivity and repeatability.Also it’s easy to operate and could save time and labour. The antigen-capture enzyme-linked immunosorbent assay was confirmed an excellent method for rapid diagnosis of RHD. In addition, 6 strains hybridoma cell secreting MAbs of RHDV stably obtained in this test would be used in the related study of RHDV ,such as screening the epitopes and discriminate detection ,in future.
引文
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