禽流感病毒H5、H9亚型单抗及ELISA鉴别诊断方法的研究和应用
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摘要
禽流感(Avian influenza,AI)是由A型流感病毒(AIV)引起的一种禽类疾病综合症,被国际兽医局确定为Ⅰ类烈性传染病。自1878年首次发现至今,世界各地都有特定的毒株引起禽流感的爆发和流行,给养禽业造成了极大的经济损失。2004年亚太地区爆发禽流感,并有传染给人甚至致人死亡的诸多报道,因而引起了我国政府及普通民众的广泛关注。鉴于我国地域广阔,禽类总数占有量大,禽产品流通频繁,一旦发生禽流感后难以从外界环境中彻底清除,而且禽流感病毒存在抗原漂移和抗原转变,低致病力的毒株也有可能突变为高致病力的毒株,这给我国养禽业造成了极大的潜在性威胁。因此,若能适时进行快速诊断,查明病原,在流行初期即能检测出鸡体是否感染了AIV,从而为快速处置预案或早期预防治疗提供诊断依据,最大限度地减少经济损失,就显得尤为重要。为此,本研究拟通过研制出AIV H5、H9亚型血凝素特异性的单克隆抗体,建立一种能快速区分高致病性禽流感H5亚型和低致病性禽流感H9亚型的夹心ELISA鉴别诊断试剂盒,主要研究工作和结果如下。
1.禽流感病毒H5、H9亚型血凝素单克隆抗体的研制
将AIV H5和AIV H9分别经9～11日龄鸡胚传代后,收获鸡胚尿囊液,经27000r/min超速离心1h,沉淀用0.01mol/L,pH7.2的PBS重悬,测得尿囊液和浓缩液的血凝效价分别为2~6和2~(11),将浓缩悬液分别与等量佐剂混合后免疫Balb/c鼠,按常规方法取免疫脾细胞与骨髓瘤细胞融合,用AIV H9、H5血凝素抗原以血凝抑制试验进行筛选和克隆,获得了七株杂交瘤细胞。其中,抗AIV H5的杂交瘤细胞株为H5284、H53C9、H55E8、,经亲和力测定单抗H5284与AIV H5亲和力最好;抗AIV H9的杂交瘤细胞株为H91G3、H92C2、H94C4、H91C6;单抗H94C4与AIVH9亲和力最好。两株单抗腹水效价分别可达2~(13-20);所有杂交瘤细胞染色体数目均在82～92条之间,高于脾淋巴细胞和骨髓瘤细胞染色体数目;各株单克隆抗体杂交瘤细胞经冻存与复苏,仍能稳定分泌抗体。所获得七种单克隆抗体腹水对相应AIV病毒液血凝抑制价效价均在2~(13)-2~(20);两种单抗互相之间以及与其他无关的禽类病毒之间无任何交叉反应;为禽流感的夹心ELISA鉴别诊断方法的建立奠定了基础。
2.禽流感病毒H5、H9亚型ELISA鉴别诊断方法的建立
用抗AIV H5亚型血凝素单克隆抗体为包被抗体,兔抗AIV IgG为第二抗体;同时用鸡抗AIV IgG为包被抗体,抗AIV H9亚型血凝素单克隆抗体为第二抗体,通过ELISA反应条件的优化,分别建立了检测禽流感病毒H5、H9亚型抗原的夹心
Avian Influenza (AI) is one of syndrome disease of birds caused by influenza A virus in avian. At present, highly pathogenic avian influenza (HPAI) is listed in A infectious diseases by World Organization for Animal Health (OIE). Since AIV was found in Italy 1878, there were breaking out and prevalence caused by different strains of AIV and caused important economic losses in the avian industry throughout the world. Especially, events that AI infected human being happened in south-eastern Asian (2003~ 2004), which shocked mankind and caused our attention to commonality sanitation of AI. Because of broad area, large quantity of birds, frequent circulation of poultry product, it is difficult to remove completely from the external environment after once the bird flu happened, The antigenic drift and antigenic shift of AIV makes it be possible that low virulence influenza shift to HPAI, and which makes potential menace to the avian industry in our country. It is importance to diagnose timely and speedily in order to win golden time for forepart prevention and therapy, and to reduce economic losses furthest. This research is aimed to develop ELISA for detection of AIV in order to provide technique to diagnoses of AI in short time, especially to differentiability diagnoses of AIV H5 and AIV H9. The results of research are summarized as following:1. Preparation of monocloneantibody(McAb) of anti-haemaglutinin of AIV H5 and AIV H9AIV H9 and AIV H9 were inoculated separately into 9 to 10day embryonating chicken eggs separately by the allantoic route, then allantoic fluid was harvested and centrifuged with 27,000r/min, and deposit was redissolved by 0.01mol/L PBS (pH7.2) which is 1/30 volume of allantoic fluid. Immune spleen cells which get from the Balb/c mouse immunized with redissolved fluid of AIV H5 and AIV H9 and Sp2/0 myeloma cells which disfigured hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were amalgamated, and seven strains of hybridized cells were obtained after being cloned. The number of chromosome of those seven strains hybridized cells is all between82 to 92, which is more than that of their parents. The cells could secrete antibody stably after being froze and anabiosis. The HI titers of ascites fluids to corresponding subtype AIV are 2~(13)-2~(20). No cross reaction of HI be found to McAb H52B4,H53C9 and H55E8 with AIV H9, EDS_(-76)V, NDV, IBV, ILTV, APV, MDV and IBDV, and to McAb H91C6, H91G3, H92C2 and H94C4 with AIV H5 and other virus. The appetence is best between McAb H52B4 and AIV H5, and between McAb H94C4 and AIV H9. Obtainment of the monocloneantibodies provided the foundation for the development of enzyme-linked
immunosorbent assay (EL1SA) for AIV H5 and AIV H9.2. Development of enzyme-linked immunosorbent assay (ELISA) for AIV H5 andAIVH9.A sandwich ELISA for detection of AIV H5 antigen was developed by using monocloneantibody (McAb) of anti-haemaglutinin of AIV H5 as the first antibody coated on the ELISA plate and rabbit anti-AIV IgG regarded as the second antibody. And another sandwich ELISA for detection of AIV H9 antigen was developed by using purified chicken anti-AIV IgG as the first antibody coated on the ELISA plate and monocloneantibody (McAb) of anti-haemaglutinin of AIV H9 regarded as the second antibody. The dilution titers of positive AIV H5, and AIV H9 sample by corresponded ELISA was 16 times higher than that by hemagglutination test Positive AIV H9 sample can not be detect by ELISA for detection of AIV H5 antigen, and positive AIV H5 sample can not be detect by ELISA for detection of AIV H9 antigen. No cross reaction was observed with other viruses of birds (Newcastle disease virus (NDV), infectious bronchitis virus (IBV), eggs drop syndrome virus (EDS-76V), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV), avian pox virus (APV) and Marek's disease virus(MDV) )by both the ways. The results illuminated that tow methods have highly specificity and sensitivity. A Sandwich Enzyme-linked immunosobent assay test Differentiability kit based on previous work was developed for detection of avian influenza virus (AIV) antigen including all AIV subtypes detecting, subtype of ATV H5 and subtype of AIV H9. The kit consists of twelve reagents and a piece of 96 well microtiter plate. It is highly specificity, sensitivity and repeatability to detecting AIV antigen with the test kit. It was stable well when storing at below -10°C for tow months up to the present. It is very easy to manipulate, convenient to schlep and the result can be reported speedily and veraciously.
1.禽流感病毒H5、H9亚型血凝素单克隆抗体的研制
将AIV H5和AIV H9分别经9～11日龄鸡胚传代后,收获鸡胚尿囊液,经27000r/min超速离心1h,沉淀用0.01mol/L,pH7.2的PBS重悬,测得尿囊液和浓缩液的血凝效价分别为2~6和2~(11),将浓缩悬液分别与等量佐剂混合后免疫Balb/c鼠,按常规方法取免疫脾细胞与骨髓瘤细胞融合,用AIV H9、H5血凝素抗原以血凝抑制试验进行筛选和克隆,获得了七株杂交瘤细胞。其中,抗AIV H5的杂交瘤细胞株为H5284、H53C9、H55E8、,经亲和力测定单抗H5284与AIV H5亲和力最好;抗AIV H9的杂交瘤细胞株为H91G3、H92C2、H94C4、H91C6;单抗H94C4与AIVH9亲和力最好。两株单抗腹水效价分别可达2~(13-20);所有杂交瘤细胞染色体数目均在82～92条之间,高于脾淋巴细胞和骨髓瘤细胞染色体数目;各株单克隆抗体杂交瘤细胞经冻存与复苏,仍能稳定分泌抗体。所获得七种单克隆抗体腹水对相应AIV病毒液血凝抑制价效价均在2~(13)-2~(20);两种单抗互相之间以及与其他无关的禽类病毒之间无任何交叉反应;为禽流感的夹心ELISA鉴别诊断方法的建立奠定了基础。
2.禽流感病毒H5、H9亚型ELISA鉴别诊断方法的建立
用抗AIV H5亚型血凝素单克隆抗体为包被抗体,兔抗AIV IgG为第二抗体;同时用鸡抗AIV IgG为包被抗体,抗AIV H9亚型血凝素单克隆抗体为第二抗体,通过ELISA反应条件的优化,分别建立了检测禽流感病毒H5、H9亚型抗原的夹心
Avian Influenza (AI) is one of syndrome disease of birds caused by influenza A virus in avian. At present, highly pathogenic avian influenza (HPAI) is listed in A infectious diseases by World Organization for Animal Health (OIE). Since AIV was found in Italy 1878, there were breaking out and prevalence caused by different strains of AIV and caused important economic losses in the avian industry throughout the world. Especially, events that AI infected human being happened in south-eastern Asian (2003~ 2004), which shocked mankind and caused our attention to commonality sanitation of AI. Because of broad area, large quantity of birds, frequent circulation of poultry product, it is difficult to remove completely from the external environment after once the bird flu happened, The antigenic drift and antigenic shift of AIV makes it be possible that low virulence influenza shift to HPAI, and which makes potential menace to the avian industry in our country. It is importance to diagnose timely and speedily in order to win golden time for forepart prevention and therapy, and to reduce economic losses furthest. This research is aimed to develop ELISA for detection of AIV in order to provide technique to diagnoses of AI in short time, especially to differentiability diagnoses of AIV H5 and AIV H9. The results of research are summarized as following:1. Preparation of monocloneantibody(McAb) of anti-haemaglutinin of AIV H5 and AIV H9AIV H9 and AIV H9 were inoculated separately into 9 to 10day embryonating chicken eggs separately by the allantoic route, then allantoic fluid was harvested and centrifuged with 27,000r/min, and deposit was redissolved by 0.01mol/L PBS (pH7.2) which is 1/30 volume of allantoic fluid. Immune spleen cells which get from the Balb/c mouse immunized with redissolved fluid of AIV H5 and AIV H9 and Sp2/0 myeloma cells which disfigured hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were amalgamated, and seven strains of hybridized cells were obtained after being cloned. The number of chromosome of those seven strains hybridized cells is all between82 to 92, which is more than that of their parents. The cells could secrete antibody stably after being froze and anabiosis. The HI titers of ascites fluids to corresponding subtype AIV are 2~(13)-2~(20). No cross reaction of HI be found to McAb H52B4,H53C9 and H55E8 with AIV H9, EDS_(-76)V, NDV, IBV, ILTV, APV, MDV and IBDV, and to McAb H91C6, H91G3, H92C2 and H94C4 with AIV H5 and other virus. The appetence is best between McAb H52B4 and AIV H5, and between McAb H94C4 and AIV H9. Obtainment of the monocloneantibodies provided the foundation for the development of enzyme-linked
immunosorbent assay (EL1SA) for AIV H5 and AIV H9.2. Development of enzyme-linked immunosorbent assay (ELISA) for AIV H5 andAIVH9.A sandwich ELISA for detection of AIV H5 antigen was developed by using monocloneantibody (McAb) of anti-haemaglutinin of AIV H5 as the first antibody coated on the ELISA plate and rabbit anti-AIV IgG regarded as the second antibody. And another sandwich ELISA for detection of AIV H9 antigen was developed by using purified chicken anti-AIV IgG as the first antibody coated on the ELISA plate and monocloneantibody (McAb) of anti-haemaglutinin of AIV H9 regarded as the second antibody. The dilution titers of positive AIV H5, and AIV H9 sample by corresponded ELISA was 16 times higher than that by hemagglutination test Positive AIV H9 sample can not be detect by ELISA for detection of AIV H5 antigen, and positive AIV H5 sample can not be detect by ELISA for detection of AIV H9 antigen. No cross reaction was observed with other viruses of birds (Newcastle disease virus (NDV), infectious bronchitis virus (IBV), eggs drop syndrome virus (EDS-76V), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV), avian pox virus (APV) and Marek's disease virus(MDV) )by both the ways. The results illuminated that tow methods have highly specificity and sensitivity. A Sandwich Enzyme-linked immunosobent assay test Differentiability kit based on previous work was developed for detection of avian influenza virus (AIV) antigen including all AIV subtypes detecting, subtype of ATV H5 and subtype of AIV H9. The kit consists of twelve reagents and a piece of 96 well microtiter plate. It is highly specificity, sensitivity and repeatability to detecting AIV antigen with the test kit. It was stable well when storing at below -10°C for tow months up to the present. It is very easy to manipulate, convenient to schlep and the result can be reported speedily and veraciously.
引文
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