抗树鼩IgG单抗制备与ELISA检测方法初步建立
摘要
树鼩(Tupaia, tree shrew)形似松鼠、分类上有羊膜类、哺乳纲、攀鼩目,被认为是最低等的灵长类动物。树鼩主要分布于亚洲东南部的热带、亚热带地区,在东南亚国家及我国的云南、广西、广东和海南等地广泛分布。我国树鼩属中缅树鼩(Tupaia belangeri)，是树鼩中分布最广的一个属。树鼩体型小,生长繁殖快,易于捕捉、驯养和繁殖成本低,其新陈代谢特点和解剖学一些特点比啮齿类动物更接近人类。随着国内外非人灵长类资源的逐渐减少和实验动物日益小型化,树鼩作为研究人类相关疾病尤其是病毒性疾病的可能动物模型,受到广泛关注。
树鼩作为多种疾病的的可能实验动物模型,有很好的应用前景。抗体水平是评价树鼩机体对疾病病原体免疫应答反应的重要指标,然而由于尚缺少有效的树鼩IgG检测手段,对树鼩抗体方面的研究尚未见报道。抗树鼩IgG单克隆抗体的有效制备及其为主要组分的酶联免疫吸附检测方法(Enzyme Link Immunosorbent Assay, ELISA)的建立,可以为树鼩抗体的检测提供有力的工具,促进树鼩作为实验动物模型各方面的研究。
本论文用分离、纯化得到的树鼩血清IgG免疫雌性BALB/c小鼠,经骨髓瘤/免疫脾细胞细胞融合及阳性细胞亚克隆筛选,获得能稳定分泌抗树鼩IgG抗体的杂交瘤细胞株。实验共获得了两株阳性杂交瘤细胞(TBS-1、TBS-2),细胞融合率为158/480=32.92%,融合阳性率为2/158=1.27%。其中,TBS-2细胞经过了4次亚克隆筛选,细胞阳性率仍未达到100%,并混有很多阴性细胞,培养液中分泌的抗体含量也不高,细胞纯度不高,抗体分泌不稳定；TBS-1细胞经过3次亚克隆筛选,细胞阳性率就达到100%,培养液中能稳定分泌高效价(1：800)的抗树鼩IgG单克隆抗体。用阳性细胞小鼠腹腔注射,收集腹水的方法获得大量单克隆抗体,腹水效价达到1：25600以上。融合细胞在培养不同代次经核型分析发现,杂交瘤细胞染色体数目和形态正常,细胞遗传特征稳定。Western blot结果显示,单克隆抗体可以有效结合树鼩血清IgG,没有产生杂带,特异性较好。
利用鼠抗树鼩IgG单抗与本实验室已制备的兔抗树鼩IgG多克隆抗体,以纯化的树鼩血清IgG为阳性样本,初步建立起检测树鼩血浆中IgG总量的三抗体夹心ELISA方法。树鼩IgG检测的最佳条件为：①包被：1/400单抗腹水100μl/孔,37℃温育2h,然后4℃过夜；②封闭：2%BSA封闭,200μl/孔,37℃温育2h；③多抗：1/2000多抗,10μl/孔,空白只加PBS,37℃温育1h；④酶标抗体：1/8000酶标羊抗兔,100μl/孔,37℃温育1h；⑤TMB底物溶液,100μl/孔,37℃避光温育15min；⑥2M H2SO4终止反应,100μl/孔,用酶标仪测定OD450值。所建立的方法检测特异性高,可重复性好,树鼩IgG在0.25μg/ml～3.0μg/ml区间内,树鼩IgG量与OD450值呈较好的线性关系(R2=0.9912)。
本研究成功制备了抗树鼩IgG单克隆抗体,初步建立了检测树鼩总IgG的三抗体夹心ELISA方法,为研究树鼩免疫应答过程中抗体水平提供了有效工具,可作为树鼩的特异性抗体、中和抗体等检测方法建立的重要参考,为树鼩作为相关疾病研究动物模型的建立奠定了基础。
Tree shrew (Tupaia), with the shape of squirrel, was classified into Mammalia, placentalia Scandenti. It had been considered as primate animal, mainly distributed in tropical and subtropical areas of Southeast Asia, and was also inhabitated in Yunnan, Guangxi, Guangdong and Hainan provinces of China. More exactly, trew shrew of China was belonged to Tupaia belangeri, the most distributed subspecies. Tree shrew has small size, grow and reproduce fastly. This animal could be easily captured, domesticated and breed with low cost. Its metabolism and the anatomic characters of were much closer with humans than rodents. Because of the limitation of non-primate animal resource and the miniaturization trend of animal model, the tree shrew, as a possible animal model of human diseases, particularly viral diseases, had attracted the comprehensive attentions.
As a novel experimental animal model, the application prospect of tree shrew was desirable. Antibody level is a important parameter to evaluate the ability of immune response of bodies to invaded pathogens. But, the records on the immune response of tree shrew were still scare because of the lake of detection methods of IgG globulin. It is urgent to obtain the anti-IgG monoclonal antibodies, and to establish Enzyme Link Immunosorbent Assay (ELISA) IgG detection method. These will predicatively provide a useful tool to promote the related research on tree shrew.
Here, the purified IgG of tree shrew was used to immune female BALB/c mice, followed with artificial cytomixis hybridization of myeloma cell and spleen derived B-cell, and then screening of positive hybridoma, which could secrete anti tree shrew IgG antibody. It was proved that two positive hybridoma (TBS-1, TBS-2) had been obtained. The cell fusion rate was 32.92%, and the positive rate of hybridoma cells was 1.27%. By sub-clony screening of 4 generation, TBS-2 cells still not below of 100% positive rate, mixed with the negative cells. The valence of secreted antibody was also not high enough. All of these indicated that this strain of hybridoma was not productive and bio-stable. In comparison, the positive rate of TBS-1 had increased to 100% after 3 generations sub-clony screening. The higher valence (1:800) of its secreted antibody had been achieved. After abdominal cavity injection with TBS-1, the valence of antibody in collected ascites had been up to 1:25600. The analysis on chromosome karotype of hybridoma had shown that the number of chromosome in different passge fusion cells was normal, and its genetic characters were stable. With Western blot assay, secreated monoclonal have been proved to hybridize with serum derived tree shrew IgG effectively and specifically, no other hybrid band had been found.
Uing this prepared anti-IgG monoclonal antibody and previously gotted anti-IgG polyclonal antibody from immuned rabbit, and purified tree shrew IgG taken as positive sample, the ELISA detection method of total IgG. in tree shrew had been established preliminarily. The optimized conditions for detection was listed as below:①Coatting: 100μl 1/400 diluted ascites was used for one hole, incubated at 37℃for 2h, then overnight at 4℃;②seal:200μl 2% BSA was incubated at 37℃for 2h in one hole;③polyclonal antibody concentration:for one hole, 100μl 1/2000 diluted rebbit serum, which containing polyclonal antibody, was incubated at 37℃for 1 hour. PBS was replaced in blank control;④enzyme-labeled antibody was used in 1/8000 diluted goat anti-rabbit IgG (HRP) for 100μl/hole, at 37℃incubation for 1h;⑤TMB substrate was used in 100μl/hole at 37℃dark incubation for 15min;⑥Terminate reaction was performed with 2M H2SO4 for 100μl/hole. The established method was evaluated as high specific, good repeatable. From 0.25μg/ml to 3.0μg/ml of tree shrew IgG, a good linear relationship (R2=0.9912) had been found between OD450 values and IgG concentration.
In summary, the anti-IgG monoclonal antibody of tree shrew was prepared succefully, and the ELISA detecte method of tree shrew total IgG was established. These will provide a useful tool to evaluate the antibodies level during immune response of tree shrew. Furthermore, these could be considered as the reference for detection of specific antibody and neutralize antibody. For development of tree shrew animal model of human diseases, these would doubtless to be the necessary foundation.
树鼩作为多种疾病的的可能实验动物模型,有很好的应用前景。抗体水平是评价树鼩机体对疾病病原体免疫应答反应的重要指标,然而由于尚缺少有效的树鼩IgG检测手段,对树鼩抗体方面的研究尚未见报道。抗树鼩IgG单克隆抗体的有效制备及其为主要组分的酶联免疫吸附检测方法(Enzyme Link Immunosorbent Assay, ELISA)的建立,可以为树鼩抗体的检测提供有力的工具,促进树鼩作为实验动物模型各方面的研究。
本论文用分离、纯化得到的树鼩血清IgG免疫雌性BALB/c小鼠,经骨髓瘤/免疫脾细胞细胞融合及阳性细胞亚克隆筛选,获得能稳定分泌抗树鼩IgG抗体的杂交瘤细胞株。实验共获得了两株阳性杂交瘤细胞(TBS-1、TBS-2),细胞融合率为158/480=32.92%,融合阳性率为2/158=1.27%。其中,TBS-2细胞经过了4次亚克隆筛选,细胞阳性率仍未达到100%,并混有很多阴性细胞,培养液中分泌的抗体含量也不高,细胞纯度不高,抗体分泌不稳定；TBS-1细胞经过3次亚克隆筛选,细胞阳性率就达到100%,培养液中能稳定分泌高效价(1：800)的抗树鼩IgG单克隆抗体。用阳性细胞小鼠腹腔注射,收集腹水的方法获得大量单克隆抗体,腹水效价达到1：25600以上。融合细胞在培养不同代次经核型分析发现,杂交瘤细胞染色体数目和形态正常,细胞遗传特征稳定。Western blot结果显示,单克隆抗体可以有效结合树鼩血清IgG,没有产生杂带,特异性较好。
利用鼠抗树鼩IgG单抗与本实验室已制备的兔抗树鼩IgG多克隆抗体,以纯化的树鼩血清IgG为阳性样本,初步建立起检测树鼩血浆中IgG总量的三抗体夹心ELISA方法。树鼩IgG检测的最佳条件为：①包被：1/400单抗腹水100μl/孔,37℃温育2h,然后4℃过夜；②封闭：2%BSA封闭,200μl/孔,37℃温育2h；③多抗：1/2000多抗,10μl/孔,空白只加PBS,37℃温育1h；④酶标抗体：1/8000酶标羊抗兔,100μl/孔,37℃温育1h；⑤TMB底物溶液,100μl/孔,37℃避光温育15min；⑥2M H2SO4终止反应,100μl/孔,用酶标仪测定OD450值。所建立的方法检测特异性高,可重复性好,树鼩IgG在0.25μg/ml～3.0μg/ml区间内,树鼩IgG量与OD450值呈较好的线性关系(R2=0.9912)。
本研究成功制备了抗树鼩IgG单克隆抗体,初步建立了检测树鼩总IgG的三抗体夹心ELISA方法,为研究树鼩免疫应答过程中抗体水平提供了有效工具,可作为树鼩的特异性抗体、中和抗体等检测方法建立的重要参考,为树鼩作为相关疾病研究动物模型的建立奠定了基础。
Tree shrew (Tupaia), with the shape of squirrel, was classified into Mammalia, placentalia Scandenti. It had been considered as primate animal, mainly distributed in tropical and subtropical areas of Southeast Asia, and was also inhabitated in Yunnan, Guangxi, Guangdong and Hainan provinces of China. More exactly, trew shrew of China was belonged to Tupaia belangeri, the most distributed subspecies. Tree shrew has small size, grow and reproduce fastly. This animal could be easily captured, domesticated and breed with low cost. Its metabolism and the anatomic characters of were much closer with humans than rodents. Because of the limitation of non-primate animal resource and the miniaturization trend of animal model, the tree shrew, as a possible animal model of human diseases, particularly viral diseases, had attracted the comprehensive attentions.
As a novel experimental animal model, the application prospect of tree shrew was desirable. Antibody level is a important parameter to evaluate the ability of immune response of bodies to invaded pathogens. But, the records on the immune response of tree shrew were still scare because of the lake of detection methods of IgG globulin. It is urgent to obtain the anti-IgG monoclonal antibodies, and to establish Enzyme Link Immunosorbent Assay (ELISA) IgG detection method. These will predicatively provide a useful tool to promote the related research on tree shrew.
Here, the purified IgG of tree shrew was used to immune female BALB/c mice, followed with artificial cytomixis hybridization of myeloma cell and spleen derived B-cell, and then screening of positive hybridoma, which could secrete anti tree shrew IgG antibody. It was proved that two positive hybridoma (TBS-1, TBS-2) had been obtained. The cell fusion rate was 32.92%, and the positive rate of hybridoma cells was 1.27%. By sub-clony screening of 4 generation, TBS-2 cells still not below of 100% positive rate, mixed with the negative cells. The valence of secreted antibody was also not high enough. All of these indicated that this strain of hybridoma was not productive and bio-stable. In comparison, the positive rate of TBS-1 had increased to 100% after 3 generations sub-clony screening. The higher valence (1:800) of its secreted antibody had been achieved. After abdominal cavity injection with TBS-1, the valence of antibody in collected ascites had been up to 1:25600. The analysis on chromosome karotype of hybridoma had shown that the number of chromosome in different passge fusion cells was normal, and its genetic characters were stable. With Western blot assay, secreated monoclonal have been proved to hybridize with serum derived tree shrew IgG effectively and specifically, no other hybrid band had been found.
Uing this prepared anti-IgG monoclonal antibody and previously gotted anti-IgG polyclonal antibody from immuned rabbit, and purified tree shrew IgG taken as positive sample, the ELISA detection method of total IgG. in tree shrew had been established preliminarily. The optimized conditions for detection was listed as below:①Coatting: 100μl 1/400 diluted ascites was used for one hole, incubated at 37℃for 2h, then overnight at 4℃;②seal:200μl 2% BSA was incubated at 37℃for 2h in one hole;③polyclonal antibody concentration:for one hole, 100μl 1/2000 diluted rebbit serum, which containing polyclonal antibody, was incubated at 37℃for 1 hour. PBS was replaced in blank control;④enzyme-labeled antibody was used in 1/8000 diluted goat anti-rabbit IgG (HRP) for 100μl/hole, at 37℃incubation for 1h;⑤TMB substrate was used in 100μl/hole at 37℃dark incubation for 15min;⑥Terminate reaction was performed with 2M H2SO4 for 100μl/hole. The established method was evaluated as high specific, good repeatable. From 0.25μg/ml to 3.0μg/ml of tree shrew IgG, a good linear relationship (R2=0.9912) had been found between OD450 values and IgG concentration.
In summary, the anti-IgG monoclonal antibody of tree shrew was prepared succefully, and the ELISA detecte method of tree shrew total IgG was established. These will provide a useful tool to evaluate the antibodies level during immune response of tree shrew. Furthermore, these could be considered as the reference for detection of specific antibody and neutralize antibody. For development of tree shrew animal model of human diseases, these would doubtless to be the necessary foundation.
引文
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