Preparation and Identification of Neutralizing Monoclonal Antibodies against Egg Drop Syndrome Virus and the Use in Sandwich ELISA

doi:10.15933/j.cnki.1004-3268.2024.02.014

Journal of Henan Agricultural Sciences ›› 2024, Vol. 53 ›› Issue (2): 128-135.DOI: 10.15933/j.cnki.1004-3268.2024.02.014

• Animal Husbandry and Veterinary Medicine • Previous Articles Next Articles

Preparation and Identification of Neutralizing Monoclonal Antibodies against Egg Drop Syndrome Virus and the Use in Sandwich ELISA

WEI Qiang¹，LI Qingmei¹，JIN Qianyue¹，SONG Yapeng¹，BAI Yilin³，ZHANG Gaiping^1,2

（1.Key Laboratory of Animal Immunology，Henan Academy of Agricultural Sciences/Henan Key Laboratory of Animal Immunology/Key Laboratory of Animal Immunology，Ministry of Agriculture，Zhengzhou 450002，China；2.College of Animal Science and Veterinary Medicine，Henan Agricultural University，Zhengzhou 450002，China；3.School of Agricultural Sciences，Zhengzhou University，Zhengzhou 450001，China）

Received:2023-09-25 Published:2024-02-15 Online:2024-03-20

鸡减蛋综合征病毒中和性单克隆抗体的制备、鉴定及在双抗体夹心ELISA 中的应用

魏蔷¹，李青梅¹，金前跃¹，宋亚鹏²，白怡霖³，张改平^1,2

（1. 河南省农业科学院动物免疫学重点实验室/河南省动物免疫学重点实验室/农业部动物免疫学重点实验室，河南郑州 450002；2. 河南农业大学牧医工程学院，河南郑州 450002；3. 郑州大学农学院，河南郑州 450001）

通讯作者: 张改平（1960-），男，河南内黄人，研究员，博士，中国工程院院士，主要从事动物免疫学研究。E-mail：zhanggaip@126.com
作者简介:魏蔷（1989-），女，河南驻马店人，助理研究员，博士，主要从事动物免疫学研究。E-mail：chrysqiang@126.com
基金资助:
国家自然科学基金青年基金项目（32002284）；河南省农业科学院优秀青年科技基金项目（2022YQ22）；河南省科技攻关项目
（232102110105）

Abstract

Abstract: Recombinant egg drop syndrome virus(EDSV)fiber protein was obtained and its hemagglutination(HA)activity was identified in order to generate neutralizing monoclonal antibodies against EDSV.The HA titer of the purified fiber protein reached 1∶2⁷.The recombinant fiber protein was used as an immunogen to immunize BALB/c mice.The indirect enzyme⁃linked immunosorbent assay(iELISA)and indirect immunofluorescence assay(IFA)titers of the mice serum were up to 1∶409 600and 1∶12 800，respectively,after four immunizations. Hybridoma cells were prepared by cell fusion using spleen cells from immunized mice. Two hybridoma cell lines,9G12 and 10E11，which could secrete neutralizing monoclonal antibodies stably，were effectively developed after subcloning and screening with iELISA and IFA.The ascites titers of monoclonal antibodies 9G12 and 10E11 detected by iELISA were 1∶128 000 and 1∶1 020 000，respectively.The titers of IFA were 1∶2 000 and 1∶8 000,espectively.Both monoclonal antibodies were unable to react selectively with the denatured EDSV fiber protein，according to the Western blot data. Viral neutralization test confirmed that 9G12 and 10E11 had neutralization activity.The neutralization titers of 9G12 and 10E11 were 1∶2⁷ and 1∶2⁴_,respectively.The heavy chains of the antibodies were IgG1 subtype,whereas the light chains were Kappa type.A sandwich ELISA method based on monoclonal antibodies 9G12 and 10E11 was applied to the detection of EDSV in clinical samples,and the coincidence rate with real⁃time PCR results was 91.7%.In conclusion，two hybridoma cell lines，9G12 and 10E11,which secreted specific anti⁃EDSV antibodies,were successfully screened in this study.Both monoclonal antibodies had the activity of neutralizing EDSV infection and could be applied to clinical detection of EDSV.

Key words: Egg drop syndrome virus（EDSV）, Fiber protein, Neutralizing monoclonal antibodies, Antibody neutralization titer, Sandwich ELISA

摘要： 为了制备针对鸡减蛋综合征病毒（EDSV）的中和性单克隆抗体，首先纯化得到重组EDSV纤维蛋白，经血凝试验（HA）鉴定纤维蛋白的血凝活性，HA 效价为1∶2⁷。之后将纤维蛋白作为免疫原免疫BALB/c小鼠，免疫4次后，小鼠血清的间接酶联免疫吸附试验（iELISA）效价最高达到1∶409 600，间接免疫荧光试验（IFA）效价最高达到1∶12 800。取效价最高免疫小鼠脾细胞进行细胞融合制备杂交瘤细胞，结合iELISA和IFA检测，经过多轮亚克隆，最终成功获得了2株稳定分泌中和性单克隆抗体的杂交瘤细胞株9G12 和10E11。iELISA 结果显示，单克隆抗体9G12 和10E11 的腹水效价分别为1∶128 000 和1∶1 020 000；IFA效价分别为1∶2 000和1∶8 000。Western blot检测结果显示，单克隆抗体9G12和10E11均不能识别变性的EDSV纤维蛋白，表明二者识别纤维蛋白的构象型表位。病毒中和试验结果表明，单克隆抗体9G12和10E11均具有中和活性，中和效价分别为1∶2⁷和1∶2⁴。亚型鉴定表明，这2种单克隆抗体的轻链均为Kappa型，重链均为IgG1亚型。将基于单克隆抗体9G12和10E11建立的双抗体夹心ELISA方法应用于临床检测EDSV抗原，与荧光定量PCR检测结果的符合率为91.7%。综上，成功筛选出9G12和10E11杂交瘤细胞株，二者分泌抗EDSV特异性抗体，且这2种抗体具有中和EDSV感染活性，可应用于EDSV的临床检测。

关键词: 减蛋综合征病毒, 纤维蛋白, 中和性单克隆抗体, 抗体中和效价, 双抗体夹心ELISA

CLC Number:

S855.3

WEI Qiang, LI Qingmei, JIN Qianyue, SONG Yapeng, BAI Yilin, ZHANG Gaiping. Preparation and Identification of Neutralizing Monoclonal Antibodies against Egg Drop Syndrome Virus and the Use in Sandwich ELISA[J]. Journal of Henan Agricultural Sciences, 2024, 53(2): 128-135.

魏蔷, 李青梅, 金前跃, 宋亚鹏, 白怡霖, 张改平. 鸡减蛋综合征病毒中和性单克隆抗体的制备、鉴定及在双抗体夹心ELISA 中的应用[J]. 河南农业科学, 2024, 53(2): 128-135.

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Preparation and Identification of Neutralizing Monoclonal Antibodies against Egg Drop Syndrome Virus and the Use in Sandwich ELISA

鸡减蛋综合征病毒中和性单克隆抗体的制备、鉴定及在双抗体夹心ELISA 中的应用

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