IBDV单克隆抗体的制备及双抗体夹心ELISA检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.07.035

Abstract

Abstract:

The aim of this study was to establish a rapid and effective method for detecting infections bursal disease virus (IBDV), monoclonal antibodies were prepared using the whole IBDV as the immunogen. Double antibody sandwich ELISA method was established using monoclonal antibody 6G3 as the capture antibody and monoclonal antibody 4C12 as the detection antibody for the detection of IBDV. The results showed that the ELISA method had good specificity and no cross-reaction with REV, AEV, ALV or EDSV. The sensitivity test showed that the minimum detectable amount of IBDV is 1.585×10³ELD₅₀. The results of inter and intra plate repeatability tests showed that this method had good repeatability. Forty-nine clinical samples were detected by the double antibody sandwich ELISA method and commercial IBDV antigen detection card, and the coincidence rate was 94.18%. In conclusion, the double-antibody sandwich ELISA method established in this study has good specificity, sensitivity and repeatability, and can be used for rapid detection of IBDV. Thus, laysing the foundation for the development of a commercial IBDV detection kit.

Key words: IBDV, monoclonal antibody, detection method, double antibody sandwich ELISA

CLC Number:

S852.65

JIANG Yanping, LIU Wei, GONG Haoyang, CAI Limeng, LI Jiaxuan, CUI Wen, ZHOU Han, HAN Jianchun, TANG Lijie. Preparation of Monoclonal Antibodies to IBDV and Establishment of a Double Antibody Sandwich ELISA Method for Detection[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3433-3441.

Figures/Tables 12

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References 22

1	张建军, 高巍, 侯军. 鸡传染性法氏囊病毒研究进展[J]. 中国家禽, 2007, 29 (12): 47-49, 52.
	ZHANG J J , GAO W , HOU J . Research progress of infectious bursal virus of chicken[J]. China Poultry, 2007, 29 (12): 47-49, 52.
2	SIVANANDAN V , MAHESWARAN S K . Immune profile of infectious bursal disease: Ⅰ. Effect of infectious bursal disease virus on peripheral blood T and B lymphocytes of chickens[J]. Avian Dis, 1980, 24 (3): 715- 725.
3	MÜLLER H . Replication of infectious bursal disease virus in lymphoid cells[J]. Arch Virol, 1986, 87 (3-4): 191- 203.
4	佟美姣, 高雪丽, 李博韬, 等. 传染性法氏囊病毒对SPF雏鸡免疫器官细胞凋亡及bcl-2 mRNA表达的影响[J]. 畜牧兽医学报, 2013, 44 (4): 617- 621.
	TONG M J , GAO X L , LI B T , et al. The apotosis and expression of bcl-2 mRNA in the immune organs of SPF chicks infected with infectious bursal disease virus[J]. Acta Veterinaria et Zootechnica Sinica, 2013, 44 (4): 617- 621.
5	BURKHARDT E , MÜLLER H . Susceptibility of chicken blood lymphoblasts and monocytes to infectious bursal disease virus (IBDV)[J]. Arch Virol, 1987, 94 (3-4): 297- 303.
6	TSUKAMOTO K , TANIMURA N , MASE M , et al. Comparison of virus replication efficiency in lymphoid tissues among three infectious bursal disease virus strains[J]. Avian Dis, 1995, 39 (4): 844- 852.
7	ASFOR A S , REDDY V R A P , NAZKI S , et al. Modeling infectious bursal disease virus (IBDV) antigenic drift in vitro[J]. Viruses, 2023, 15 (1): 130.
8	CUI P , MA S J , ZHANG Y G , et al. Genomic sequence analysis of a new reassortant infectious bursal disease virus from commercial broiler flocks in central China[J]. Arch Virol, 2013, 158 (9): 1973- 1978.
9	STOUTE S T , JACKWOOD D J , SOMMER-WAGNER S E , et al. Pathogenicity associated with coinfection with very virulent infectious bursal disease and Infectious bursal disease virus strains endemic in the United States[J]. J Vet Diagn Invest, 2013, 25 (3): 352- 358.
10	ZHANG W , WANG X , GAO Y , et al. The over-40-years epidemic of infectious bursal disease virus in China[J]. Viruses, 2022, 14 (10): 2253.
11	FENG X F , ZHU N , CUI Y Q , et al. Characterization and pathogenicity of a naturally reassortant and recombinant infectious bursal disease virus in China[J]. Transbound Emerg Dis, 2022, 69 (4): e746- e758.
12	ISLAM M R , NOORUZZAMAN M , RAHMAN T . A unified genotypic classification of infectious bursal disease virus based on both genome segments[J]. Avian Pathol, 2021, 50 (2): 190- 206.
13	WANG W W , HUANG Y , ZHANG Y , et al. The emerging naturally reassortant strain of IBDV (genotype A2dB3) having segment A from Chinese novel variant strain and segment B from HLJ 0504-like very virulent strain showed enhanced pathogenicity to three-yellow chickens[J]. Transbound Emerg Dis, 2022, 69 (4): e566- e579.
14	刘静静. IBDV双抗体夹心ELISA检测方法的建立和胶体金试纸条的研制[D]. 泰安: 山东农业大学, 2013.
	LIU J J. Establishment of a double antibody sandwich ELISA and colloidal gold immunochromatographic strip for the detection of infectious bursal disease virus[D]. Taian: Shandong Agricultural University, 2013. (in Chinese)
15	JIANG Y P , LIN Q Y , HAN B , et al. Molecular Differentiation of different pathogenic phenotypes of infectious bursal disease viruses by RT-PCR combined with restriction fragment length polymorphism (RFLP) assay[J]. Journal of Northeast Agricultural University (English Edition), 2019, 26 (1): 37- 45.
16	杨坤, 马静文, 周新瑞, 等. 三株传染性法氏囊病病毒重组毒株的致病性研究[J]. 畜牧兽医学报, 2024, 55 (6): 2550- 2559. doi: 10.11843/j.issn.0366-6964.2024.06.026
	YANG K , MANG J W , ZHOU X R , et al. Pathogenicity of three recombinant strains of infectious bursal disease virus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55 (6): 2550- 2559. doi: 10.11843/j.issn.0366-6964.2024.06.026
17	姜楠. IBDV新型变异株与超强毒株致病性和抗原性差异及其分子机制研究[D]. 哈尔滨: 中国农业科学院, 2022.
	JIANG N. Study on the differences of pathogenicity and antigenicity between novel variant IBDV and very virulent IBDV and its molecular mechanism[D]. Harbin: Chinese Academy of Agricultural Sciences, 2022. (in Chinese)
18	林彦星, 翁巧玉, 吴江, 等. 检测非洲猪瘟病毒抗原的双抗体夹心ELISA方法的建立[J]. 中国兽医学报, 2024, 44 (1): 7- 11.
	LIN Y X , WENG Q Y , WU J , et al. Development of double antibody sandwich ELISA assay for detection of African swine fever virus[J]. Chinese Journal of Veterinary Science, 2024, 44 (1): 7- 11.
19	杨帆. 抗H7N9禽流感病毒HA蛋白单克隆抗体的制备及初步应用[D]. 杭州: 浙江大学, 2020.
	YANG F. Preparation and preliminary application of monoclonal antibodies against HA protein of H7N9 avian influenza virus[D]. Hangzhou: Zhejiang University, 2020. (in Chinese)
20	纪品品. 基于纳米抗体的Fenobody和RANbody禽新城疫病毒夹心ELISA检测方法的建立[D]. 杨凌: 西北农林科技大学, 2020.
	JI P P. Development of Newcastle disease virus sandwich ELISA detection assay based on Fenobody and RANbody of nanobody[D]. Yangling: Northwest A&F University, 2020. (in Chinese)
21	NI T , PENG D , WANG Y , et al. Development of a broad-spectrum monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for the multi-residue detection of avermectins in edible animal tissues and milk[J]. Food Chem, 2019, 286, 234- 240.
22	刘赫. 鸡源呼肠孤病毒检测方法的建立及其σC蛋白单克隆抗体的制备[D]. 泰安: 山东农业大学, 2019.
	LIU H. Development of Detection method and preparation of monoclonal antibodies to σC protein of chicken-derived avian orthoreovirus[D]. Taian: Shandong Agricultural University, 2019. (in Chinese)

亚型Subtype	单抗Monoclonal antibody
IgG1κ	3D7、4C12、6G3
IgG2aκ	9D11、8F7
IgGMκ	2A5

杂交瘤细胞Hybridoma cell	3D7	4C12	6G3	9D11	8F7	2A5
3D7	-	28.68%	25.31%	20.26%	18.45%	25.92%
4C12	28.68%	-	37.69%	40.7%	29.98%	44.82%
6G3	25.31%	37.69%	-	23.6%	34.42%	15.53%
9D11	20.26%	40.7%	23.6%	-	13.63%	7.92%
8F7	18.45%	29.98%	34.42%	13.63%	-	28.54%
2A5	25.92%	44.82%	15.53%	7.92%	28.54%	-

项目 Item	双抗体夹心ELISA Double antibody sandwich ELISA	IBDV抗原检测卡 IBDV antigen test card
阳性样品数量 Number of positive samples	16	17
阴性样品数量 Number of negative samples	33	32

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Preparation of Monoclonal Antibodies to IBDV and Establishment of a Double Antibody Sandwich ELISA Method for Detection

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