鸭短喙矮小综合征病毒单克隆抗体制备及竞争ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2026.02.039

Abstract

Abstract:

Objective This study aimed to establish a rapid serological method for detecting antibodies against Short beak and dwarfism syndrome virus （SBDSV） in ducks， providing technical support for the development of SBDSV antibody detection and diagnostic kits. Method BALB/c mice were immunized with purified and inactivated SBDSV M15 strain. Splenocytes from the immunized mice were fused with SP2/0 myeloma cells. Hybridoma cell lines that could secrete SBDSV monoclonal antibody were screened by indirect ELISA. Ascites was prepared from the mice inoculated with screened hybridoma cells. The titers of monoclonal antibodies were determined by indirect ELISA. The characteristics of the monoclonal antibodies were identified by indirect immunofluorescence assay （IFA） and latex particle agglutination （LPA） assay. The neutralizing activity of monoclonal antibodies was determined by neutralization test. Monoclonal antibodies subclasses were tested by commercialized kit. The reaction system was optimized by using the checkerboard titration method， and a competitive ELISA detection method based on SBDSV monoclonal antibody was established. The cut-off value of this method was determined by detecting negative sera. The specificity， sensitivity and repeatability of the established competitive ELISA detection method were performed. The clinical sera were detected by this method， which results were compared with the results of latex particle agglutination inhibition （LPAI） assay. Result Three hybridoma cells stably secreting SBDSV monoclonal antibody were obtained by indirect ELISA， which were named D5-3， G11-23 and H2-27， respectively. The results of indirect ELISA showed that the antibody titers in the supernatant of three hybridoma cells D5-3， G11-23 and H2-27 were 1∶2⁶， 1∶2⁵ and 1∶2⁶， respectively， and the antibody titers in the ascites were 1∶10⁵， 1∶10⁴ and 1∶10⁶， respectively. The IFA results showed that the monoclonal antibodies D5-3， G11-23 and H2-27 all had good binding activity and could be used for IFA detection. The results of LPA assay showed that only monoclonal antibody H2-27 had the characteristic of sensitizing latex. The results of neutralization test showed that all three monoclonal antibodies had the ability to neutralize SBDSV， and monoclonal antibody H2-27 had the strongest neutralizing activity. The identification results of monoclonal antibody subclasses showed that monoclonal antibody D5-3 and G11-23 belonged to IgG1 subclass， while monoclonal antibody H2-27 belonged to IgM subclass. A competitive ELISA method for detecting SBDSV antibody was established by using purified and inactivated SBDSV （M15 strain） as coating antigen and monoclonal antibody H2-27 as competitive antibody. When inhibition rate （PI）≥23.70%， serum sample was determined to be positive for SBDSV antibody， when PI≤15.91%， it was determined to be negative. This method demonstrated good specificity to SBDSV positive sera and had no cross-reaction with the positive serum of Muscovy duck parvovirus （MDPV）， Muscovy duck reovirus （MDRV）， Duck paramyxovirus （DPMV）， Duck hepatitis virus-1 （DHV-1） and Duck Tembusu virus （DTMUV）. The test result of the hyper immune serum （LPAI titer 1∶2⁸） which was diluted at 1∶25 600 was remained positive. The coefficients of variation for inter-assay and intra-assay were both less than 6%， which indicated good repeatability of this method. The consistency between the established competitive ELISA and LPAI assay was good， which reached 89.10% （188/211）. Conclusion Three monoclonal antibodies against SBDSV were successfully prepared in this study. The competitive ELISA method based on monoclonal antibody H2-27 had strong specificity， high sensitivity and good repeatability， could detect SBDSV antibodies in high throughput， providing technical support for monitoring the prevalence of SBDSV in China and for evaluating maternal antibody levels in ducklings.

Key words: duck; Short beak and dwarfism syndrome virus （SBDSV）; monoclonal antibody; competitive ELISA

CLC Number:

S852.65

ZHU Xiaoli, WANG Shao, DONG Hui, CHENG Xiaoxia, JIANG Dandan, XIAO Shifeng, CHEN Shaoying, CHEN Shilong. Preparation of Monoclonal Antibodies Against Duck Short Beak and Dwarfism Syndrome Virus and Establishment of a Competitive ELISA Detection Method[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(2): 948-958.

Figures/Tables 10

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References 33

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抗原包被浓度	单克隆抗体稀释度 Dilution of monoclonal antibody
Coating antigen concentration/（mg/mL）	1∶1 000	1∶2 000	1∶4 000
0.2	1.929	1.465	1.088
0.1	1.546	1.018	0.936
0.05	1.333	0.913	0.821

项目	血清稀释度Serum dilution
Items	1∶50	1∶100	1∶200	1∶400
阳性Positive	0.133	0.127	0.115	0.155
阴性Negative	1.042	0.973	0.933	1.172
PI/%	83.21	89.72	94.86	72.10

项目 Items	封闭液 Blocking solution
	1% BSA	5%脱脂奶粉
	1% BSA	5% skimmed milk powder
阳性Positive	0.104	0.185
阴性Negative	0.993	1.172
PI/%	90.23	69.54

项目	酶标二抗稀释度 Dilution of enzyme labeled secondary antibody
Items	1∶4 000	1∶5 000	1∶6 000	1∶7 000
阳性Positive	0.133	0.122	0.192	0.205
阴性Negative	1.142	0.954	0.923	0.916
PI/%	75.92	92.03	87.54	86.80

待检血清 Serum to be tested	D_{492 nm}值D_{492 nm} value	PI/%	结果 Results
MDPV⁺	1.005	13.46	－
MDRV⁺	1.106	4.69	－
DPMV⁺	1.111	4.26	－
DHV-1⁺	1.097	5.51	－
DTMUV⁺	1.103	4.98	－
SBDSV^－	1.139	1.91	－
SBDSV⁺	0.1536	86.77	+

Preparation of Monoclonal Antibodies Against Duck Short Beak and Dwarfism Syndrome Virus and Establishment of a Competitive ELISA Detection Method

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血清样本 Serum samples		批内试验 Intra-assay		批间试验 Inter-assay
血清样本 Serum samples		平均值±标准差 Mean±SD	变异系数 CV/%	平均值±标准差 Mean±SD	变异系数 CV/%
阳性血清 Positive serum	1	87.79±4.12	4.69	87.53±3.45	3.94
	2	85.50±4.86	5.68	86.02±3.09	3.59
	3	88.75±3.46	3.90	87.97±3.46	3.93
阴性血清 Negative serum	4	4.44±0.19	4.36	9.04±0.47	5.18
	5	6.85±0.37	5.41	6.06±0.25	4.21
	6	3.22±0.18	5.46	11.29±0.63	5.61

样品来源 Sample source	LPAI	竞争ELISA cELISA		总计 Total
样品来源 Sample source	LPAI	阳性 Positive	阴性 Negative	总计 Total
人工感染 Experimental infection	阳性 Positive	100	0	100
	阴性 Negative	10	9	19
	总计 Total	110	9	119
临床采样 Clinical sampling	阳性 Positive	63	1	64
	阴性 Negative	12	16	28
	总计 Total	75	17	92

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