牛嵴病毒单抗制备及双抗体夹心ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2025.10.028

Abstract

Abstract:

This study aimed to prepare monoclonal antibody against bovine kobuvirus (BKV) and establish a double antibody sandwich ELISA method for detection. The pET-30a (+) vector was utilized to express the BKV structural proteins VP0, VP3, and VP1 in a prokaryotic expression system. Three structural proteins were purified and used for immunization in BALB/c mice respectively. After three immunizations, the spleen cells of the mice were isolated and fused with SP2/0 cells to prepare hybridoma cells. Then the hybridoma cells were screened by the indirect ELISA method, the prepared monoclonal antibodies were used as capture antibody and detection antibody respectively, the conditions such as antibody concentration and incubation time were optimized, and a double antibody sandwich ELISA method for specifically detecting BKV was established. Results were as follows: Two hybridoma cell lines stably secreting monoclonal antibodies were obtained, designated 2A12 and 5E11, both of which were anti-VP1 protein antibodies; The results of the superimposed ELISA and antibody variable region sequence analysis indicated that they recognized different antigen sites. The results of monoclonal antibody specificity detection showed that the two monoclonal antibodies were specifically bound to BKV and were not bound to other bovine diarrhea viruses. One hundred and seven clinical samples were detected by RT-PCR method and the double antibody sandwich ELISA method established in this study. The results demonstrated that the positive coincidence rate between the two methods was 92.2%, the negative coincidence rate was 95.3%, and the overall coincidence rate was 93.5%. In conclusion, two monoclonal antibodies against BKV were prepared in this study, and a double antibody sandwich ELISA method for BKV was established. This method has good specificity, repeatability and sensitivity, which lays a foundation for the rapid diagnosis, prevention and control of BKV.

Key words: bovine kobuvirus, structural protein, monoclonal antibody, double antibody sandwich ELISA

CLC Number:

S852.659.6

CAO Hengzhi, MA Qianyue, JIANG Yanping, CUI Wen, LI Jiaxuan, QIAO Xinyuan. Preparation of Monoclonal Antibody against Bovine Kobuvirus and Establishment of Double Antibody Sandwich ELISA Method[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5084-5094.

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References 20

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	扩增片段大小/bp Length of amplified fragment	退火温度/℃ Annealing temperature
BKV-VP0-F	CGGGATCCATGGGCACGAGCCAGACC	1 104	56
BKV-VP0-R	CGAGCTCTCACTGCTTGGTCACATGCC
BKV-VP3-F	CGGGATCCATGCACTGGAAGACTCGAGCC	669	56
BKV-VP3-R	CGAGCTCTCATTGGAGAGCTAGCGTCGG
BKV-VP1-F	CGGGATCCATGGCTGGTGAGGGCATCG	801	54
BKV-VP1-R	CGAGCTCTCATTGGCGCCGTACCAT

引物名称 Primer name	引物序列(5′→3′) Primer sequence	扩增片段大小/bp Length of amplified fragment
VH-F	GGCCTCGAGATGCAGGTGCAGTTGATGGAGTCAG	340
VH-R	GTGAATTCTGAGGAGACGGTGACTGAGGT
VL-F	AAGCACGCGTAGACATTGTGCTGACCCAATCTC	340
VL-R	CCAGCGGCCGCGGATACAGTTGGTGCAGCATC

单克隆抗体 monoclonal antibody	说明 Description	相似性/% Per.identity	登录号 Accession No.
2A12 VL	Mus musculusstrain BALB/c isolate 6A truncated immunoglobulin light chain variable region (IgKappa) mRNA, partial cds	99.39	FJ233898.1
5E11 VL	Mus musculusisolate ms6_0280_IGKV3-4_IGKJ1_IGKC immunoglobulin light chain variable region mRNA, partial cds	97.41	PP322127.1
2A12 VH	Mus musculusclone Flu002_d8-1_GC_IgH-305 immunoglobulin heavy chain variable region (Igh) mRNA, partial cds	95.85	KU257208.1
5E11 VH	Mus musculus clone 8G2.6 anti-phosphocholine immunoglobulin heavy chain variable region gene, partial cds	99.28	AY194185.1

组别Group	HRP-2A12
2A12	0.239±0.032
5E11	0.509±0.013
PBS	0.516±0.015

样本 Samples	板内变异系数 Intra-plate variation coefficient		批间变异系数 Inter batch variation coefficient
样本 Samples	${\bar x}$ ± s	CV/%	${\bar x}$ ± s	CV/%
阳性样本 Positive samples	0.763 1±0.027 2	3.57	0.748 5±0.045 8	6.12
	0.739 2±0.060 7	8.21	0.745 3±0.054 6	7.32
	0.741 7±0.037 7	5.08	0.753 7±0.048 3	6.41
	0.748 0±0.041 9	5.60	0.749 2±0.049 6	6.62
阴性样本 Negative samples	0.135 0±0.007 2	5.35	0.128 9±0.010 6	8.24
	0.123 5±0.010 1	8.20	0.136 1±0.008 7	6.36
	0.121 9±0.004 7	3.89	0.124 1±0.010 1	8.12
	0.126 8±0.007 3	5.76	0.129 7±0.009 8	7.56

Preparation of Monoclonal Antibody against Bovine Kobuvirus and Establishment of Double Antibody Sandwich ELISA Method

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检测方法 Method		RT-PCR
检测方法 Method		阳性 Positive	阴性 Negative	合计 Total
双抗体夹心 ELISA Double antibody sandwich ELISA	阳性Positive	59	2	61
	阴性Negative	5	41	46
	合计Total	64	43	107
符合率/% Coincidence rate		92.2	95.3	93.5

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