基于非洲猪瘟病毒p30与p54蛋白表位串联多肽的间接ELISA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2022.12.018

Abstract

Abstract: Here, we report the development of an indirect ELISA antibodies detection method for African swine fever virus (ASFV). Two purified monoclonal antibodies (mAbs) against ASFV p30 and p54 protein were used as targets and a phage-displayed 12-mer peptide library was used to conduct four rounds of biopanning to screen peptide epitopes, then amino acids GGG was used as a linker to synthesize tandem-epitope peptide of ASFV p30 and p54 protein which was used as coating antigen. The optimum reaction conditions of indirect ELISA were determined by chessboard titration, and clinical serum samples were used to evaluate the specificity, sensitivity, stability and conformity of this method. The biopanning experiment indicated that ¹⁴⁶PAEPYTT¹⁵² was a core domain of the B cell linear epitope of p54 protein. The optimization results of ELISA reaction conditions showed that the tandem-epitope peptide coupled with ovalbumin (OVA) at N-terminal had low background of non-specific serum reaction. And the optimum reaction effect was obtained when the polypeptide antigen was coated with carbonate buffer in 2 μg·mL^-1, the serum was diluted 100-fold with blocking solution (1% gelatin solution), and the HRP-antibody was diluted 5 000 times with 0.05% PBST solution. The cut-off value was determined to be 0.339. Furthermore, the results of specificity, sensitivity and stability tests showed that there is no cross-reaction in positive serum samples of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV), the detection limit of ASFV positive sera is 1∶1 600, and the method had high repeatability. Finally, Total 320 swine serum samples were detected simultaneously by the present established method and commercial ASFV antibody detection kit. The results showed that the relative specificity and sensitivity of the two methods were 97.6% and 97.3%, respectively. And the coincidence rate was 97.5%. In conclusion, this method showed good specificity, sensitivity, repeatability and coincidence rate, that had the potential value of developing clinical diagnostic kit.

Key words: African swine fever virus, p30 protein, p54 protein, biopinning, tandem-epitope peptide, indirect ELISA

CLC Number:

MA Jun, WANG Zhiyuan, LIANG Xingling, ZHENG Zezhong, YANG Hanchun, ZHANG Guihong, WANG Heng. Development of an Indirect ELISA Antibodies Detection Method on Tandem-epitope Peptide of African Swine Fever Virus p30 and p54 Proteins[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4325-4336.

References 26

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Development of an Indirect ELISA Antibodies Detection Method on Tandem-epitope Peptide of African Swine Fever Virus p30 and p54 Proteins

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