Journal of AOAC International · 2026
Development of Blocking ELISA Kit Based on Recombinant Monoclonal Antibody for the Serological Diagnosis of Peste Des Petits Ruminants Virus.
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Feng Chunyan, Li Runze, Wang Caixia, Geng Yujing, Wu Bin, Yu Haoyang, Li Haoxuan, Zhang Zhou, Yang Zhen, Wu Shaoqiang, Lin XiangMei
Аннотация
BACKGROUND: Peste des petits ruminants (PPR) is a highly contagious and economically devastating disease affecting sheep and goats. The performance and cost of serological diagnostic kits remain a major challenge for PPR eradication.
OBJECTIVE: The nucleocapsid (N) protein of PPR virus and its monoclonal antibody (mAb) were engineered for development of a blocking ELISA.
METHODS: The N protein was truncated and expressed by the eukaryotic expression system. The mAb 65A8 was recovered, followed by sequencing and cloning of its variable regions into expression vector. The recombinant mAb was expressed in 293F cells. Key characteristics of the recombinant mAb were evaluated. A blocking ELISA was developed and validated. Comparison between the blocking ELISA and a commercial blocking ELISA kit was performed.
RESULTS: The truncated N protein exhibited a higher yield compared to the full-length N protein. Recombinant mAb 65A8 demonstrated the same blocking efficacy as that of ascites-derived mAb. The yield of recombinant mAb reached 5.3 mg/100 mL. The affinity constant of recombinant mAb 65A8 was comparable to that of the ascites-derived 65A8 (3.48 × 109 L/mol versus 6.76 × 109 L/mol). Based on the truncated N and recombinant mAb, a blocking ELISA was established. The detection limit of blocking ELISA was 1:128 and 1:32 based on strong positive serum and weak positive serum, respectively. Inter- and intra-assay coefficients of variation were <10%, indicating robust reproducibility. The blocking ELISA kit showed high coincidence with the commercial ELISA kit recommended by the World Organization for Animal Health reference laboratory.
CONCLUSIONS: This study successfully developed a reliable and cost-effective blocking ELISA by protein engineering of the antigen and mAb.
HIGHLIGHTS: The mAb of PPR Virus was recombinantly expressed in 239F cells, which was employed in the development of a reliable blocking ELISA. This work provided novel ideas and methods for the development of the blocking ELISA kits.
OBJECTIVE: The nucleocapsid (N) protein of PPR virus and its monoclonal antibody (mAb) were engineered for development of a blocking ELISA.
METHODS: The N protein was truncated and expressed by the eukaryotic expression system. The mAb 65A8 was recovered, followed by sequencing and cloning of its variable regions into expression vector. The recombinant mAb was expressed in 293F cells. Key characteristics of the recombinant mAb were evaluated. A blocking ELISA was developed and validated. Comparison between the blocking ELISA and a commercial blocking ELISA kit was performed.
RESULTS: The truncated N protein exhibited a higher yield compared to the full-length N protein. Recombinant mAb 65A8 demonstrated the same blocking efficacy as that of ascites-derived mAb. The yield of recombinant mAb reached 5.3 mg/100 mL. The affinity constant of recombinant mAb 65A8 was comparable to that of the ascites-derived 65A8 (3.48 × 109 L/mol versus 6.76 × 109 L/mol). Based on the truncated N and recombinant mAb, a blocking ELISA was established. The detection limit of blocking ELISA was 1:128 and 1:32 based on strong positive serum and weak positive serum, respectively. Inter- and intra-assay coefficients of variation were <10%, indicating robust reproducibility. The blocking ELISA kit showed high coincidence with the commercial ELISA kit recommended by the World Organization for Animal Health reference laboratory.
CONCLUSIONS: This study successfully developed a reliable and cost-effective blocking ELISA by protein engineering of the antigen and mAb.
HIGHLIGHTS: The mAb of PPR Virus was recombinantly expressed in 239F cells, which was employed in the development of a reliable blocking ELISA. This work provided novel ideas and methods for the development of the blocking ELISA kits.