Development of a IPMA Method Applicable for Screening mAb Targeting PRRSV

doi:10.7668/hbnxb.20190199

Abstract

Abstract: To develop a high-throughput cell culture detection method for screening specific and sensitive monoclonal antibodies against Porcine reproductive and respiratory syndrome virus(PRRSV). A single layer of Marc-145 cells covered with 96-well plates was inoculated with a virus amount of about 100 cells infected per well. After 12 h, cells were fixed with 3% H₂O₂ in methanol to prepare an immunoperoxidase monolayer cell assay(Immunoperoxidase monolayer assay, IPMA) reaction plate. The culture supernatant of the hybridoma cells cultured for 10 days after the fusion was added to the IPMA reaction plate in an amount of 100 μL/well, and horseradish peroxidase-labeled goat anti-mouse IgG-HRP was used as the secondary antibody and 3-amino-9-ethylcarbazole(AEC) was used as a chromogenic substrate, then observed under an inverted microscope. The results showed that 39 PRRSV monoclonal antibodies, such as 1D1 and 7G8, were screened, and these 39 monoclonal antibodies were able to specifically stain Marc-145 cells infected with high-viral strain HN07-1 and classical strain BJ-4 PRRSV. There was no cross-staining of Marc-145 cells infected with Classical swine fever virus, Pseudorabies virus, and Porcine epidemic diarrhea virus. Therefore, this IPMA method constructed by the study can capture PRRSV monoclonal antibodies sensitively and accurately.

Key words: Porcine reproductive and respiratory syndrome virus, IPMA, Screening, Monoclonal antibody

CLC Number:

S852.651

YANG Yanyan, QIAO Songlin, LI Rui, GUO Junqing, LI Qingmei, TENG Man, WANG Li, ZHAO Dong, LI Xuewu, DENG Ruiguang, ZHANG Gaiping. Development of a IPMA Method Applicable for Screening mAb Targeting PRRSV[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(3): 217-223. doi: 10.7668/hbnxb.20190199.

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