Development of Whole-Porcine Monoclonal Antibodies with Potent Neutralization Activity against Classical Swine Fever Virus from Single B Cells

Abstract

Classical swine fever (CSF) is a highly contagious swine disease found worldwide that has caused devastating economic losses. However, there are few efficacious mAbs against the CSF virus (CSFV) that can be used for treatment because most mAbs against CSFV are derived from mouse hybridoma cells and these murine mAbs have disadvantages of inefficient effector functions elicitations and high immunogenicity in vivo. Accordingly, we characterized whole-porcine anti-CSFV neutralizing mAbs (NAbs) isolated directly from single B cells sorted from a CSFV-vaccinated pig using the fluoresceinated conserved linear neutralizing epitope of the CSFV E2 protein and fluorophore conjugated goat anti-pig IgG.Immunoglobulin (Ig) genes were isolated via nested PCR, and two porcine mAbs termed HK24 and HK44 were produced. We determined that these mAbs can bind to E2 protein and recognize sites within this major antigenic epitope. In addition, we found that mAbs HK24 and HK44 exhibit potent neutralizing activity against CSFV, and they can protect PK-15 cells from infections in vitro with potent IC 50 values of 9.3 μg/ml and 0.62 μg/ml, respectively.Notably, we demonstrated that these two mAbs can be used as novel reagents for detecting virus infection. These data suggest that our results not only provide a method for efficiently obtaining mAbs against CSFV but also offer promising mAb candidates for development of antibody-based diagnostic and antiviral agents.

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“…Equal quantities of samples were separated and immunoblotted as previously described. 34 The primary antibodies used were mouse anti-CTR1 (Cell Signaling Technology) and mouse anti-β-actin (Proteintech).…”

Liquid chromatography–tandem mass spectrometry glycoproteomic study of porcine IgG and detection of subtypes

“…Pig antibodies also hold promise in the development of therapeutic agents against viral diseases. With this aim, Dong et al isolated two pig IgG variants specific to the classical swine fever virus 7 . The quantitative analysis of pig antibodies in this regard has become of importance, as emphasized in a report by Zhao et al who developed a new method for the detection of pig IgG based on fluorescence via an immuno‐sensing strategy using selective pull‐down by magnetic beads 8 …”

AbSE Workflow: Rapid Identification of the Coding Sequence and Linear Epitope of the Monoclonal Antibody at the Single-cell Level

“…Recombinant mAb is Successfully Expressed and Exhibit High Affinity to the Antigen. To produce the recombinant 2F10 mAb (r2F10), the identified Igh and Igk genes were synthesized and cloned into the expression vector separately as described previously 39 (Figure S3 and Table S2). The full-length recombinant mAb was produced in HEK 293T cells and purified from the cell culture supernatants with protein G, and then characterized by SDS-PAGE, native-PAGE, and Western blot.…”

“…The resulting full-length HC and LC sequences of 2F10 were synthesized by Genscript and were sequenced before being cloned into the pEGFP-C1 expression vector as previously described. 39 The plasmids expressing the HC or LC genes were co-transfection into HEK293T cells using the Lipofectamine 3000 Transfection Reagent (Invitrogen, USA). Cells were cultured in 6-well plates at 37 °C in 5% CO 2 .…”