Generation of Antibodies in Agricultural Species Using Classical and Novel Platforms

Abstract

Research, disease diagnosis, and treatment benefit from antibodies, and monoclonal and polyclonal antibodies are valuable tools. Mice, rats, and humans are the only species used to generate monoclonal antibodies (mAbs). The strict use of these species to generate mAbs has led to a lack of reagents against highly conserved epitopes. Thus, we sought a means of using both poultry and horses as a host species to generate mAbs. Activated caspase-3 was chosen due to a lack of high-affinity anti-p17 antibodies. Through the development of single B-cell cloning technology we generation of a chicken mAb, designated 2G3, specific against both the p17 peptide that was used for immunization and cell lysate stimulated to undergo apoptosis while not recognizing the procaspase-3 peptide or non-induced cell lysate. This demonstrated that we indeed developed a monoclonal antibody that specifically recognizes p17. Hyper-immunized anti-PNAG serum has been shown to be efficient in treating pneumonia in foals caused by Rhodococcus equi (R. equi). We propose the development of an equine anti-PNAG mAb as a treatment for R. equi due to the variability in serum lots and the high volume of serum required for treatment. Three equine anti-PNAG mAbs were developed using a modified version of single B-cell sequencing technology. Our criteria required at least a threefold increase in PNAG recognition, and the dominant clone B11 only increased it by a factor of 2.5 over the control. These data suggest that we did not generate a therapeutic equine anti-PNAG mAb. This was the first time that the generation of equine mAbs had been reported in scientific literature. Recently, bovine ultralong (UL) CDRH3 antibodies have been utilized to generate broadly reactive antibodies. The lack of reagents for the recognition of bovine UL antibodies has resulted in a paucity of data regarding these antibodies. A chicken polyclonal reagent against the CTTVHQ motif in bovine CDRH3 stalk regions recognizes the heavy chain of UL CDRH3 mAb, bovine B-cells expressing ultralong BCRs, and determines the concentration of bovine UL CDRH3 from a bovine serum sample. This novel reagent can be used for studying these unique yet relatively undescribed antibodies.