Production and Characterization of Monoclonal Antibodies against Bovine Leukaemia Virus using Various Crude Antigen Preparations: a Comparative Study

Abstract

A total of 59 monoclonal antibodies (mAbs) specific against the bovine leukaemia virus (BLV) using different antigen preparations was produced. The five antigen preparations for immunizing BALB/c mice were: live cells (CEL), sonicated and ultracentrifuged cells (SOC), cell lysates (LYS), semi-purified BLV (PV), and formalin-treated cells (FOR) from two cell lines permanently infected with BLV (FLK-BLV and BLV-bat2). These viral component presentations were selected to obtain mAbs against specific BLV proteins: located on the cell surface (FOR and CEL), in free virus particles (PV) and intracellular viral proteins (SOC and LYS). Two antigen preparations (SOC and LYS) were lethal to the mice following the intravenous and intrasplenic routes. Six fusions were performed in this study that rendered specific antibodies against BLV. The highest number of hybridomas was produced with SOC; however, the majority of the hybridomas produced (> 90%) were against cellular proteins. Even though immunization with PV gave the lowest number of hybridomas, the majority of them were specific against BLV. Based on the reactivity of the mAbs in Western blot (WB), we classified the mAbs into five groups, namely anti-gp51SU (39 mAbs), anti-gp30TM (six mAbs), anti-Pr72env (nine mAbs), anti-Pr66gag-pro (one mAb) and anti-Prgag (four mAbs). A very high percentage of the mAbs produced (48 of 59) reacted with gp51SU, suggesting that this is the most immunogenic and accessible BLV protein presented in the different antigen preparations. The majority of our mAbs recognized more than one band in WB, suggesting that, aside from reacting with mature proteins, the mAbs also recognized viral precursors.

Production, Characterization, and Use of Monoclonal Antibodies Against gp51 Protein to Diagnose Bovine Leukemia Virus Infection

“…2) verified the specificity of the mAbs against gp51, suggesting that this is the most immunogenic protein of BLV and that the isotype IgM (60%) is predominant. Our results corroborate those reported by Llames et al, 19 who showed that 66.1% of the hybridomas they produced reacted to gp51 of BLV and 42.3% were the isotype IgM, using different antigen preparations.…”

Morphologic and Functional Changes in Bovine Monocytes Infected In vitro with the Bovine Leukaemia Virus

“…Western blot analysis Western blot analysis was performed to detect BLV‐related proteins using polyclonal sera from naturally BLV‐infected animals [28] Briefly, cells were harvested and treated with lysis buffer. After centrifuging at 14 000 × g for 30 min at room temperature, the supernatant was collected and kept at − 20 °C for further use.…”

“…After polymerization, samples were maintained at 60 °C for 1 h, stored at room temperature, and cut into 60‐nm ultrathin sections (Ultracut E, Reichert‐Jung, Leica, Lausanne, Switzerland) for further use. To localise BLV particles/proteins in infected cells, sections were incubated with one or two BLV‐specific MoAbs as recently described [28]. Hybridoma culture supernatant was used undiluted; MoAb generated from ascites fluid were diluted 1 : 50 in PBS‐Tween 20.…”

Characterization and application of recombinant Bovine Leukemia Virus Env protein

“…Gp51 is the most immunogenic subunit of Env and induces massive expression of specific antibodies in infected animals 20 , 21 . The N-terminal region of gp51 (the first 173 aa 22 ) contains putative receptor binding domain (RBD 16 ) together with conformational epitopes namely F, G 23 and H, involved in infectivity and syncytia formation and two neutralization domains 23 – 28 .…”