They produce rMAb18BG3 from the Hybridoma Cells. First they isolated 30 µg of RNA from 1×10^6 hybridoma cells and used as a template in a one-step RT-PCR performed with the enzyme SuperScript III. Two independent PCRs were performed with primers using VH and VL cDNAs as templates. Both PCRs were performed with the FastStart Taq polymerase according to the manufacturer setting conditions. Finally, the resulting amplified products were employed in an overlap extension PCR to assemble the single-chain variable fragment (scFv) construct connecting 3′VH to 5′VL through the linker. This scFv was cloned by HindIII/Xho I digestion and subsequently ligated into the expression vector pCMV6-AC-Fc-S that adds a mouse Fc tag at the 3′-end of the cloned sequence. Hence, the final format of the rMAb was a scFv-Fc. The cloned sequence was corroborated using automatic sequencing, and the plasmid was purified and transfected in suspension cultures of HEK293 FreeStyle cells. The DNA was added at 1 µg of plasmid/mL of cell culture using the cationic polymer FectoPro. After four days at 37 °C, 5% CO2, and 125 rpm, the culture was collected, the supernatant separated with centrifugation, and the rMAb purified from the supernatant with affinity chromatography to protein A. The recombinant antibody was analyzed using SDS-PAGE and an indirect ELISA to assess its purity and activity, respectively.

African swine fever (ASF) is a viral disease of swine with a huge impact due to its high mortality. Lately, the disease has actively spread around the world, affecting new areas from which it had been eradicated long ago. To date, ASF control is carried out by the implementation of strict biosecurity measures such as the early identification of infected animals. In this work, two fluorescent rapid tests were developed to improve the sensitivity of point-of-care diagnosis of ASF. For antigen (Ag) detection in blood, a double-antibody sandwich fluorescent lateral flow assay (LFA) was developed, employing a newly developed recombinant antibody to the VP72 of the virus. To complement the diagnosis, a double-recognition fluorescent LFA was developed using the VP72 for the detection of specific antibodies (Ab) in sera or blood. Both assays statistically improved the detection of the disease when compared to the commercial colorimetric assays INgezim® ASFV CROM Ag and INgezim® PPA CROM Anticuerpo, respectively, with higher statistical significance between 11 and 39 days post-infection. From the observation of results, it can be concluded that the combination of both Ag-LFA and Ab-LFA assays would facilitate the identification of infected animals, regardless of post-infection time.