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In this work it was developed marker-free transgenic indica rice plants (cv J-104) by biolistic co-transformation and segregation approach. We attempted to express the NmDef02 antifungal defensin. Primary transformants were regenerated from embryogenic callus on culture medium with 50 mg/L hygromycin. Screening of hpt-marker-free transgenic lines was made by PCR in T 1 progeny lines, germinated on semisolid medium without hygromycin. Relative expression of NmDef02 mRNA was examined by quantitative RT-PCR in marker-free T 1 plants. In vitro antifungal test was performed by disk diffusion assay against Sarocladium oryzae. PCR assay verified that 15.12% of T 1 plants were marker-free (NmDef02+/hpt−). RT-PCR analysis indicated that NmDef02 gene was successfully transcribed and the transgenic lines displayed different expression levels of the NmDef02 cDNA. Protein extracts of marker-free lines with high relative expression of NmDef02 inhibited fungus mycelial growth around disks. In contrast, it was confirmed fungus proliferation on disks impregnated with protein extracts of non-transgenic plants. The results of the present work demonstrated that the expression of the NmDef02 defensin in transgenic rice plants is effective against the phytopathogenic fungus Sarocladium oryzae under in vitro conditions. Thus, NmDef02 defensin could be a useful tool for J-104 rice improvement.

Heberprot-P® is a therapeutic product approved in Cuba for the treatment of diabetic foot ulcer. In spite of its ample clinical use, its immunogenicity in patients has not been evaluated. Regulatory agencies have developed guidelines for the validation of immunoassays designed at the detection of anti-drug antibodies to biologicals. The aim on this work was to validate a method for the detection of binding antibodies in human serum to recombinant human epidermal growth factor (rhEGF), the active ingredient of Heberprot-P®. A two-tiered enzyme-linked immunosorbent assay (ELISA) format was used. First tier was a screening assay, based on immobilized rhEGF and anti-human polyvalent alkaline-phosphatase conjugated as development reagent. Positive samples were then tested with a confirmatory assay which used a competitive soluble rhEGF in solution. As FDA and EMA guidelines recommend, minimum required dilution, quality controls, screening and confirmatory cut points, sensitivity, recovery, precision, specificity, selectivity, robustness and stability of samples were determined. The assay was precise and its sensitivity was calculated to be 214.2 ng/mL. The majority of evaluated parameters fulfilled the figures recommended by current guidelines. The immunoassays described in this work could be reliable tools for the evaluation of immunogenicity of Heberprot-P® in well-designed clinical studies.

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Studies were conducted to improve the production of murine ascites with monoclonal antibodies that recognize SARS-CoV-2 proteins. BALB/c mice were primed with 0.5 mL of mineral oil into the abdominal cavity. Seven days after priming, mice were divided in two groups: the group 1 was inoculated intraperitoneally with 2x106 cells/mL of MAb-secreting hybridomas against the nucleocapsid and spike proteins of SARS-CoV-2; the group 2 was injected simultaneously with the same inoculum of hybridoma cells and mineral oil, 18 days after priming. No disturbances or suffering signals were observed in mice from both groups, suggesting that double administration of mineral oil did not produce significant distress with respect to the single dose used for priming, and that none of the hybridoma cell lines were particularly aggressive for the inoculated mice. Ascites was collected in 90.48% and 97.68% of mice from groups 1 and 2, respectively. Ascites was not collected in 7.42% of all mice. The main cause was they never developed ascites tumors but no solid tumors were observed either. The volume of ascitic fluid per mouse was increased significantly in mice from group 2, and there were no significant differences between groups in terms of the concentration of IgG in clarified ascites. According to these results, to obtain higher amounts of MAb the strategy applied in group 2 should be used, since it showed the best results in the development of ascites tumors and it significantly increased the volume of ascites fluid per mouse. This could allow the use of fewer animals for ascites production, which is an ethical and economic benefit.