BackgroundThe highly pathogenic avian influenza viruses of the H5 subtype, such as the H5N1 viral strains or the novel H5N8 and H5N2 reassortants, are of both veterinary and public health concern worldwide. To combat these viruses, monoclonal antibodies (mAbs) against H5 hemagglutinin (HA) play a significant role. These mAbs are effective diagnostic and therapeutic agents and powerful tools in vaccine development and basic scientific research. The aim of this study was to obtain diagnostically valuable mAbs with broad strain specificity against H5-subtype AIVs.ResultsWe applied the hybridoma method to produce anti-HA mAbs. The cloning and screening procedures resulted in the selection of 7 mouse hybridoma cell lines and their respective antibody clones. Preliminary immunoreactivity studies showed that these newly established mAbs, all of the IgG1 isotype, had high specificity and broad-range activities against the H5 HAs. However, these studies did not allow for a clear distinction among the selected antibodies and mAb-secreting hybridoma clones. To differentiate the analyzed mAbs and determine the exact number of hybridoma clones, peptide mapping of the Fc and Fab fragments was performed using a Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF/TOF) mass spectrometer. Detailed analyses of the acquired MS and MS/MS spectra confirmed that the Fc fragments constituted highly conserved species- and isotype-immunoglobulin components, whereas the Fab fragments exhibited considerable variation in the sequences that determine antibody specificity. This approach enabled unambiguous characterization of the selected mAbs according to their peptide composition. As a result, 6 different clones were distinguished.ConclusionsOur work provided a unique panel of anti-H5 HA mAbs, which meets the demand for novel, high-specificity analytical tools for use in serologic surveillance. Applications of these mAbs in areas other than diagnostics are also possible. Moreover, we demonstrated for the first time that peptide mapping of antibody fragments with mass spectrometry is an efficient method for the differentiation of antibody clones and relevant antibody-producing cell lines. The method may be successfully used to characterize mAbs at the protein level.Electronic supplementary materialThe online version of this article (10.1186/s12985-017-0886-2) contains supplementary material, which is available to authorized users.

Our research aimed to compare the effects of four cryoprotectants and four slow freezing programs on the viability and apoptosis of primordial germ cells (PGCs) in vitro. PGCs were collected from chicken embryonic blood at Hamburger and Hamilton (HH) stages 14-16 and purified by Percoll density gradient centrifugation and then subjected to cryopreservation. We applied microscopy to determine the survival of PGCs after trypan blue staining and flow cytometry to examine apoptosis and viability after annexin V kit staining. We also examined the functionality of cryopreserved PGCs in vivo. Significant differences in viability of PGCs determined via microscopy and flow cytometry were observed. The most unfavorable combination for slow freezing PGCs was program 3 and MIX H (10% DMSO and 5% glycerol in Hank's solution supplemented with 10% FBS) as the cryoprotectant (48.43 and 15.37% live and early apoptotic PGCs, respectively). The highest average percentage of live PGCs (93.1%) and the lowest percentage of early apoptotic PGCs (6.5%) were achieved by slow freezing PGCs in the presence of DMSO F (10% DMSO in FBS) via program 1. Therefore, this method was chosen for the in vivo test. Cryopreserved (group 1) and freshly isolated (group 2) PGCs were transfectedwith a pEGFP-N1 plasmid, cultured under antibiotic selection, and then injected into 3-day-old embryos. After 5 days of incubation, we identified the EGFP marker gene in the gonads of 40 and 45% of recipients in groups 1 and 2, respectively. This is the first study to apply flow cytometry to examine the apoptosis and viability of cryopreserved PGCs. The in vitro and in vivo findings showed that the developed PGC cryoconservation method, depending on slow freezing at the rate of 2°C/min (program 1) in the presence of 10% DMSO F, is an improvement over previous cryoconservation methods and may be a useful tool for the ex situ strategy of poultry biodiversity preservation.

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Algae are employed commonly in cosmetics, food and pharmaceuticals, as well as in feed production and biorefinery processes. In this study, post-fermentation leachate from a biogas plant which exploits stillage and maize silage was utilized as a culture medium for Chlorella vulgaris. The content of polyphenols in hydrophilic extracts of the Chlorella vulgaris biomass was determined, and the extracts were evaluated for their antioxidant activity (DPPH assay), antibacterial activity (against Escherichia coli, Lactobacillus plantarum, Staphylococcus aureus, Staphylococcus epidermidis) and antifungal activity (against Aspergillus niger, Candida albicans, Saccharomyces cerevisiae). The use of the post-fermentation leachate was not found to affect the biological activity of the microalgae. The aqueous extract of Chlorella vulgaris biomass was also observed to exhibit activity against nematodes. The results of this study suggest that Chlorella vulgaris biomass cultured on post-fermentation leachate from a biogas plant can be successfully employed as a source of natural antioxidants, food supplements, feed, natural antibacterial and antifungal compounds, as well as in natural methods of plant protection.

The goal of this study was to determine the effectiveness of methane fermentation of dairy wastewaters in terms of the volume and qualitative composition of biogas produced in an anaerobic reactor with magneto-active filling (MAF) mounted in the zone of hydrolysis and acidogenesis. Experiments were conducted in the fractional-technical scale in a vertical fermentation reactor with a labyrinth flow. Synthetic dairy wastewaters prepared based on milk powder were used as a substrate. The analyzed filling was made of plastic covered with iron and copper powders and was additionally equipped in fluid magnetizers (FM) in the form of neodymium magnets. The study demonstrated that MAF incorporation into the technological system significantly improved effectiveness of biogas production, increased methane concentration and lowered content of hydrogen sulfide in gaseous metabolites of fermentative bacteria. A significant increase was also observed in the effectiveness of COD removal from dairy wastewaters. Reported experiments proved a correlation between the effectiveness of methane fermentation and number of MAF elements introduced into the anaerobic reactor.