Presentation on theme: "Production of Novel Monoclonal Antibodies Against Extracellular Polysaccharides from Pleurotus ostreatus by Using Hybridoma Technology ISEL Magda Semedo."— Presentation transcript:
1 Production of Novel Monoclonal Antibodies Against Extracellular Polysaccharides from Pleurotus ostreatus by Using Hybridoma TechnologyISELMagda Semedo 1, Sónia Martins 1, Amin Karmali 1* and Luís Fonseca21 Chemical Engineering and Biotechnology Research Center and Departmental Area of Chemical Engineering of Instituto Superior de Engenharia de Lisboa,R. Conselheiro Emídio Navarro 1, , Lisboa, Portugal.2 Department of Bioengineering, Centre for Biological and Chemical Engineering of Instituto Superior Técnico , Av. Rovisco Pais 1, Lisboa, Portugal*ABSTRACTBasidiomycete mushrooms are very rich in nutritional biomolecules with a wide spectrum of therapeutic and prophylactic properties. Among these compounds, polysaccharides play an important role in immunomodulation, antitumor activity, antiviral and antibacterial activities.Hybridoma technology was used to raise monoclonal antibodies (Mabs) against extracellular polysaccharides from Pleurotus ostreatus. According to the literature search, these are the first Mabs produced against polysaccharides which were purified by chromatographic techniques and characterized in terms of specificity, selectivity, affinity and epitope analysis. These Mabs have been shown to be useful for detection and quantification of intracellular and extracellular polysaccharides from other mushroom strains.production of exopolysaccharides from Pleurotus ostreatusin vitro hybridomaproduction and clone selectiondetection of mushrooms polysaccharides by ELISAMATERIALS AND METHODSRESULTS AND DISCUSSIONMaterials : Young fruiting bodies of Pleurotus ostreatus, Lentinula edodes and Cordyceps sinensis were obtained from Mycology Research Laboratories Ltd, United Kingdom.As far as production of Mabs is concerned, 33 % of the total culture wells exhibited hybrid growth, 5 % and 6 % of the total wells contained anti-exopolysaccharide IgG and IgM production, respectively. One positive hybrid cell line (2.58) was cloned twice by limiting dilution, 15 clones were obtained that secreted Mabs of IgM class and 13 clones secreted Mabs of IgG class against exopolysaccharides. Two clones were selected for Mab production in vitro: 3F8_3H7 secreting Mabs of IgM class and 1E6_1E8 secreting Mabs of IgG class. However, subsequent research work was focused on 1E6_1E8 for characterization purposes. This hybridoma cell line was subsequently recloned and named 1E6_1E8_B5.Production and Isolation of Exopolysaccharides from Pleurotus ostreatusExopolysaccharides were isolated from supplemented whey permeate medium after 10 days of P. ostreatus growth in an aerated 5 L batch reactor, by submerged fermentation, at 28 ºC and 150 rpm. Biomass growth, protein and polysaccharide production were monitored by analysing aliquots of fermentation broth taken after 4, 7 and 10 days of fermentation . The exopolysaccharides present in the fermentation broth were precipitated with four volumes of 95 % (v/v) ethanol overnight, broth and dissolved in 15 mM phosphate buffered saline pH 7.2.Cell viability was measured by the trypan blue method and by MTT method. Figure 1 shows 1E6_1E8_B5 cells proliferation during 11 days of incubation at 37 ºC and 5 % CO2. Exponential phase was reached after the 1st day of incubation and the maximal antibody activity of cell culture supernatants was detected after de 6th day.Production of Mabs against Exopolysaccharides from Pleurotus ostreatusPrecipitated exopolysaccharide sample was used as antigen and Mabs were produced by hybridoma technology. Female BALB/c mice (4 weeks old) were immunized by intraperitoneal injections on day 0 with 50 μg of exopolysaccharides. Subsequently, three immunizations were carried out at three weeks intervals with the same amount of antigen. Three days later, after the last immunization, mice were bled and the antibody titer was determined by ELISA. Mabs were produced from 1E6_1E8_B5 clone in culture in vitro (RPMI % (v/v) fetal bovine serum) at 37 ºC and 5 % CO2.Indirect Enzyme-Linked ImmunoSorbent Assay (ELISA)Culture supernatants from wells with cell growth were screened by ELISA and hybridomas from positive wells were cloned twice by limiting dilution. PolySorp ELISA plate wells were incubated with exopolysaccharides from P. ostreatus as the antigen (50 μg/well), rabbit antimouse IgG-alkaline phosphatase conjugate as the second antibody and p-nitrophenyl phosphate as the substrate . One antibody unit is defined as the amount of Mab required to give a change in absorbance of 1.0 per 30 min at 415 nm due to the action of rabbit antimouse IgG-alkaline phosphatase conjugate on p-nitrophenyl phosphate under the standard conditions of ELISA.Figure 1: Cell growth curve and Mab production. 1E6_1E8_B5 cells were seeded on 96-well tissue culture plate. Analysis of cell viability was carried out by trypan blue and MTT methods. Antibody activity of cell culture supernatants was determined by indirect ELISA.Figure 2: Purification of 1E6_1E8_B5 against exopolysaccharides from Pleurotus ostreatus by hydroxyapatite chromatography.The elution profile of Mab 1E6_1E8_B5 from hydroxyapatite type II column (figure 2) shows no significant loss of Mab during the adsorption step and three major peaks revealing antibody activity during the gradient.Chromatographic Behavior of Culture Supernatant Containing IgG on Hydroxyapatite ChromatographyAB-+12 kDa20 kDa30 kDa45 kDa60 kDa110 kDa12354Fractions with antibody activity were further investigated by electrophoretic analysis. Native PAGE in figure 3A reveals only a band of Mr of aprox. 155 kDa for fraction 15, suggesting no presence of the major contaminant albumin from fetal bovine serum. This result was also confirmed by SDS-PAGE analysis (figure 3B). Fraction 15 exhibits only the band corresponding to high chain Mab with a Mr of aprox kDa. Fractions 55 and 67 didn`t exhibited any band neither by native nor by SDS-PAGE (data not shown).In order to develop an alternative method to protein A and G chromatography, culture supernatant containing Mab activity (5 mL diluted 1:2) was applied to a column (1×5 cm) packed with hydroxyapatite type II previously equilibrated with 10 mM sodium phosphate buffer at pH 7.2. The Mab was eluted from the column with a linear gradient of sodium phosphate buffer ( mM) and fractions (1.5 mL) were assayed for antibody activity and protein concentration.132 kDa66 kDa45 kDa1234590 kDaElectrophorectic AnalysisSDS-PAGE and native PAGE of eluted samples were carried out as mentioned previously and stained for protein with silver nitrate .Detection and Quantification of Exopolysaccharides Production from P. ostreatus by Indirect ELISAP. ostreatus was first grown on PDA and then transferred into culture medium supplemented with whey permeate as mentioned above. Cultures were grown in 500 mL flasks containing 100 mL of culture medium at 28ºC and 140 rpm for 10 days. Polysaccharide production was monitored daily by analysing the fermentation broth of each flask by indirect ELISA using Mab 1E6_1E8_B5. Flasks containing culture medium without inoculum were maintained at the same conditions and were used as controls.Figure 3: Electrophoretic analysis of eluted fractions of Mab 1E6_1E8_B5 from hydroxyapatite type II column. (A) Native PAGE of purified Mab and cultured supernatant using a 7.5% separating gel. Lanes: 1 – culture supernatant; 2 to 4 – eluted fractions 14, 15 and 16. (B) SDS-PAGE of purified Mab and cultured supernatant using a 12.5% separating gel. Lanes: 1 to 3 – eluted fractions 16, 14 and 15; 4 - culture supernatant. The margins represent molecular weight markers.Analysis of precipitated exopolysaccharides from P. ostreatus by indirect ELISA exhibited a maximum antibody activity for the medium sample collected at the 9th day of growth (figure 4).Determination of Cross-Reactivity of Mabs with Different PolysaccharidesThe binding of Mab to several polysaccharides was investigated by indirect ELISA. Intracellular polysaccharides (IPS) isolated from Pleurotus ostreatus, Lentinula edodes, Cordyceps sinensis, Amanita ponderosa, Ganoderma carnosum, commercial -1,3 glucan from Euglena gracillis, starch, cellulose, chitosan from shrimp, chitin azure, laminarin (from Laminaria digitata) or -D-glucan from barley were used as the antigen (75 μg/well), culture supernatant containing Mab activity as the first antibody, rabbit anti-mouse IgG alkaline phosphatase conjugate as the second antibody, and p-nitrophenyl phosphate as the substrate.REFERENCESFigure 4: Production of exopolysaccharides from P. ostreatus growing in culture medium supplemented with whey permeate by indirect ELISA.Figure 5: Comparative analysis of crossreactivity of Mabs for different polysaccharides from different mushroom strains by indirect ELISA.. S. Silva, S. Martins, A. Karmali, E. Rosa, Journal of the Science of Food and Agriculture, 92 (2012) 1826–1832.. S. Martins, J. Andrade, A. Karmali, L. Serralheiro, Molecular Biotechnology, 30 (2005). W. Wray, T. Boulikes, V.P. Wray, R. Hancock, Analytical Biochemistry, 118 (1981)Studies of Mab selectivity revealed that MAb 1E6_1E8_B5 also binds intracellular polysaccharides isolated from L. edodes, C. sinensis and A. ponderosa fruiting bodies (figure 5). Polysaccharides isolated from A. ponderosa mycelium biomass and exopolysaccharides isolated from this basidyomycete strain were also recognized by the Mab revealing that these polysaccharides also have the same epitope recognized in exopolysaccharides from P. ostreatus. Surprisingly, IPS from P. ostreatus were not recognized by the Mab neither the other commercial polysaccharides tested (data not shown). These data strongly suggest that these monoclonal antibodies also bound to beta-glucans from different mushroom strains which revealed that they recognized the triple helix tertiary structure of beta glucans.Acknowledgements: We thank Fundação para a Ciência e a Tecnologia/MCTES (Portugal) for financial support (PTDC/AGR-AAM/74526/2006; Unit 702 and PEst-OE/EQB/UI0702). Some experimental work carried out by the MSc. student Carla Rodrigues is gratefully acknowledged.