The half-maximal effective concentration (EC50) values for those antibodies varied between 16

The half-maximal effective concentration (EC50) values for those antibodies varied between 16.4?ng?mL?1 and 21.3?ng?mL?1. The clones showed binding specificity not only for schizophyllan but also for other beta-(1,6)-branched beta-(1,3)-D-glucans of comparable macromolecular structure. Denaturation of the secondary structure led to a reduced antibody binding, indicating an epitope requiring the correct conformation of the triple helical structure of the glucans. 1. Introduction A variety of fungal beta-(1,3)-D-glucans are described as substances with antitumoral and immunomodulating activities [1]. One example is usually schizophyllan (SCH) that is produced as extracellular polysaccharide by the basidiomyceteSchizophyllum commune[2C5]. Its bioactivity is based on the enhancement of cell-mediated immune response with stimulation of T lymphocytes and macrophages and improving cytokine production [6C9]. The primary molecular structure of SCH consists of a beta-(1,3)-D-glucan main chain with single beta-(1,6)-linked glucose molecules at approximately every third glucose monomer of the backbone [10, 11]. In aqueous solution it forms the secondary structure of a triplex composed of three twisted chains stabilized by hydrogen bonds with single beta-(1,6)-linked glucose residues protruding outside the helix backbone [12C15]. Differences in branching grade and molecular weight are considered to be responsible for variations in bioactivity [16C18]. In addition to its bioactivity, SCH also has physical properties favorable for a use in food and oil industry. Aqueous solutions of SCH are viscous and show pseudoplastic flow behavior [19]. Furthermore, they are stable at high temperature and in a broad pH range, starting to denature at pH > 12 or >135C [15, 20, 21]. Since PROTAC ER Degrader-3 SCH can be used for many applications, the generation of antibodies would be of great value because they could be used for quantitative trace analysis or for the investigation of the role of particular conformations for glucan bioactivity. The objective of this study was the generation of recombinant PROTAC ER Degrader-3 monoclonal antibodies (rAbs) against the beta-D-glucan schizophyllan. Therefore, we constructed an antibody phage display library from the lymphocytes of three mice which had been immunized with proteinase K treated SCH (SCH-PK). After panning of this library for SCH-PK binding antibody phage, we were able to derive three rAbs PROTAC ER Degrader-3 specificity binding beta-(1,6)-branched beta-(1,3)-D-glucans with the same secondary structure as SCH. 2. Materials and Methods 2.1. Chemicals All chemicals were purchased from Sigma Aldrich if not mentioned otherwise. 2.2. Preparation of beta-(1,6)-Branched beta-(1,3)-D-Glucans Following beta-(1,6)-branched beta-(1,3)-D-glucans were prepared from biomass-free and stabilized (5?g?L?1 formic acid) culture supernatants: SCH (S. communewas diluted to 1 1.0?g?L?1 glucan in 100?mL with water and adjusted to pH 7.5 with NaOH. 2?mL of 0.5?mol?L?1 CaCl2 in 1?mol?L?1 Tris-HCl (pH 7.4) and 1?mL of 10% (w/v) sodium dodecyl sulfate were added and PROTAC ER Degrader-3 the solution was incubated at 80C for 4 hours. After cooling to 40C, 10?mg of proteinase K was solved and the solution incubated for 24?h at 40C under slight shaking. The proteinase K treatment was stopped by incubation at 80C for 4 hours. The solution was diluted Rabbit Polyclonal to RFA2 to 1 1 L with water, cleared by centrifugation with 13,000?g for 1?h at 16C and then processed as described above. The same procedure was applied for culture supernatant ofS. rolfsii.(variable light) and (variable heavy) and their cloning into the phage display vector pHAL30 was carried out as described [25, 26]. The derived sublibraries for lambda (V-LAMBDA) and kappa (V-KAPPA) of each mouse were kept separately. The six sublibraries were packaged with hyperphage [27]. 2.5. Ethics Statement The experimental protocols were carried out in accordance with the Directive 2010/63/EU of the European Parliament and the Council of the European Union of 22 September 2010 and all procedures were approved by guidelines from the Animal Committee on Ethics in the Care and Use of Laboratory Animals of TU Braunschweig, Germany (Az 5 (02.05) TschB TU BS Az:33.42502-14-005/08). 2.6. Immobilization of Schizophyllan onto Multiwell Plates For the antibody selection and ELISA experiments, schizophyllan was immobilized to Carbo-BIND multiwell plates (Corning, Corning, USA). The wells were filled with 100?E. coliclones.