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Customer Report DYNALogue 02/2001 Use of magnetic beads for isolation of antigenspecific human B cells producing monoclonal antibodies Ekaterina Sidorova Institute for Viral Preparations of Russian Academy of Medical Sciences, Moscow, Russia and Anders Rosén Linköping University, Dept of Biomedicine and Surgery, Division of Cell Biology, Linköping, Sweden. References 1. Milstein C. With the benefit of hindsight. Immunol. Today 2000; 21(8); 359-363. 2. Herzenberg L.A., De Rosa S.C. and Herzenberg L.A. Monoclonal antibodies and the FACS: complementary tools for immunobiology and medicine. Immunol.Today 2000; 21(8); 383-390. Introduction Methods Monoclonal antibodies play an enormous role in the development of highly specific tests for disease-associated marker biomolecules. Mainly murine monoclonal antibodies from immune splenocytes fused with myeloma cells have been used. These antibodies are now being used for therapy (1-4) but for many reasons, human or humanised monoclonals are more effective in activating human effector functions, to eliminate antigens and also avoid reactions against murine antibodies. Antigens Obtaining human monoclonal antibodies is important in cases where in vivo immune response is very weak or when animals do not respond to the antigen. For example, EBNA-1 of Epstein-Barr virus (EBV) is low immunogenic in mice. This is explained by its structural composition (numerous Gly-Ala repeats), which does not fit into MHC class 1 groove. After prolonged infection there is formation of EBNA-1 antibodies in infected people (5), thus human monoclonal antibodies to EBNA-1 may be obtained. To date, one exceptional anti-EBNA-1 antibody with specificity against residues 408-446 outside the repeat region is described (6). Many different approaches have been used to develop human monoclonal antibodies. The most successful approach so far involved B lymphocyte transformation from infected seropositive patients by EBV (7) with subsequent homo- or hetero-hybridisation to develop long-lived antibody-producing hybridomas. However, this procedure involves the use of infectious material requiring special safety laboratories for infections like HIV. To avoid contact with the infectious material, immunisation in vitro was suggested (8). This procedure is sophisticated and the number of resulting antibody-producing cells is relatively low (9). 3. van Spriel A.B., van Ojik H.H. and van de Winkel J.G.J. Immunotherapeutic perspectivefor bispecific antibodies. Immunol. Today 2000; 21(8); 391-3974. We have developed a novel approach based on the positive selection of B cells specific for antigens from normal human tissue or from blood with the subsequent transformation of isolated cells by EBV. 4. Fisk R.B. Anti-IgE as novel therapy for the treatment of asthma. Curr. Opin. Pulm. Med. 1999; 5(1); 76-80. To isolate B cells bearing those antigen-specific receptors, magnetic beads (Dynal Biotech), were used. 6 Synthetic peptides MN-24 corresponding to V3 loop of gp120 of HIV-1 strain (residues 302-322) and peptide 107 (p107) from EBNA-1 were dissolved in phosphate-buffered saline (PBS), sterilised and stored at -20°C. The Po/myelin protein was isolated from bovine peripheral nerve tissue. Preparation of sensitised magnetic beads Dynabeads ® M-450 Tosylactivated were used to couple viral peptides and Po protein. Briefly, magnetic beads were washed in 0.1 M phosphatebuffered saline (PBS), pH 7.4 and incubated for 24 hours at 22°C with 200 mg of peptides or Po protein (1 ml), rotating end-over-end. After removal of supernatant, magnetic beads were washed three times, last time overnight at 4°C using buffer containing stabilising/blocking protein, suspended in the same buffer and stored at 4°C. Culture media and lymphocyte preparation For B cell culture RPMI 1640 medium mixed (1:1) with OptiMem medium, or Iscove medium mixed (1:1) with OptiMem medium (GibcoTM, Invitrogen, Sweden) and supplemented with 10% of fetal calf serum (FCS), glutamin and antibiotics (complete media) were used. Total lymphocytes were prepared from freshly removed tonsils obtained from University Hospital, Linköping, ENT-Department or from blood of a patient with polyneuropathy associated with monoclonal gammopathy of uncertain significance (PN-MGUS). Mononuclear cells were separated by density centrifugation in Ficoll-Paque (Amersham Pharmacia Biotech, Sweden). The interface fraction was collected, washed and depleted for macrophages (in the case of blood cells) by adherence-to-plastic for 1 hour at 37°C or for T cells (SRBC-rosetting) and macrophages (in the case of tonsil cells). Selection of B lymphocyte specific for MN-24 and p107 peptides and to Po protein Magnetic beads coated by viral peptides (107 beads/ ml) were washed just before the cell separation by PBS-bovine serum albumin and then by RPMI 16401% FCS (3 times each). Human B cells (108/ml) were mixed (10:1) with the washed magnetic beads and incubated for 2 hours www.dynalbiotech.com Customer Report at 4°C on a rotator. After incubation, the target cells with 3-5 attached magnetic beads (rosetted cells) were separated from non-rosetted cells using a magnetic device (Dynal MPC®-1), washed five times with cold complete medium, counted and used for transformation. Normal B lymphocytes untouched by magnetic beads were used as a control. Determination of human immunoglobulins and antibodies Human immunoglobulins (Ig), anti-peptide and anti-Po/myelin antibodies were determined by ELISA (10). Results Incubation of 100 x 106 of tonsil B lymphocytes with magnetic beads coated by MN-24 or by p107 peptides resulted in 365 x 103 and 270 x 103 rosetted cells, respectively. Incubation of 38 x 106 or 8.4 x 106 of blood B lymphocytes with magnetic beads coated by Po protein yielded 0.8 x 106 and 1 x 105 rosettes, respectively. In all plates, the first growing clones became visible after one week. In a month, practically all wells contained transformed B cell clones. Antibodies to MN-24 peptide were produced in 52 cultures of 384 in MN-24 master plates, and antibodies to p107 in 24 cultures of 192 in p107 master plates. None of growing clones in control plates containing unselected B lymphocytes were positive for antibodies to either MN-24 or p107. Antibodies to Po/myelin were produced in all cell cultures from preselected cells. All secreted antibodies were of IgM isotype. It was not unexpected, since it is known that EBV transforms mainly B lymphocytes expressing surface IgM. DYNALogue 02/2001 Of all colonies obtained, the most positive for anti-MN-24, anti-p107 and anti-Po protein (one for each) were selected and expanded. Cells have continuously secreted antibodies during 3 years. It was shown that initially clones of M2D6 cells (selected by MN-24-beads), E2C5 cells (selected by p107-beads) and TJ99D cells (selected by Po protein-beads) were CD5-positive, thus belonging to the B1-lymphocytes subpopulation. After cultivation during 4 months, cells gradually lost CD5 expression. Antibodies produced by the B cell clones M2D6 and E2C5 were monoclonal but polyreactive, whereas antiPo/myelin antibodies were monospecific. Discussion The results obtained in this study confirm and expand our previous data [11], and prove that isolation of B lymphocytes bearing specific Igreceptors with antigen-coated magnetic beads is a convenient and simple method for developing human antibody-producing cultures for human monoclonal antibodies. The method described here permits specific B lymphocyte isolation from normal human tissues in quantities sufficient for subsequent transformation and culturing. Conclusion An approach was developed for the isolation of human B cells based on the positive selection of B cells bearing specific Ig-receptors with magnetic beads coated by respective antigens. This approach seems to be more economic, timesaving and safe compared to more traditional methods. Ordering Information Product Product No. Content Dynabeads® M-450 Tosylactivated Dynabeads® M-450 Tosylactivated 140.03 140.04 2 ml 10 ml www.dynalbiotech.com 7 5. Dillner J., Kallin B. The Epstein-Barr virus proteins. Adv. Cancer Res. 1988; 50; 95-158. 6. Chen M.R., Huang H., Fen C.Y. and Chen J.Y. A novel EBNA-1 tag system for high level expression and efficient detection of fusion proteins in vitro and in vivo. J. Virol. Methods 2000; 85(1-2); 35-41. 7. Rosén A., Gergely P., Jondal M, Klein G. and Britton S. Polyclonal Ig production after EpsteinBarr virus infection of human lymphocytes in vitro. Nature 1977; 267; 52-54. 8. Borrebaeck C.A.K. Strategy for the production of human monoclonal antibodies using in vitro activated B cells. J. Immunol. Methods 1989; 123; 157-165. 9. Borrebaeck C.A.K. Antibodies in diagnosticsfrom immunoassays to protein chips. Immunol. Today 2000; 21(8); 379-382. 10. Sidorova E., Borisova T., Lyakisheva L. and Rosén A. Human monoclonal antibodies to viral peptides. Human Antibodies, 1997; 8(2); 65-69. 11. Lundkvist A., Hörling J., Athlin L., Rosén A. and Niklasson B. Neutralizing human monoclonal antibodies against Puumala virus, causative agent of nephropathia epidermica: a novel method using antigencoated magnetic beads for specific B cell isolation J. Gen. Virol. 1993; 74; 13031310.