Monoclonal Antibodies to Osteopontin: Generation and Characterization David Denhardt, Josephine Cassella, Yao Li, Cassandra Louis, Tanya Gordonov, Melissa Weidner, Christian Kazanecki, Aaron Kowalski, and Esben Sørensen Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, New Jersey Abstract Methods, Procedures, Typical Data Results – Overall Summary We have generated monoclonal antibodies (MAbs) to OPN using several strategies. OPN-deficient mice have been immunized with Western Blotting Coomassie Blue Gels Our progress so far has led to the identification of many recombinant (unmodified) human and mouse OPN, with native new and different monoclonal antibodies that bind to OPN. A human milk OPN (Christensen et al., 2005), and with peptides subset of them have been characterized for their ability to bind containing phosphorylated serines representative of selected, to native (modified) or recombinant (unmodified) human or well-conserved phosphorylated sequences in OPN. Using well- A mouse OPN using four different approaches, examples of established procedures, we fused cells from the spleens of which are shown on the left. We have also been able to locate L immunized mice with SP2/0 myeloma cells and then distributed the epitopes that some of them recognize. The cartoon of the the fusion products into the wells of 96-well plates. After several OPN molecule at bottom left (adapted from Sodek et al. 2000) weeks, cells from those wells that contained growing hybridomas S shows the approximate location of the epitopes recognized by were expanded into progressively larger cultures in rich medium; some of these MAbs. For the most part, epitope identification many died out along the way. Hybridomas showing good growth is consistent with the location of the epitope relative to the were weaned away from serum into serum-free hybridoma thrombin cleavage site. Our preliminary data so far suggest: Native/recombinant OPN WB. N/C thrombin fragments 1) That some of the MAbs recognize only native OPN (e.g. growth medium. Conditioned media were prepared and aliquots → : Full length OPN PC: polyclonal antiserum of the cells frozen down. The media samples were filtered, Samples of concentrated, conditioned serum- 66A7), or only recombinant OPN (e.g. 73A3), and 2) that some concentrated using Centripreps, and immunoglobulins purified Electrophorese proteins in an SDS-PAGE gel. free media are electrophoresed on SDS-PAGE of the MAbs (e.g. 45B5) are detected only when OPN is freely on Sepharose Protein A/G columns. Anti-OPN monoclonal Transfer proteins in the gel onto a PVDF membrane. gels and stained for protein using CB. accessible, not bound to a support. Add antibodies to the strips of the membrane. L (~55kD) and S (~25kD) represent the antibodies produced by these hybridomas are being characterized Add secondary antibody (Goat-anti-mouse HRP). approximate positions of the heavy and light as described herein. Identify MAb location by enhanced chemiluminescence. IgG chains; A is an unknown protein. Future Work – Long Term Goals Peptide Assay (similar to an ELISA) Overview Magnetic Bead Assay Used to test which specific OPN sequence, if Our goals in this research are two. On the one hand we want to 1. Protein G coupled to magnetic beads (InVitrogen). any, the monoclonal antibody binds. identify MAbs that will allow the presence or absence of Our approach is to characterize the IgG produced by each 2. MAbs are incubated with the protein G/magnetic Wells of 96-well neutravidin-coated plates are specific post-translational modifications, or exons for that hybridoma using four different strategies so as not to bias the beads (protein G binds IgG at the Fc portion incubated with biotinylated OPN peptides. matter, to be defined. This may allow functionally specific selection in favor of only MAbs that bound OPN in one particular leaving the antigen binding site available). 18-mer peptides (19 in all covering the entire OPN regions of the OPN molecule to be identified. On the other 3. Beads are washed, and differentially fluorescently molecule) in separate wells. assay, for example western blotting or ELISAs. This is because hand we want to identify MAbs that inhibit OPN function labeled native and recombinant OPN are added. The wells are washed and goat anti-mouse IgG certain epitopes might be sequestered and unable to bind an MAb conjugated to Alexafluor 594 is added. either in cell culture or in the animal. Functional inhibitors of because they were bound to cell culture plastic or the PVDF 4. The antibody/antigen complex is dissociated from After washing, the fluorescence (EX: 584 nm; EM: native OPN in mammals might be useful therapeutic agents in the beads by low pH citrate buffer. membrane. Thus we use two additional screening methodologies 590) is read. Readings above background the treatment of OPN-dependent pathologies; these include 5. The fluorescence of the supernatant is read at the to detect antigen-antibody associations free in solution (a peptide suggest the MAb binds that peptide. autoimmune disease, cancer metastasis, osteoporosis, stress- two wavelengths indicative of the 2 forms of OPN. assay and a magnetic bead assay). We use both native (modified) induced immune disfunction, and certain inflammatory and recombinant (unmodified) OPN to identify MAbs that conditions (sepsis?). specifically recognize either the native or the recombinant form. Cartoon illustrating locations of know epitopes Antibodies specific only for the native form would be candidates OPN molecule courtesy Jaro Sodek for MAbs recognizing an epitope that is post-translationally modified; MAbs specific only for the recombinant form would References presumably recognize peptide sequences that are post- Christensen B, Nielsen MS, Haselmann KF, Petersen TE, translationally modified. Sorensen ES. (2005) Post-translationally modified residues The peptide assay makes use of peptides that collectively of native human osteopontin are located in clusters: cover the entire OPN molecule with 2-4 amino acid overlaps; in identification of 36 phosphorylation and five O- some cases both a phosphorylated peptide and its non- glycosylation sites and their biological implications. phosphorylated counterpart are used. These peptides possess a Magnetic beads coupled to Protein-G Biochem J. 390:285-92. biotin at the C terminus coupled to the peptide by two glycine Sodek J, Ganss B, McKee MD. (2000) Osteopontin. Crit Rev residues; via the biotin, the peptides bind to the surface of Magnetic Bead Assay Oral Biol Med. 11:279-303. NeutrAvidin-coated wells in a 96-well format. The magnetic . bead assay employs two different forms of OPN, the highly post- 0.25 Optical Density (OD) Native translationally modified native OPN purified from human milk 0.2 Recombinant ELISA and the unmodified form synthesized in E. coli. In this novel assay the two forms (differentially labeled with fluorescent tags) 0.15 2 Acknowledgements Optical Density (OD) Native 0.1 1.5 are mixed and allowed to react in solution with a specific MAb. 0.05 Recombinant 1 The antigen-antibody complexes are then captured on magnetic This research was supported by funds from the NIH, a 0 beads coupled to protein G. After the beads are washed, the 2A1 1 2D6 2 66G3 3 45B5 4 7E3 5 71A10 6 0.5 Busch Biomedical Research Award, the Rutgers Technology -0.05 adsorbed IgG is eluted at low pH, and the fluorescence at the two Antibodies 0 Commercialization Fund, and a grant from the National 2A1 1 10H4 2 48F3 3 49C12 4 wavelengths determined. MAbs that selectively bind the native -0.5 Multiple Sclerosis Society. The support of Aresty Summer protein are presumed to be antibodies that recognize post- Antibodies Fellowships to Tanya Gordonov and Cassandra Louis are translational modifications. We believe this novel method is 2A1 is a positive control for native and recombinant gratefully acknowledged. Yao Li was supported by a SURF OPN. 2D6 appears to bind both forms strongly; 66G3 important in that it detects the antibody-antigen reaction in 2A1 is a positive control for native and award. We thank Avni Shah for technical assistance and Dr. binds more to native; 7E3 binds more to recombinant; solution. This is in contrast to the methods currently in use which recombinant OPN. 10H4 also binds strongly to Larry Steinman for his generous contributions of the peptides 71A10 binds to neither; 45B5 shows a strong affinity for entail the immobilization of the antigen on a plastic surface native OPN only in the magnetic bead and peptide both forms, while 48F3 binds preferentially to and other reagents used in this research. (ELISA) or membrane (Western blots). assay; it has always appeared negative on Westerns and native OPN whereas 49C12 binds more ELISAs. (IgG background subtracted.) strongly to recombinant OPN.