Custom Antibody Services

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1 Custom Antibody Services

2 Custom service offerings DNA sequence Plasmid Peptide Structure Protein Peptide Small molecule Cells Spleen Lymphocytes Antigen Preparation Immunization Fusion & Subcloning Expansion & Delivery Production & Purification Antigen and screening materials Antisera, Purifled polyclonal antibody Supernatants Supernatants Frozen cells Supernatants, Purified monoclonal antibody - Core service - Additional service - Input - Output Assays Assays Antibody Cloning & Engineering Assay Development Immunogen design and preparation Peptide design, synthesis, and conjugation cdna cloning and recombinant protein production in bacterial or mammalian systems Hybridoma development Fusion and screening of supernatants by ELISA Differential screening for phospho-, point mutation-, cleavage- or isoform-specific projects RabMAb production Small scale (<10 mg): hybridoma/integra Mid scale (10 to 500 mg): recombinant/transient Large scale (500 mg or more): recombinant/cho stable cell lines Stable cell line development Heavy and light chain cloning into single, proprietary vector Expression in suspension CHO cells Multiple rounds of subcloning for optimized production Polyclonal antibody services Immunization strategy design Antisera production and titer testing Affinity purification and/or affinity depletion Optional: Spleen harvest for RabMAb development Humanization Novel patented humanization method Other humanization technologies also available Assay development Sandwich and competition ELISAs Receptor-ligand binding, phosphorylation and functional cell-based assay Tissue microarray IHC profiling PK, PD, antigenicity assays

3 Why a custom rabbit monoclonal antibody from Abcam? Abcam s custom antibody service utilizes RabMAb technology, Abcam s patented antibody technology for the development high quality rabbit monoclonals. Benefits of Abcam s rabbit monoclonal antibody service includes: Novel epitope recognition Picomolar affinity High specificity Excellent IHC staining results Detection of subtle changes such as post-translational modifications Human protein and mouse ortholog cross-reactivity Maintain continuity in your antibody projects Comparison of rabbit and mouse monoclonal Comparison Rabbit monoclonals Mouse monoclonals Antigen recognition High success rates with a variety of antigens, including small molecules and peptides Good responses to rodent antigens Broad epitope recognition Small molecules and peptides often non-immunogenic Less responsive to rodent antigens Limited epitope recognition Affinity Picomolar (K D = M) possible Typically nanomolar (K D = 10-9 M) or lower Specificity Higher Lower IHC / ICC usage Excellent results in IHC and ICC Not always suitable for IHC or ICC CDX2 Paraffin-embedded human colonic carcinoma tissue stained with CDX2 rabbit monoclonal (ab76541) and Vendor A s CDX2 mouse monoclonal (both at 1:1000 dilution factor) E-Cadherin Paraffin-embedded human breast carcinoma tissue stained with E-Cadherin rabbit monoclonal (ab40772) and Vendor A s E-Cadherin mouse monoclonal (both at 1:50 dilution factor) HER2 / ErbB2 Paraffin-embedded human breast carcinoma tissue stained with HER2 / ErbB2 rabbit monoclonal (ab134182) and Vendor A s HER2 / ErbB2 mouse monoclonal (both at 1:500 dilution factor) Rabbit MAb Mouse MAb Rabbit MAb Mouse MAb Rabbit MAb Mouse MAb

4 Antibody generation process Polyclonal Polyclonal Antigen Immunization Polyclonal antisera Purified polyclonal Detection Polyclonal Antigen : A rabbit Immunization is injected with Polyclonal antigen antisera and mounts Purified an immune polyclonal response. The collected Detection antisera contain a mixture of and other components. The are typically affinity-purified, and they recognize multiple epitopes on the antigen. Monoclonal Monoclonal Antigen Immunization Lymphocytes Partner cells Fusion mixture + + Antigen Immunization Lymphocytes Fusion mixture Partner cells Frozen hybridomas Subclones Multiclones Frozen hybridomas Subclones Multiclones Production Purified monoclonal Detection Production Purified monoclonal Detection Monoclonal : Following immunization with the antigen of choice, lymphocytes are isolated from the rabbit s spleen. Fusion with a myeloma cell line results in immortal hybridomas that secrete. These cells are plated, at which point many wells contain multiple cell types or multiclones. The supernatants are then screened for the presence of specific, and the best multiclones are diluted and replated to isolate single lines or subclones. After further screening, the subclones are expanded and frozen for delivery. Monoclonal, which recognize a single epitope on the antigen, can then be produced in bulk by the hybridomas or by exogenous cell lines.

5 Antibody generation timeline Go/No-go Go/No-go Go/No-go Month Immunization Customer Testing Fusion & Customer Testing Subcloning Customer Testing Expansion & Delivery Order Invoice Invoice Invoice Invoice Polyclonal - 3 months Monoclonal - 7 to 8 months Legend: - Process stages - Abcam sends processed antibody for customer testing - You may choose to proceed with the service (Go) or end it (No-go) if you are already satisfied at this point - You receive an invoice from us at these stages In the last 6 years we have developed a number of very useful rabbit monoclonal. For our research work such sensitive and specific tools had not been available before. Without the project planning and management of the group in Vienna and without the excellent scientists in California this success story would not have been possible. - Dr. Pidder Jansen-Duerr, Institute for Biomedical Ageing Research of the Austrian Academy of Sciences

6 USA +1 (888) Canada +1 (877) Germany, Austria, and Switzerland +43 (0) UK and rest of Europe +44 (0) Japan +81 (0) Hong Kong (852) China (toll-free) Rest of world +1 (888) _14_DG Discover more at abcam.com/custom Copyright Our RabMAb technology is covered under the following U.S. Patents, No. 5,675,063 and 7,429,487

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