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Epitope Mapping Service Epitope Mapping Service Identification of antibody or other protein:protein binding epitopes and their characterization at the amino acid level Understanding antibody specificity at the molecular level provides the key to optimizing their use as research or diagnostic tools - it forms the basis of their application as therapeutic agents. Arrays of protein sequence-derived short peptides have emerged as a powerful tool to identify and characterize such binding epitopes. LC Sciences combines flexibility of customizable parallel synthesis and high data quality of microfluidic technologies to create high density peptide microarrays for high throughput detection, concentration titration, and screening applications. IMMUNOLOGICAL STUDIES BIOMARKER SCREENING Identify specific substrate for a known antibody Detect autoimmune-response antibodies. Identify immunodominant regions in antigens Screen biomarkers from biological samples Make quantitative measurement of binding curves (i.e. blood serum or cell lysates) Perform multiplex antigen binding assays Develop immunological tools for clinical applications Study immune networks VACCINE DEVELOPMENT Develop specific antibodies by pre-screening cross-reactivity Map antibody cross-reactivity Develop new antigen agents for novel antibodies Develop epitope-vaccine therapies Develop inhibitor of antigen-antibody www.lcsciences.com 1-888-528-8818 PepArray™ Biochip Technology LC Sciences provides a comprehensive epitope peptide microarray assay service to make both qualitative and quantitative measurements of antibody/epitope binding. Screening on a peptide microarray (PepArray™) offers the opportunity to study thousands (up to 30K peptides/chip) of potential epitopes in a single experiment utilizing only sub-µg quantity of protein. Using a tiling method, we can systematically map the binding sites on a pathogen/protein at single amino acid resolution. COMPREHENSIVE SAMPLE TO DATA SERVICE This is a comprehensive epitope mapping service - send us your sample - we’ll synthesize a custom designed array, carry out the sample binding assays you request, perform data collection and analysis, and deliver a results report to you. Our cost effective 1-stop solution can save you tremendous time and money and the use of a microarray titer plate format will increase your workflow to 40X that of a single microtiter plate. MICROFLUIDIC ARRAY PLATFORM These are not spotted arrays! A proprietary µParaflo® microfluidic biochip is used and custom peptide sequences are synthesized on-chip. The microfluidic technology produces a uniform distribution of the sample solutions on the array, ensures efficient sample-peptide contact and enhances binding reactions and stringency wash processes. The microarray chip consists of thousands of three-dimensional chambers and is a closed system. Under these conditions multiplex protein assays are carried out in a way much like in thousands of pico-liter tubes, enclosure keeps the proteins in a stable environment, in solution and protected from air– oxidation/contamination. The miniaturized system provides automation, sample/reagent-savings and simplicity in operation. HIGH THROUGHPUT FORMAT Thousands of different peptide sequences are synthesized on one array (1 cm2) 1 4K peptide microarray = 40 X 96-well microtiter plates. We have chip designs up to 30K . Target thousands of peptide sequences at once - perform thousands of multiplex parallel assays at addressable chip locations. Generate hundreds of binding curves in a single experiment. QUANTITATIVE RESULTS Generate information about specific probe sequences - sequences can be defined to each single amino acid residue and the interpretation of the assay results are taken directly from digital image read out of an addressable microarray. Achieve reliable and quantifiable results through the highly stringent design and use of negative and positive control references. False positive readings are minimized. FLEXIBLE DESIGN Completely customizable design of peptides according to your needs. All arrays are synthesized to order. Choose your own customer specified sequences and layout, or sequences custom designed by LC Sciences. Quickly revise microarray design and content to keep experiments moving forward based on previous results. ULTRA-LOW SAMPLE CONSUMPTION Miniaturized array features reduces consumption of valuable samples to the sub-nanoliter to picoliter level per reaction. Perform thousands of peptide assays per array using only sub-µg of protein. www.lcsciences.com 1-888-528-8818 30-10A-02 Application Examples EPITOPE DISCOVERY x 1,000 x 1,000 12 12 Chip Chip 9 9 6 6 3 3 Signal (AU) Signal Binding Signal Binding (normalized) 0 0 YA YP DVP DYA YPY PDYA YPYD YPYDV VPDYA YDVPD YPYDVP DVPDYA PYDVPDY YDVPDYA YPYDVPD PYDVPDYA YPYDVPDY YPYDVPDYA YPYDVPDYA APYDVPDYA YPADVPDYA YPYDAPDYA YPYDVPDAA YPYDVPDYA YPYAVPDYA YPYDVPAYA YAYDVPDYA YPYDVADYA Antibody: anti-HA N-truncation C-truncation N,C-truncation Ala-scanning Epitope (known): YPYDVPDYA YPYDVPDYA YPYDVPDYA Chip #1: HA and flag epitope and vari- blue: residues dispensable from both ends red: important residues ant sequences Chip #2: HA, c-myc, VSVG, and flag Anti-HA Epitope Binding Array—Measure relative binding affinity to epitope sequence epitope and other peptide sequences variants (N-truncation, C-truncation, Ala scan, tiling). Achieve single amino acid resolution for the binding core. Identify the minimum molecular structure that is recognized by the Assay solution: 200 ml TBS (pH 6.8) antibody. Temperature: 4 ˚C Results: HA epitope is a hexapeptide DVPDYA. Experimental Time: 1 hour Three amino acids (D4, D6 and Y7) are essential. MEASUREMENT OF ASSOCIATION CONSTANTS Make high-throughput quantitative measurements of antibody binding curves and calculate association constants. Association curves measured with protein concentration titration on an epitope peptide microarray Microfluidic design enables multiple concentration bindings on the same chip to generate not only a single titration curve for the known epitope but also titration curves for all the epitope variants. Anti-HA varied from 0.1 to 60,000 ng/ml, signal intensities acquired at different scanning gains were scaled according to a calibration curve, curve fitting used Origin (Origin Lab). Kd values measured are in the range of 2-4 ug/ml. Association curves measured with peptide concentration (density) variation on an epitope peptide microarray Unique synthesis chemistry makes it possible to vary the substrate density across the array. “Pico-titer” plate. Thousands of binding curves can be measured simultaneously by one assay incubation. Equivalent to 40 microtiter plates. In situ synthesis and innovative chemistry can provide more than 25-fold change in peptide density on the surface. Measured a 31-fold change in anti-HA binding for highest density vs regular peptide density. Electron micrograph view of a Plate 1 ® µParaflo biochip Seq 001 002 003 004 005 006 007 008 Plate 2 Repeat Seq 011 012 013 014 015 016 017 018 Plate 3 Spots Density Repeat Seq 101 102 103 104 105 106 107 108 Spots Density Repeat Spots Density www.lcsciences.com 1-888-528-8818 www.lcsciences.com 1-888-528-8818 30-10A-03 Epitope Mapping Service PepArray™ Publications 1. Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, Shively J, Cannon GC, Kerfeld CA. (2015) Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component. Life 5(2), 1141-1171 [abstract]. 2. Wang W, Woodbury NW. (2014) Unstructured interactions between peptides and proteins: Exploring the role of sequence motifs in affinity and specificity. Acta Biomaterialia 11(88-95) [abstract]. 3. Aloisio GM, Nakada Y, Saatcioglu HD, Peña CG, Baker MD, Tarnawa ED, Mukherjee J, Manjunath H, Bugde A, Sengupta AL. (2014) PAX7 expression defines germline stem cells in the adult testis. Journal of Clinical Investigation 124 (9), 3929-3944 [abstract]. 4. Assis DN, Leng L, Du X, Zhang CK, Grieb G, Merk M, Garcia AB, McCrann C, Chapiro J, Meinhardt A. (2013) The role of macrophage migration inhibitory factor (MIF) in autoimmune liver disease. Hepatology [Epub before print] [abstract]. 5. Reichmann D, Xu Y, Cremers CM, Ilbert M, Mittelman R, Fitzgerald MC, Jakob U. (2012) Order out of Disorder: Working Cycle of an Intrinsically Unfolded Chaperone. Cell 148(5), 947-57 [abstract]. 6. Butterfield K, Caplan M, Panitch A. (2010) Identification and Sequence Composition Characterization of Chondroitin Sulfate-Binding Peptides through Peptide Array Screening. Biochemistry 49(7), 1549-55 [abstract]. 7. Williams BA, Diehnelt CW, Belcher P, Greving M, Woodbury NW, Johnston SA, Chaput JC. (2009) Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds. J Am Chem Soc 131(47), 17233-41 [abstract]. 8. Zhu, Q., Hong, A., Sheng, N., Zhang, X., Matejko, A., Jun, K.-Y., Srivannavit, O., Gulari, E., Gao, X. and Zhou, X. (2007) µParaflo Biochip for Nucleic Acid and Protein Analysis. Methods in Molecular Biology 382, 287-12 [abstract]. @LCSciencesLLC @LCSciences LC Sciences LLC www.lcsciences.com 1-888-528-8818 30-10A-04 .
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