ProArray Ultra Ligand Binding Assays as a Service

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1 ProArray Ultra Ligand Binding Assays as a Service Why do ELISAs yourself when you can outsource them for less than your in-house labor and consumable costs miniaturize them to require just microliters of precious samples scale up your assays cost effectively to ensure that you achieve statistical power We offer a unique new service to carry out ligand binding assays powered by ProImmune s ProArray Ultra customizable protein microarrays. The Turnkey Solution to Your Epitope Discovery Program Flexibility, scalability and sensitivity are the hallmarks of the ProArray Ultra platform. Adding real value to your research projects is the additional element brought to you by our customer service team of PhD immunologists and biochemists. Discuss your project with us and we will design an assay and array format tailored to achieve your objectives. The ProArray Ultra technology is the turnkey solution to your epitope mapping and discovery programs. It has a similar principle to ELISA but with better sensitivity, lower costs and you do not have to do the work! Figure 1: ProArray Ultra ligand binding assays can be produced in line with typical configurations for microtiter plates. Antisera or plasma can be used In place of antibodies and any protein-protein interaction can be explored. Linker or biotin-streptavidin linked formats can also be used. The customer retains the freedom to set up and optimize the assays for their specific purposes. Key Features of the ProArray Ultra Platform The ProArray Ultra microarray has more than 30,000 features per slide, compared to 96 wells in a microtiter plate. Reagent consumption at all stages is vastly reduced, slashing the cost of the analytes and allowing a greater number of samples to be analysed. Detection is carried out using a fluorescence microarray reader, resulting in better sensitivity than standard ECL ELISA. The array can be split into 1/2/4/8 or 24 subarrays. A 24 subarray format still has nearly 1000 features available per sub-array. Figure 2: ProArray Ultra schematic in 24 sub-array configuration. Each sub-array offers nearly 1000 features which can be incubated separately

2 The Design Objective: ProArray Ultra has been specifically developed as a combined peptide and protein array platform; combining the epitope/interaction site resolving power of scanning peptide arrays with that of high throughput and protein interaction screening. ProArray Ultra achieves full compatibility for overlapping peptide libraries and proteins, and uses a flexible peptide synthesis platform to enable printing of arrays optimized for specific applications. Moreover, our design improvements result in greater uniformity of spot morphology between peptides and proteins, reduced background signal and improved assay performance in terms of sensitivity, dynamic range, and variance. It is far more cost effective as you do not need to supply consumables or allocate equipment and bench space for the assay. ProImmune will use the reagents that you supplied, ensuring reagent reaction is minimal bringing you significant cost benefits as Luminex and MSD consumables can still be quite expensive. What is the result? ProImmune can carry out your assays at high quality and low cost with rapid turnaround. For larger projects we can often deliver the full assay and report for less than your inhouse labor and consumable costs. Even better savings for follow on projects using the same materials. How to use our comprehensive assay service: it's as easy as Contact us with your specific requirements. Our PhD qualified representatives will understand your project quickly and provide you with a fully-costed proposal, usually within one day. 2. Our customer service team will provide instructions on sending your samples to ProImmune for analysis. i. ProImmune will receive and store your samples and obtain any additional peptides or proteins (as agreed in advance) that may be needed for the assay. ii. iii. iv. All samples will be stored in our fully tracked sample banks. ProImmune will carry out all testing. A full analysis report and data tables will be compiled. 3. The report, along with accompanying data files, are available for download from our secure web-server

3 How does the ProArray Ultra service compare to platforms, such as Luminex and MSD? Our assays are provided as a comprehensive service, so there is no need for you to supply consumables or allocate equipment and bench space for the assay. Luminex and MSD consumables can still be quite expensive. With our service, ProImmune can use your supplied reagents ensuring reagent consumption is absolutely minimal and bringing you significant cost benefits. In some cases ProImmune may be able to carry out an entire project for less than the consumable costs on another platform. Luminex offers up to 50-plex reactions per well and MSD offers up to 100-plex for some 24 well plates. In contrast ProArray Ultra can accommodate up to 38,000-features per array and has very significant scale economies, especially for larger projects. Cost comparison between ELISA, MSD, Luminex and our assay service. Table 1: Example comparison of the consumable-only cost per ELISA plate equivalent for ELISA, MSD and Luminex with the all-in service cost of our assay service. Taking all aspects of your project into account, from labour costs to the equipment and software required, including all the overhead charges that may develop such as equipment and software maintenance or lab space rental, it is not a question of whether you can afford to outsource, rather can you afford not to? Applicable Program Areas The ProArray Ultra technology is appropriate for a range of programs and projects, including: Serum screening Screen sera for multiple protein reactivities. If you have access to large scale, historical serum banks 1000s of sera could be screened against dozens of proteins using minimal sample volumes. Antigen discovery Reverse immunology on proteins or entire pathogen proteomes. Screen sera from disease state or convalescent donors against overlapping peptides. Biomarker discovery and screening Cancer, infectious diseases, cardiovascular, metabolic, CNS your imagination is the limit. Mapping of monoclonal antibody binding sites - 3 -

4 Mapping is linear in the first instance (unless conformational peptides are used), but strong conformational epitopes will be detected in linear sequences too. The platform also allows for the use of constrained peptides, e.g. cyclized peptides. Understanding the epitopes presented and recognized Disease progression cycle. Pathogen life cycle and recovery phases. Batch release testing: vaccines, biologics Cytokine arrays Figure 3: ProArray Ultra was used for measuring release of the cytokine IL-6 in whole blood caused by the therapeutic antibody Campath in a dose escalation study. ProArray Ultra has been specifically developed as a combined peptide and protein array platform; combining the epitope/interaction site resolving power of scanning peptide arrays with that of high throughput and protein interaction screening. ProArray Ultra achieves full compatibility for overlapping peptide libraries and proteins, and uses a flexible peptide synthesis platform to enable printing of arrays optimized for specific applications. Moreover, our design improvements result in greater uniformity of spot morphology between peptides and proteins, reduced background signal and improved assay performance in terms of sensitivity, dynamic range, and variance. The result is a protein array platform that is comparable in analytical performance to MSD technology and that outperforms ECL ELISA assays, while at the same time tens of thousands of interactions can be detected in a single experiment. More importantly, only microliter amounts of sample are required for each analysis, enabling projects using limited amounts of sample that are simply impossible to carry out with 96-well plate-based assays

5 Performance Data The design of ProArray Ultra overcomes the compromise between the analytical quality of multi-well immunoassay platforms and the scalability of existing protein and peptide array technology. Evaluation of our new design features against other more conventional assay methodologies, such as optimized ECL ELISA and the MSD technology shows that ProArray Ultra compares favourably to both plate-based platforms, and has lower background and better overall signal to noise performance (Figure 4). Figure 4: Results of a direct comparison of ProArray Ultra (blue bars) and high performance 96 well MSD assay technology (red bars) for identical peptide libraries derived from the well characterized Hepatitis B Virus surface Antigen (HBVsAg) incubated with the same anti HBVsAg seropositive serum sample. All data include error bars reflecting standard error of the mean (SEM) values. ProArray Ultra compares favourably to the MSD plate based platform, and has lower background and better overall signal to noise performance. The peptides identified as epitopes by ProArray Ultra correlate with results from the MSD assay. Figure 5: Limit of Detection (LOD) comparison for MSD and ProArray Ultra using a specific monoclonal antibody. A saturation curve profile was obtained with a linear range of approximately 2.5 logs for both ProArray Ultra and MSD assay. Very low signal variation was found for both technologies in terms of signal detected. The limit of physical detection can be seen as ~0.5ng/ml

6 Flexible Array Design Our printing technology confers complete flexibility for design around your requirements. The ProArray Ultra microarray setup is based on five printing layouts of 1, 2, 4, 8 or 24 identical sub arrays per slide. The slides can be overlaid with a separation gasket allowing each sub array to be incubated with an individual antibody sample, i.e. up to 24 separate samples can be analyzed per slide. The exact print setup is based on the number of peptides or proteins being probed, the number of sample repeats required, and the number of technical repeats required (at least 3 are suggested, up to 6 are recommended). All these factors are taken into account to create the optimal printing layout. The five different print setups can accommodate a large range of custom printed features (peptides & proteins) alongside standard control spots. Figure 6: The diagram shows 5 possible layouts of the microarray printing design. The first number in each pair indicates the number of samples that can be spotted in triplicate; the second number indicates the number of spots in sextuplicate. The subarrays can be physically separated from each other using specially fitted gaskets, allowing incubation of each discrete sub array with a different sample. Consistent High Performance for Peptides and Proteins ProArray Ultra has been developed to minimize spotting variation in terms of spot size and morphology, and further optimized to ensure comparability between peptides and proteins on the same slide (a major concern unsatisfactorily addressed by first generation peptide microarrays). Figure 5 emphasizes the level of consistency achieved across a range of titrations for the ligand protein

7 ProArray Ultra : example of spot quality Figure 7: ProArray Ultra peptide microarray imaged at 5μm resolution. Peptides and protein samples are titrated and spotted in quadruplicate rows, in duplicate sub grids, and detected with purified antibody. The array photograph shows peptide antibody interactions with signal intensities from high to low, reflected by gradations in colour from red (high signal) to black/dark red (low signal). The change in signal intensity (high to low) reflects a decrease in the amount of analyte spotted onto the array. The image confirms that both peptide and protein spots are of a consistent high quality, reinforcing the suitability of the platform for multiple application. Performance data from alternative microarray technology Figure 8: Peptide microarray image from a leading alternative A. Alternative A peptide microarray imaged at 5μm resolution. Triplicate grids are shown together for clarity and cross grid comparison purposes. These image data reflect 78 overlapping peptides derived from a single immunogenic protein printed with triplicate control protein features (yellow box) in triplicate grids. Significant variations are clearly visible in terms of spot size and morphology (white boxes)

8 Technology Comparison Considerations and Capabilities ProArray Ultra MSD ECL ELISA Very high throughput ~ 1µl serum typically > 30,000 features possible Up to 24 serum samples per slide or a full dilution range of 1 or more serum samples. Modified/constrained/ long sequences possible 0.5 μl 2.5 μl serum for up to 10 unique features per well Low throughput (~10 epitopes per plate) Limited epitope characterization; fixed configuration, limited to pre determined test markers Good for multiple serum samples 5 μl serum volume per feature analyzed Low throughput (~ 30 epitopes per plate in triplicate) Sensitivity * 0.5 ng/ml s of ng/ml 100s of ng/ml CV (%) at high signal level < 10% 5 10 % ~ 20% Dynamic range* Binding or Spotting of Peptides and Proteins 2.5 logs Compatible for proteins and peptides Peptides individual physicochemical properties are adaptively maintained by the protein conjugation approach logs maximum range Proteins and peptides System is not specifically designed for immobilization of peptides Table 2: Detection of antibodies (0.5-1NG/ML) Provides clinical relevance 1 2 logs Proteins and peptides Can be adapted to accept any peptide - 8 -

9 Case study: Peptide Epitopes for Diagnosing Lyme Disease Clin Vaccine Immunol. 2013;20(4): [PubMedID: ] Outer Surface Protein C Peptide Derived from Borrelia burgdorferi Sensu Stricto as a Target for Serodiagnosis of Early Lyme Disease. Arnaboldi PM, Seedarnee R, Sambir M, Callister SM, Imparato JA, Dattwyler RJ. Dr. Paul Arnaboldi and his colleagues have used ProImmune's ProArray Ultra assay service to identify the bacterial peptide epitope OspC1, which could form the basis of a new and more accurate assay for diagnosing early-stage Lyme disease. Lyme disease is endemic in many regions of North America and Europe, spread by the Borrelia bacteria carried by ticks. It can be resolved with a course of antibiotics but if untreated can lead to neurological and musculoskeletal damage. Lyme's varying symptoms limits diagnosis, it instead relies on detecting antibodies against the bacterium or bacterial proteins in serological assays. The current two-tier assay used for Lyme diagnosis, an ELISA followed by a western blot, is unreliable at detecting early infection. This fails to identify the presence of early Lyme in up to 50% of cases. Arnaboldi's team hope to develop a sensitive multipeptide diagnostic assay which will detect the presence of human-generated antibodies against Lyme. The team focused on the Borrelia surface protein OspC as a potential source of epitopes. Arnaboldi chose to use the ProArray Ultra assay service to investigate whether the OspC protein contained any epitopes which were highly conserved in Borrelia and provoked an antibody response from sera of Lyme-infected patients. ProArray Ultra rapidly identified potential epitopes ProImmune synthesised a library of 37 overlapping 15-mer peptides from the OspC sequence and performed linear epitope mapping using ProArray Ultra. The peptides were screened against sera samples from eight patients which were known to contain antibodies against Borrelia, and positive antibody binding was detected using flurochrome-labelled secondary antibodies. From the data generated from ProArray Ultra, Arnaboldi and collegues were able to identify three peptides which bound to over 50% of the Lyme samples, with the entire process taking place over just five weeks. Subsequent ELISAs confirmed that one of these epitopes, OspC1, positively detects Lyme in over 75% of early Lyme patients, therefore a good candidate to form part of an assay to reliably detect early-stage Lyme. Arnaboldi hopes to identify several more of these highly specific, conserved Borrelia epitopes to increase positive patient outcomes. Copyright ProImmune Limited All Rights Reserved. ST42 V1.2 Last revision November