Technology
How are the proteins on the i-Ome protein array selected?
The i-Ome protein array, with more than 1600 proteins, is designed to enable autoantibody discovery. The proteins on this array were selected on the basis of their involvement in the immune response, representing different classes and various types of proteins such as kinases, cytokines, interleukins, and transcription factors. Some of the cancer proteins were selected based on “A Census of Human Cancer Genes,” published in 2004 by Michael R. Stratton and co-workers from the Wellcome Trust Sanger Institute in Hinxton, UK.
How are protein arrays created with KREX protein folding technology different from other protein arrays?
Other protein arrays utilize peptides, protein fragments or proteins produced with non-eukaryotic expression systems. Peptides and protein fragments only expose linear epitopes and are typically hydrophobic, promoting non-specific binding. With KREX technology, full-length, functional proteins are produced. This ensures that the conformational epitopes, to which the vast majority of antibodies bind, remain intact. This allows highly specific binding and reproducible results. Because KREX proteins are expressed in a eukaryotic expression system, they are appropriately post transcriptionally modified. Our proprietary array surface chemistry allows our proteins to remain in their native conformation.
How do I access your technology?
In addition to fee-for-service and off-the-shelf product offerings, we are open to exploring partnerships and collaborations. To start the discussion, contact us at [email protected].
How do I extract the data from the protein array?
Array images can be extracted/analyzed by many image quantification software products including analysis software provided by scanner manufacturers, third-party software or open source programs. A GenePix Array List (GAL) file for each array is generated to aid image analysis. Please note that GAL files are grid file-specific to the Genepix software and may not be compatible with any other software. GAL automatically generates grids on the array slide for auto spot detection supporting image analysis.
What scanners are compatible with the i-Ome protein array?
The protein array slides can be scanned using any open-format microarray scanners. However, we recommend using the Agilent scanner as we’ve found that it is more stable over time than other scanners we have tested.
At what resolution are the arrays scanned?
The array is scanned at 10 µm using any open format scanner.
What samples can be run on the i-Ome protein array?
Depending on the assay type, whether it is a biomarker discovery or interaction study, the samples listed below can be run on the i-Ome protein array. However, samples other than serum/plasma may require optimization.
- Serum/plasma
- Protein lysate
- Monoclonal antibodies
- Culture supernatant
- Ascites
- CSF
- Urine
- DNA/RNA
What proteins are found on the i-Ome protein array?
Cancer antigens, signaling proteins, transmembrane proteins, kinases, and other immunologically relevant proteins. View the full list of proteins.
Besides Cy-3 and Cy-5, what other fluorescent tags can be used for the i-Ome protein array?
Dyes similar to Cy-3 and Cy-5, such as Alexa 647, 555 and 660 dyes, can also be used.
Is it possible to include my proteins of interest that are not included in the i-Ome protein array?
Yes. We can custom produce proteins based on your requirements. To discuss further, contact us at [email protected].
What kind of studies are ideal using the KREX technology?
KREX technology is ideally suited for two main applications: autoantibody profiling and interaction profiling. In autoantibody profiling, different applications include biomarker discovery, patient stratification, treatment monitoring, ADR prediction, and immune-related adverse events (irAE) prediction. In interaction profiling, we profile the interaction of various analytes with the proteins on the array, such as DNA, RNA, protein, peptide, etc.
When a biomarker is found using the Sengenics KREX platform, what other methods can be used to ensure that the biomarker is transferable to a routine clinical setting?
ELISA is the gold standard for the measurement of proteins/immunoassays in a clinical setting. Other methods are available for results validation, such as IP-MS and other MS-based techniques. As ELISAs are potentially less sensitive than protein arrays (although most researchers agree there are no significant differences between biomarkers detected using a protein array and ELISA), there is the possibility of not being able to detect low-abundant biomarkers using ELISA.
Bioinformatics
How do I extract the data from the protein array?
Array images can be extracted/analyzed by any image quantification software including analysis software provided by scanner manufacturers, third-party software, or open source programs.
A GenePix Array List (GAL) file for each array is generated to aid image analysis. Please note that GAL files are grid file-specific to the Genepix software and may not be compatible with any other software. GAL automatically generates grids on the array slide for auto spot detection supporting image analysis.
Are there any publications on your data analysis methodology?
Our data processing and normalization methods are well described in Duarte et al. (2013). Biomarker discovery using our data analysis pipeline is reported in Liew et al. (2015), Suwarnalata et al. (2016), and Soe et al. (2018).
How is your method different to other data analysis methods?
Functional protein microarrays differ in many respects from DNA or RNA microarrays. Unlike DNA microarrays, functional protein microarrays often aim to discover global interactions of a single probe (protein) in a single color-channel, which results in a relatively small selection of specific proteins showing strong signals for a given sample. In this regard, we have designed a robust data pre-processing method to ensure that each reported signal intensity is highly accurate and significant.
Each replica spot on the array is subject to multiple threshold variables for quality control purposes. These quality control steps ensure replica spots from proteins showing high variance are flagged to report outliers.
Our normalization step uses both quantile-based and total intensity-based methods which utilizes common underlying distribution of control probes on the array to correct for any technical variance while conserving the biological differences between samples.
The biomarker discovery step implements protein-specific threshold calculated based on mean signal intensities from healthy controls, thus highlighting case-specific responses.
Each step of data analysis pipeline maintains the quality of data to report only true positive signals.
What is the spotting distance between the protein spots on the array?
The horizontal distance between protein spots is 405μm and the vertical distance between protein spots is 360μm.
What is the diameter of the protein spots in the array?
The diameter of individual protein spots is 150μm.
How is the quality of the protein array data verified?
Data extracted using the image quantification software is subjected to pre-processing where the percentage of coefficient of variation (CV %) of intra-protein, intra-slide and inter-array for each replica spot is calculated. CV% > 20% is used as quality benchmark for the replica spots.
What are the positive and negative controls on the array?
There are two positive controls to ensure the quality of experiment. The IgG dilution series acts as a positive control for assessing the binding capacity of fluorescent-conjugated secondary antibody. Accurate serial dilution quantification is used as a benchmark for ensuring that labeling efficiency and spot detection pass quality control thresholds.
Cy3-BSA controls act as positive controls for each array on the slide. 23 Cy3-BSA markers are present on each slide and each of their concentrations are kept constant throughout the experiment. Hence, it is considered a housekeeping probe for normalization of the signal intensities.
Why are age- and gender-matched controls important for study design?
Age-matched healthy controls are important to determine baseline immune responses which can differentiate disease-specific responses. KREX technology is sensitive enough to highlight age-specific immune responses in healthy controls which can be used as background threshold to identify biomarkers in case samples.
Lab
I would like to send in samples. How do I proceed?
Once the order has been confirmed by providing us the Purchase Order or signed Order Form, our Support team will contact you via email with samples submission and shipment information, as well as the KREX Project Form and Sample Annotation Form (if applicable) that must be filled out prior to submitting samples. We recommend using World Courier for samples shipment, as they have experience in handling dry ice shipments.
What if I want to purchase the slides only?
Yes, you may purchase our slides to be run in your own lab. Please email us at [email protected].
Is it possible to strip and reprobe/reuse slides?
We do not recommend stripping the arrays and reprobing, as this may compromise the integrity of the proteins.
How long can the finished arrays be stored before they are read on a scanner?
If it is not feasible to scan the slides immediately after running (recommended), the finished arrays can be stored overnight, in the dark to avoid photobleaching.
Protein Expression
What type of cell line is used for the production of KREX proteins?
KREX protein expression system utilizes an insect cell line.
Is it possible to express the proteins in a bacterial cell?
Yes, we have successfully expressed proteins using different protein expression systems. Please email us at [email protected] for further discussion.
What if I want to purchase the lysates only?
Please email us at [email protected] for further information.
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