Technology
What types of studies can be conducted using Sengenics microarrays?
Which isotypes are available for screening on Sengenics microarrays?
Standard analysis includes dual color isotype screening (IgG/IgA, IgG/IgM) as well as single color screening (IgG, IgA, IgM).
For custom subclass screening, along with other possible isotype screening capabilities, contact Sengenics at [email protected].
How are Sengenics protein microarrays different from other protein arrays?
Sengenics microarrays deliver reproducible and biologically relevant biomarker signatures, thanks to our multi-faceted approach:
Protein Expression System: We use a higher eukaryotic recombinant expression system (insect cells) to produce full-length proteins. This system, with molecular machinery more similar to humans than yeast or bacteria, ensures that our proteins exhibit native-like folding and post-translational modifications.
Array Surface: Our microarrays utilize a non-denaturing surface that preserves in vivo-like protein folding and optimizes accessibility for antibody binding.
KREX® Technology: Our proprietary technology guarantees that only correctly folded proteins are immobilized on the array, maintaining the integrity of conformational epitopes, which are crucial as 90% of antibodies bind to these specific structures.
Bioinformatics Services: Our expert bioinformatics team offers tailored and comprehensive support, from study design to data analysis, ensuring that your data is translated into actionable insights.
In contrast, other arrays often rely on protein fragments or lack control over the proper folding of immobilized proteins, resulting in missing epitopes, non-specific binding, and high background noise. For instance, denatured proteins can expose hydrophobic regions, which increase non-specific binding. Moreover, many arrays use recombinant proteins produced in yeast or bacteria, where folding and/or post-translational modifications differ significantly from those in humans.
What is KREX technology?
KREX technology employs a proprietary surface chemistry approach to immobilize full-length, functional proteins onto a non-denaturing array surface. This process ensures that conformational epitopes, which are the primary binding sites for most antibodies, remain intact, leading to highly specific and reproducible results.
What post-translational modification screening options does Sengenics offer?
How do I access your technology?
We offer a range of kits and services, available through Sengenics and our global network of distributors and certified service providers.
We’re also eager to explore partnerships and collaborations. To start a conversation, reach out to us at [email protected].
Can you create custom protein microarrays?
Yes, we can create custom protein microarrays tailored to your research objectives. You can either select proteins from our expansive library or we can create custom recombinant proteins. To explore this further, please contact us at [email protected].
Which methods can be used to validate a candidate biomarker signature discovered using the Sengenics platform?
Sengenics microarrays facilitate antibody profiling and the identification of protein antigens targeted by the immune system. To validate these findings, you can use a custom Sengenics microarray that includes the biomarkers of interest or employ orthogonal techniques such as enzyme-linked immunosorbent assay (ELISA) or more advanced multiplexed protein profiling methods like mass spectrometry.
Is it possible to strip and reprobe/reuse slides?
The arrays should not be stripped and reused, as doing so may compromise the integrity of the proteins and affect the accuracy of the subsequent data.
What is the shelf-life of Sengenics microarrays?
If properly stored at -20°C, the arrays have a shelf life of 12 months.
Microarray Proteins
How are the proteins on your microarrays selected?
Our library of over 2,000 human proteins is carefully curated, drawing from peer-reviewed research and the expertise of leading immunologists and opinion leaders. It is enriched with disease-associated and immunogenic proteins, covering a vast spectrum of protein functions and disease categories.
On our most comprehensive microarray, i-Ome® Discovery, you’ll find proteins spanning key functional classes, including:
- Kinase / Phosphatase
- Scaffold / Adaptor
- Transcription Factor
- Transmembrane Signal Receptor
- Chromatin / Chromatin-Associated
- Ribosomal
- Ubiquitin
- Chaperone
- GTPase
- Protease / Protease Inhibitor
- And many more
Our microarray also addresses a wide range of disease categories, such as neuronal, dermatologic, hepatorenal, hematologic, immune, endocrine, cardiovascular, gastrointestinal, respiratory, and reproductive disorders.
We offer more specialized microarrays focused on specific disease areas. For instance, i-Ome® Cancer features proteins closely tied to cancer, including those involved in key cancer pathways, targeted by monoclonal antibodies and other cancer treatments, frequently mutated in various cancers, linked to poor outcomes in cancer patients, and more.
Which proteins are included on Sengenics microarrays?
Do proteins maintain their functionality once they are printed on the array?
Yes, Sengenics has verified that the proteins printed on the array retain their functionality by measuring the on-array activity of specific protein classes. For example, the autophosphorylation of tyrosine kinases was confirmed in the presence of ATP, demonstrating their retained activity. Additionally, the DNA-binding function was validated by assessing the DNA binding affinity of native TP53 and known TP53 mutants directly on the array.
How does Sengenics handle transmembrane proteins, given the aqueous environment of the assay?
In vivo, the correct (functional) conformation of transmembrane proteins is driven by interactions between the aqueous environment, lipid membranes, and the hydrophilic and hydrophobic regions of the protein. However, research shows that optimized solubilization conditions can preserve the native folding and functionality of transmembrane proteins even without membrane remnants. Sengenics has successfully expressed multiple integral membrane proteins by using selective detergent extraction to gently dissociate these proteins from their membrane environment without disrupting their conformation. When this approach is not feasible, we express and print the intra- and extracellular regions of transmembrane proteins separately. Examples of integral membrane proteins in the Sengenics library include ACE2, EGFR, GRM3, and SLC06A1.
We advise against approaches that rely on transmembrane proteins embedded in virion membranes (e.g., VirD) for two reasons: viral expression systems may lack the molecular machinery required for correct human protein folding and functionality, and there is a risk of pre-existing seroreactivity against the virions themselves.
What type of cell line is used for the production of proteins used on Sengenics microarrays?
Sengenics utilizes an insect cell line to express the recombinant proteins. This system enables stable protein expression at lower temperatures, as well as gentle cell lysis for protein recovery. Since the expression system is eukaryotic, it contains the molecular machinery to support human-like post-translational modifications. There is virtually no baseline human seroreactivity to the insect species used to produce the proteins, which results in clear data.
Can you produce proteins in other expression systems?
Yes, we have successfully expressed proteins using different protein expression systems. We only recommend expressing bacterial proteins with a bacterial (prokaryotic) expression system.
Please email us at [email protected] for further discussion.
What types of post-translational modifications are found on Sengenics proteins?
Given that human post-translational modifications (PTMs) vary in response to stimuli, biological compartments, and cell types, and are highly dynamic, our insect cell expression system is designed to provide human-like PTMs. This system ensures that modifications occur at the correct sites and with adequate complexity, such as the addition of complex glycan moieties. This level of accuracy is not achievable with yeast, bacterial, or cell-free expression systems. However, while our insect cell system produces PTMs that are human-like, the specific glycan architectures may not be identical to those found in humans.
For truly human-like glycosylation architectures and profiles, custom expression of antigens in human cell systems is possible (subject to custom project qualification). However, it’s important to note that even with this approach, the PTMs may still not fully replicate the condition-, compartment-, and cell type-specific modifications found in vivo.
What if I want to purchase the lysates only?
Please email us at [email protected] for further information.
Samples and Buffers
Which sample types can be run on Sengenics microarrays?
Our service lab accepts serum or plasma. However, any antibody-containing biological sample may be custom-assayed, associated with additional optimization steps. Such samples include: CSF, urine, ascites fluid, tear fluid, saliva, tissue lysate, culture supernatant, therapeutic antibodies, etc. If your sample type does not appear here, please contact Sengenics at [email protected] and we’d be happy to assist.
What is the required sample volume?
Our dual color IgG/IgA or IgG/IgM assays require 100 µL or 150 µL per sample, respectively. Our single color IgG requires 50 µL per sample.
Does the choice of serum versus plasma impact assay results?
No.
Plasma and serum samples typically yield highly-similar antibody profiles and do not cluster separately during data analysis. Literature from the tuberculosis domain supports our in-house observations (e.g. https://doi.org/10.1128/CVI.00325-10).
Nonetheless, we recommend using only one sample type in a study, unless unavoidable.
Does sample hemolysis impact our immunoassay?
Hemolysis releases intracellular contents from red blood cells into the sample, including hemoglobin, which has intrinsic peroxidase activity. This can be problematic in assays that rely on exogenous peroxidase to develop the fluorescence signal, such as ELISA. However, our platform does not depend on peroxidase, as the detecting antibody is directly conjugated to the reporting fluorophore.
Theoretically, proteins with globin domains, like hemoglobin, could bind non-specifically to various antigens and interfere with antigen-antibody interactions. However, in practice, we have not observed any unusual antibody signals from hemolyzed samples on our platform, even in cases of severe hemolysis (as indicated by the sample color). Literature also suggests that mild to moderate hemolysis typically does not interfere with immunoassays, and caution may only be needed in cases of severe hemolysis.
Are Sengenics microarrays incompatible with any common buffers or additives?
Data Analysis
What type of readout is your assay?
We utilize open-format scanners to detect fluorescent signals using software such as GenePix Pro 7 to extract numerical data from the array images.
What scanners are compatible with Sengenics microarrays?
Compatible microarray scanners must be able to hold a standard glass slide, with dimensions of 25 x 75 x 1 mm. Additional parameters include, but are not limited to, the following:
- Resolution: 10 μm or lower
- Excitation: 532 nm (Cy3-equivalent or Green) and 635 nm (Cy5-equivalent or Red)
- Scanned Area: 22 mm x 73 mm
- Focus: Autofocus or manually adjustable in increments of 200 μm or less
- Detection Output: 16-bit TIFF
Please contact our technical support team at [email protected] to determine whether your microarray scanner will be compatible with this kit.
What is the lower limit of detection (LOD) for antibody profiling?
The LOD corresponds to ~140-190 pg/mL for IgG. Please refer to Beeton-Kempen et al.Int. J. Cancer: 135, 1842–1851, 2014. [DOI: 10.1002/ijc.28832] for details.
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.
How do I extract the data from the protein array?
Array images can be extracted and analyzed using various image quantification software, including tools provided by scanner manufacturers, third-party software, or open-source programs.
For each array, a GenePix Array List (GAL) file is generated to assist with image analysis. Please note that GAL files are specific to the GenePix software and may not be compatible with other programs. The GAL file automatically creates grids on the array slide, enabling auto spot detection for accurate 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?
Sengenics protein microarrays are fundamentally different from DNA or RNA microarrays. While DNA microarrays often measure thousands of probes simultaneously, our protein microarrays focus on detecting global interactions of a single probe (e.g., antibody) with the immobilized proteins in a single or dual color channel. This targeted approach typically yields a smaller, more specific selection of proteins with strong signals for a given sample. To ensure the accuracy and significance of each reported signal intensity, we have developed a robust data pre-processing method.
For quality control, each replica spot on the array is evaluated against multiple threshold variables. This process flags replica spots from proteins that show high variance, identifying potential outliers.
During normalization, we apply both quantile-based and total intensity-based methods. These techniques leverage the common underlying distribution of control probes on the array to correct for technical variance while preserving the biological differences between samples.
In the biomarker discovery phase, we use protein-specific thresholds calculated from the mean signal intensities of healthy controls, allowing us to pinpoint case-specific responses.
At every step of the data analysis pipeline, rigorous quality control measures are in place to ensure that only true positive signals are reported.
What is the spotting distance between the protein spots on the array?
The horizontal distance between protein spots is 340 μm and the vertical distance between protein spots is 385 μm.
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 undergoes a rigorous post-assay quality control process, which includes evaluating assay linearity, performance consistency, and replicate measurement concordance. Fewer than 1% of samples fail to meet quality standards.
For samples that do not meet quality requirements, the microarray image is manually inspected. If technical artifacts are identified as the cause, the sample should be rerun. If the data for that sample still do not improve, the issue is typically attributed to sample matrix factors unrelated to the assay itself.
What quality controls are included on the array?
The array incorporates several quality controls to ensure the accuracy and reliability of the experiment:
- IgG Dilution Series: This control evaluates the binding capacity of the fluorescent-conjugated secondary antibody. The precise quantification of the serial dilution serves as a benchmark to verify that labeling efficiency and spot detection meet quality control standards.
- Cy3-Equivalent BSA: Over 20 bovine serum albumin (BSA) controls are evenly distributed across the slide. The concentration of these markers remains constant throughout the experiment, serving as reliable housekeeping probes for normalizing signal intensities.
- BCCP-cMyc: Each protein on the array is immobilized via this tag. This control ensures that any observed antibody signals are not due to nonspecific binding to the tag.
- Buffer Only Control: This negative control is included to ensure that no true antibody signals are detected, alongside the use of pooled normal samples.
These controls are critical for validating the experiment’s performance and ensuring the generated data is both accurate and reliable.
Why are age- and gender-matched controls important for study design?
Age-matched healthy controls are crucial for establishing baseline immune responses, enabling the differentiation of disease-specific responses. Sengenics microarrays are sensitive enough to detect age-specific immune responses in healthy controls, which can then be used as background thresholds to identify biomarkers in case samples.
Can you help me analyze my data?
Yes, data analysis tailored to your project is included in the standard services offering. To find our more about our data analysis tiers and methods, please contact Sengenics at [email protected].
At what resolution are the arrays scanned?
The array is scanned at 10 µm using a compatible scanner.
Are data qualitative or quantitative?
The data generated from Sengenics microarrays are both qualitative and semi-quantitative. Qualitative data indicate the presence of detectable antibody specificities, limited to the protein antigens included on the specific microarray. Semi-quantitative data are represented by relative fluorescent units, which correspond to the antibody concentration in the source sample.
The Sengenics platform offers a broad linear range, spanning over four orders of magnitude, allowing most fluorescent signal intensities to accurately reflect the antibody concentrations in the sample. Currently, quantification is relative rather than absolute, which allows for effective comparison between different clinical classes.
Services
I would like to send in samples. How do I proceed?
First, contact us.
Our team will work closely with you to ensure you fully understand our offerings and process, helping you best meet your research objectives.
Once your order is confirmed by providing a Purchase Order or signed Order Form, our Support team will reach out to you via email with sample submission and shipment details. They will also provide you with the KREX Project Form and Sample Annotation Form (if applicable), which must be completed before submitting your samples. We recommend using World Courier for sample shipments, as they are experienced in handling dry ice shipments.
How are infectious samples handled?
Viral deactivation of human and animal biopsies is required before running our arrays and must be performed by our customers prior to sample shipment. This process involves adding Triton X-100 to the serum or plasma sample to a final concentration of 1%, followed by an incubation period of two hours at room temperature. This procedure should be conducted in an appropriately-rated biosafety cabinet.
Which isotypes are available for screening on the Sengenics microarrays?
Our service lab offers dual color isotype screening of IgG/IgA and IgG/IgM as well as single color screening of IgG, IgA, and IgM.
For custom subclass screening, along with other possible isotype screening capabilities, contact Sengenics at [email protected].
Can you help me analyze my data?
Yes, data analysis tailored to your project is included in the standard services offering. To find our more about our data analysis tiers and methods, please contact Sengenics at [email protected].
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].
Ready to accelerate your program?
