1. How are the proteins on the IMMUNOME protein array selected?
The standard 1600+ IMMUNOMETM protein array is designed to enable a comprehensive inspection of the immune system. In this regard, 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.
2. How is your KREX protein array different from other protein arrays?
Other protein arrays utilise peptides, fragment of proteins or purified proteins immobilised on glass slides. Peptides or fragments of proteins only make available a few epitopes per protein and therefore typically miss both discontinuous and numerous linear epitopes, as well as lacking post-translationally modified epitopes. Purified proteins, on the other hand, can be denatured due to aberrant interactions with the array surface, impairing the availability of linear epitopes and disrupting conformational epitopes of the proteins. In this regard, in order to maximise the interaction between autoantibodies and proteins on the array, our KREXTM-based protein array utilises only full-length, native proteins to be immobilised on glass slides where all the epitopes will be present for binding with autoantibodies.
3. What is your business model? How do I access your technology?
1. Funded Discovery:
Sengenics mainly offers its KREXTM technology in a collaborative, cost sharing model, in which both Parties contribute funds, share IP and share publication authorship. Sengenics shall own the commercialisation rights to any shared IP generated with royalty terms. If you are interested in our Funded Discovery option, leveraging the KREXTM technology, please contact us at [email protected].
2. Licensing Option:
The KREXTM technology may also be accessed through the Sengenics IMMUNOMETM Technology Access Program (iTAP) whereby iTAP partners will be granted a license to fully leverage the KREXTM technology , with open rights to utilise, commercialise and patent discoveries. For more licensing options through iTAP, please contact us at [email protected].
4. How are the proteins immobilised on the glass slide?
The proteins are immobilised on the glass slide via affinity attachment. Correctly folded, biotinylated proteins are spotted on streptavidin-coated glass slides where the biotin occupies the binding sites on the streptavidin. Affinity capture is a particularly advantageous way to immobilise proteins as the orientation of the immobilised protein is controlled via the folding marker which aids in preserving the structure and function of the arrayed proteins. The folding marker also provides a single point of attachment which negates the problems of protein unfolding, random orientation and non-specific binding that can occur with other protein attachment methods.
5. Where is the BCCP folding marker positioned?
It is present at the C-terminal of the protein of interest. However, upon request, it can also be positioned at the N-terminal for certain proteins especially when important/major sites of interactions of the protein are present at the C-terminal.
6. Why is it positioned at the C-terminal of protein?
The folding of the BCCP and its subsequent biotinylation is dependent on the correct folding of the protein from the N-terminal to the C-terminal. This is also the natural direction that mRNA is translated in vivo: 5′ to 3′, N -> C-terminal. The BCCP is positioned at C-terminal so that it can act as a reporter of correct folding of protein, whereby the BCCP will only fold correctly and become biotinylated if the fusion protein if correctly folded from the N-terminal to C-terminal.
7. How does the BCCP drive the correct folding of the fusion protein?
The mechanism by which BCCP aids the folding of downstream protein domains is by recruitment of chaperones to the nascent polypeptide and co-overexpression of chaperones or by increasing the overall solubility of the fusion protein.
8. How is biotin attached to the BCCP?
The attachment of biotin to the lysine residue of the BCCP is catalysed by the enzyme biotin ligase of the host cell in which expression of the BCCP is taking place. Efficient in vivo biotinylation has been observed across different cell types including bacterial, insect and mammalian cells.
9.How large (how many amino acids) is the BCCP folding marker?
E. coli BCCP is an 80-amino acid domain that folds autonomously into a compact, all beta strand structure and is biotinylated post-translationally in vivo on a single, specific lysine residue by the host cell biotin ligase.
10. Are the proteins modified prior to expression?
Modifications to the protein sequence prior to expression is dependant on the protein structure and localisation. For certain proteins, signal peptides may be added to the existing protein sequence to guide post-translational modification. Also, linkers could be added in between protein of interest and the BCCP folding marker to provide conformational flexibility to the protein.
11. What degree of misfolding will prevent a protein from attaching to the slide?
During translation of large proteins, folding begins in a co-translational manner. Co-translational folding of a fusion partner means that in the majority of cases, the N-terminal domain will start to fold first. If this initial folding results in a properly folded domain, then it is reasonable to assume that the C-terminal domain will also be able to fold correctly. Alternatively, if the N-terminal domain misfolds and starts to present aberrant hydrophobic surfaces, there is a strong likelihood that initiation of the normal folding pathway of the C-terminal domain will be affected, leading to aberrant folding of the C-terminal domain. As we positioned BCCP at C-terminal domain, it would be expected that mis-folding of a N-terminal fusion partner will perturb the proper folding of BCCP and therefore prevent downstream biotinylation of BCCP and immobilisation onto slide.
12. What is the volume of protein lysate spotted on the array?
9 nanolitres of crude protein lysate are spotted in quadruplicate on the array.
13. What is the concentration of spotted proteins?
One of the major advantages of using the streptavidin-biotin interaction as the basis for array fabrication is that they have very high binding affinity. The high affinity of streptavidin-biotin interaction means that we quickly start to saturate the available biotin-binding sites on the slide surface so a crude normalisation of protein loading can be achieved without pre-adjusting the concentrations of the protein lysates to compensate for differences in the individual expression levels of all 1,600 different proteins.
14. What robotic spotting method is used for the IMMUNOME slides?
We use non-contact piezo printing technology to dispense nanolitres of protein onto the array.
15. Are the proteins purified before immobilisation onto slides?
No. The addition of biotin to the BCCP acetyl co-A domain permits purification of the proteins by surface capture. The proteins can be simultaneously purified from cellular lysates and immobilised in a single step via the high affinity and specificity exhibited by a streptavidin surface. We term this simultaneous purification and immobilisation as “surface capture”.
16. Since you don’t purify your protein, can we expect competition between endogeneously biotinylated insect cell proteins and biotinylated recombinant proteins for binding site?
Insect cells have five endogenous proteins that have streptavidin binding sites. Under native conditions, we have observed that these proteins do not compete efficiently with biotinylated recombinant proteins for binding to streptavidin. This perhaps reflects low natural expression levels and the fact that in endogenous biotinylated protein, the biotin is typically less solvent accessible in the native protein meaning that the biotin may not be physically accessible to bind to streptavidin.
17. How do you know if a spot has recombinant protein attached to it?
The proteins on the IMMUNOME protein array are cloned in frame with c-myc tag, which can be used to visualise the proteins on the array (anti-myc assay). To ensure ﬁdelity, clones are sequence-veriﬁed immediately prior to expression in insect cells and western blot analysis is performed after protein expression.
18. How do you prove that the proteins on the array are functional?
There are over 400 kinases on the IMMUNOMETM platform and to prove the functionality of the proteins, we measure the ability of tyrosine kinases to autophosphorylate in the presence of ATP, which is only possible when the kinases are functional and correctly folded or present in their native conformation. The tumour protein, TP53 present on the array has been proven to be able to bind DNA, which is only possible when the protein is in an active (functional) form.
19. Do proteins expressed with KREX form complexes or multimers?
Yes, homodimeric proteins are expressed autonomously as homodimers, while heterodimeric proteins are expressed as monomers and subsequently co-arrayed to form heterodimers.
20. Are there any transmembrane proteins on the IMMUNOME protein array? If yes, how do you provide lipidic environment for the proteins?
Yes. The array contains separate extracellular and intracellular domains, excised from the transmembrane region in every case. There is currently no deliberate lipid environment on the array, although we do lyse the insect cells in detergent, so if there are insect cell lipids strongly associated with the antigens then they (as well as the detergent) will co-purify on the array.
19. What type of slide is used for the IMMUNOME protein array and how is it manufactured?
Our slides are manufactured using high quality, low auto-fluorescence borosilicate glass.The resulting surface has a very high binding capacity combined with a low signal variance across a single slide, between slides and between batches. The slides have a proprietary surface coating that ensures that spotted proteins remain in an aqueous environment that retains protein stability and prevents proteins from binding to the underlying glass surface.
Proteins also retain localised conformational flexibility thus preserving the proteins and preventing lateral diffusion of proteins across the array surface.
21. How do I extract the data from the protein array?
Array images can be extracted/analysed 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.
22. What are the scanners that are compatible with the IMMUNOME protein array?
The protein array slides can be scanned using any open-format microarray scanners. We would, however, recommend using the Agilent scanner as we’ve found that it is more stable over time than other scanners we have tested.
23. At what resolution are the arrays scanned at?
The array is scanned at 10 µm using any open format scanner.
24. What samples can be run on the IMMUNOME protein array?
Depending on the assay type whether it is a biomarker discovery or interaction study, these samples can be run on the IMMUNOME protein array:
25. What proteins are found on the IMMUNOME protein array?
Cancer antigens, signalling proteins, transmembrane proteins, kinases and other immunologically relevant proteins. For the full list of proteins, please click here.
26. What is the half-life of autoantibodies?
The half-life of autoantibodies is 3 weeks. Levels of autoantibodies would be expected to halve in about 20 days. Accumulation of aberrant PTMs will give rise to autoantigens that stimulate re-production of autoantibodies.
27. Besides Cy-3, what are other fluorescent tags that can be used for the IMMUNOME protein array?
Besides Cy-3 and Cy-5, dyes similar to Cy-3 and Cy-5, such as Alexa 647, 555 and 660 dyes, can also be used.
28. Is it possible to include my proteins of interest that are currently not included on the IMMUNOME protein array?
Yes. We can custom produce proteins based on your requirements. Please contact us at [email protected] to discuss this further.
29. What kind of studies are ideal using the KREX technology?
The KREXTM 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, immunotherapy and irAE prediction. In interaction profiling, we profile interaction of various analytes with the proteins on the array, such as DNA, RNA, protein, peptide, vaccine etc.
30. When a customer finds a biomarker using our platform, the platform is highly sensitive, what other methods can we use to ensure that the biomarker is not just specific to our platform, so therefore could be transferable to a routine clinical setting?
ELISA is the gold standard for the measurement of proteins/immunoassays in a clinical setting . There are also other methods 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 protein array and ELISA), there is the possibility of not being able to detect low-abundant biomarkers using ELISA.
1. How do I extract the data from the protein array?
Array images can be extracted/analysed 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.
2. Are you able to share your data analysis pipeline or software?
Yes, we share our data analysis pipeline with our customers and the pipeline is written in R language which includes pre-processing and normalisation of data as well as algorithm for biomarker discovery.
3. Are there any publications on your data analysis methodology?
4. 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 colour-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 normalisation step uses both quantile-based and total intensity-based methods which utilises common underlying distribution of control probes on the array to correct for any technical variance whilst 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.
5. Which scanners are compatible with Sengenics' protein arrays?
Our protein array slides can be scanned on any microarray scanner which is able to detect Cy3 fluorescence e.g. Perkin-Elmer, Axon etc. We would, however, recommend using the Agilent scanner as we’ve found that it is more stable over time compared to the other scanners we have tested.
6. 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 360 μm.
7. What is the diameter of the protein spots in the array?
The diameter of individual protein spots is 150μm.
8. What type of proteins are present on the IMMUNOME protein array?
There are more than 403 Kinases, 600 cancer related proteins, 380 transcription factors, 190 signalling proteins, 20 cytokine/chemokines and 360 proteins from other classes.
9. How is the quality of the protein array data verified?
Data extracted using the image quantification software is subjected to preprocessing 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.
Please refer to the IMMUNOME protein array protocol for more information on how the data is preprocessed.
10. How is the data normalised?
The data is normalised using a combination of quantile and intensity-based methods. Please refer to the IMMUNOME protein array protocol for detailed calculations.
11. How is data from the protein array analysed?
Data was analysed via a combination of penetrance fold change and penetrance frequency. Please refer to the IMMUNOME protein array protocol for detailed calculations on how the penetrance fold change is derived.
12. What are the criteria for identifying possible biomarkers?
With the availability of sample cohorts i.e Case and Control, we adapt penetrance-based fold change (pFC) analysis method to identify proteins with high intensity in each case sample. Utilising this method will eliminate any false positive signals from the analyses. This is achieved by implementing a protein-specific threshold i.e. background threshold.
The per-protein background threshold is calculated based on the signal intensities for each specific protein measured for a given cohort of healthy control samples.
Putative biomarkers are identified and ranked according to the following criteria:
1. Penetrance fold change for case ≥ 2
2. % Frequency for case ≥ 10%
13. Why isn't the t-test used to compare case vs control?
The t-test takes into account the intrinsic variability in the positive signals across the samples as a function of the fold change between case and control. However, we’d expect the intrinsic variability in true positive signals to be quite high, even though the intrinsic variability in the signals from the controls should be low, so it may be that a t-test isn’t the most appropriate statistical test to run here.
Multiple testing correction then adds a further layer of stringency, essentially trying to turn the initial p-value into a false discovery probability. Multiple testing correction assumes that the individual tests are independent but pathway analyses often show that the identified antigens are in fact not truly independent i.e. their expression is linked somehow.
14. 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 incubation. Accurate serial dilution quantification is used as a benchmark for ensuring that labelling 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 as a housekeeping probe for normalisation of the signal intensities.
For a detailed explanation about controls, please refer to the IMMUNOME README.
15. 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.
1. How long does it take to run an array in the laboratory?
It will take approximately 7 hours. Please refer to the protocol here
2. Can you provide us with the KREX protein array protocol?
Yes, the protocol can be found here
3. What buffer is used for diluting the samples and for washing the array?
Serum Assay Binding (SAB) Buffer is used thorughout the experiment, both for diluting the samples as well as washing. Please refer to <link> for the recipe of SAB buffer.
4. How do you handle the KREX protein array slides?
Please wear gloves when handling the slides. Hold the slides only at the barcoded area. Please do not touch the active side of the array. Make sure the active side of the array is facing upwards and submerged in the appropriate solutions during incubation.
5. How are serum samples prepared for KREX protein array run?
Please refer to the protocol of sample preparation here
6. What is the shelf life of the KREX protein array slides?
If properly stored, the arrays have a shelf life of at least 18 months for most applications
7. Can we use a tilting shaker during incubation and washing?
No, you cannot use a tilting shaker. Please use a horizantal shaker. This is to prevent mixing of solutions between chambers.
8. What is the storage condition for the slides?
The slides should be stored at -20°C
9. Can we store the SAB buffer for some time before using it?
It is advisable to prepare fresh SAB buffer before running the slides . If that is not feasible, the buffer can be stored at 4°C up to 1 week.
10. Where do you store the used slides?
The used slides are stored in a slide box in a cool dry environment.
11. Can I rescan the used slides?
Yes, however, from our experience the maximum storage time is one week. The intensities decrease over time.
12. 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 on Samples Submission and Shipment SOP. You will also need to fill in the KREX Project Form and Sample Annotation Form (if applicable) which will be attached in the email before proceeding with the samples submission. We recommend the customer to always use World Courier for samples shipment as they have experience in handling dry ice shipments.
13. What if I want to purchase the slides only?
Yes you may also purchase our slides to be run in your own lab. Please email us at [email protected]
14. Is it possible to strip and reprobe slides (reuse)?
We do not recommend stripping the arrays and reprobing, as this may compromise the integrity of the proteins.
15. 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 run (recommended), the finished arrays can be stored overnight, in dark to avoid photobleaching.
1. What protein expression platform are used to produce KREX™proteins?
KREX™ utilises baculovirus expression system for the production of proteins of interest in insect cells. Although producing these recombinant baculoviruses are technically laborious, it may offers many advantages over other traditional systems: 1) Safety – baculoviruses are nonpathogenic, 2) Ease to scale-up – insect cell suspension cultures allows large-scale protein production in bioreactors, 3) Size – accomodate large genes, 4) High levels of protein expression – variety of efficient gene promoters and 5) Post-translational modification – proper protein folding, glycosylation, phosphorylation, acetylation and acylation.
2. What type of cell line is used for the production of KREX proteins?
KREX protein expression system utilises insect cell known as Sf9, a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE.
3. Is it possible to express the proteins in bacterial cell?
Yes, we have successfully expressed proteins using different protein expression systems. Please email us at [email protected] for further discussion.
4. We have our own proteins of interest. How long does it take to produce these proteins from the DNA sequence provided?
Depending on the size and complexity of the protein, the duration may vary between 3-6 months. Please email us at [email protected] for further discussion.
5. How much does it cost to produce a protein from DNA sequence provided?
The cost is largely dependent on the size and the nature of the protein. Please email us at [email protected] for further discussion.
6. What is the laboratory assay used to validate the infectivity of baculoviruses upon protein production?
Plaque assay is carried out to determine viral titre.
7. What is the laboratory assay used to validate expressed proteins of interest?
Crude lysates are subjected to SDS-PAGE and the biotinylated proteins were detected via Western blot.
8. What is the purity of the proteins produced?
Our final product is crude protein lysates. However purified proteins are also available at an additional cost, please contact us at [email protected].
9. What if I want to purchase the lysates only?
Please email us at [email protected] for further information.