What is the recommended approach to validate Immunome results?
You can validate the results using ELISA by applying one of this techniques listed below:
- Streptavidin coated microtitre plates to purify and immobilise each antigen via the BCCP tag
- Pre-purify each antigen off-line and immobilise onto a conventional ELISA plate (which will be at least partially denaturing)
- Use an antigen-specific antibody to capture the antigen onto an ELISA plate
What is an alternative method to validate the Immunome results?
The alternative methods are by carrying out Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot to validate the protein array results on a patient-by-patient basis. The selected individual crude lysates or known as â€˜antigenâ€™ is separated by SDS-PAGE and then carried out a Western blot using the relevant patient serum sample. The â€˜antigen-autoantibodyâ€™ binding are detected by using horseradish peroxidase (HRP)-labelled anti-human IgG.
What are the advantages of using SDS-Page and Western Blot?
The advantages are:
- Able to verify the signals measured on the arrays arise from the specific recombinant proteins and are not due to any spurious cross-reactivity.
- Serve to confirm the approximate molecular weight of the recombinant protein, whilst separate blotting with streptavidin-HRP confirms biotinylation; with those two pieces of information per lane on the Western blot, we have not felt the need to carry out mass spec analysis on the immuno-reactive band.
Can I get the detailed outline of SDS-Page and Western Blot protocol?
The protocols as per mentioned below:
- Prepare crude insect cell lysates for the specific antigens which have been identified as significantly expressed from the Immunome assay and also empty vector insect cell lysates (i.e. insect cells expressing the BCCP tag only) as a control.
- Load 20 Âµg of each total insect cell lysate and control into separate lanes of a 10% SDS-PAGE gel and separate the proteins by electrophoresis.
- Transfer the separated proteins onto a PVDF or nitrocellulose membrane using an electroblotter.
- Block the membrane in 5% milk powder (w/v) in Phosphate Buffer Saline (PBS) for an hour at room temperature.
- Probe the blocked membrane using a 1:200 dilution of a serum sample from the patient in question for an hour at room temperature (NB. dilute in 5% milk powder (w/v) in PBS).
- Wash the membrane three times for 5 minutes each in PBS.
- Incubate the membrane with a 1:2,000 dilution of a HRP-conjugated anti-human IgG for an hour at room temperature (NB. Dilute in 5% milk powder (w/v) in PBS/0.1% Tween).
- Wash the membrane 3 times for 5 minutes each in PBS/0.1% Tween.
- Develop the blot using chemiluminescent or colorimetric HRP substrates.
What is the sample requirement for Immunome?
30-50ul of plasma, serum or purified monoclonal antibody samples.
What are the scanners that are compatible with Immunome arrays?
The 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 because it is much more stable over time than other scanners we have tested. In other words if you run control arrays over a long period of time they appear very similar to each other on the Agilent scanner but on other scanners the intensities can vary over time as the laser power seems to vary from one day to another. This means that normalisation between the overall intensities on each array becomes more important so it is just easier to make comparisons over time with the Agilent scanner.
How do I extract the data from the arrays?
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 GAL (GenePix Array List) file for each array is generated to aid image analysis. Please note GAL files is grid file specific to Genepix software and may not be compatible with any other software. GAL automatically generate grids on the array slide for auto spot detection supporting image analysis.
What is the amount or 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 a 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 normalization 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 1,631 different proteins.
What type of slide is used for Immunome and how is it manufactured?
NEXTERIONÂ® Slide H-S is manufactured using high quality, low auto-fluorescence borosilicate glass.The specially developed two-stage coating process produces a very high density, as well as an extremely uniform coating of streptavidin molecules. The resulting surface has a very high binding capacity combined with a low signal variance across a single slide, between slides and between batches. These factors make Slide H-S an ideal surface for developing diagnostic applications.
What type of pathogens samples have been run on the Immunome array?
Human serum infected with Plasmodium knowlesi and Patient with Parkinson Disease infected with Helicobacter pylori.
How do you make sure that every single protein on the array is correctly folded?
We perform Anti-c myc assay to determine whether all the proteins are spotted onto the slide. Only correctly folded protein+BCCP tag will be biotinylated and attached on streptavidin coated array.