Genepix pro 7

To extract quantitative data from the slides, image analysis was utilized to evaluate the number of autoantibodies present in each serum sample by measuring the median intensities of all the pixels within each probed spot. A raw .tiff image file was generated for each slide (sample). Automatic extraction and quantification of all the pixels in each spot on the array were performed using GenePix Pro 7 software (Molecular Devices), which provides statistics for each probed spot on the array. This includes the mean and median of the pixel intensities within a spot and its local background. A GAL (GenePix Array List) file for the array was generated to aid the image analysis. This file contains the information of all probed spots and their positions on the array. Following data extraction, a GenePix Results (.GPR) file, which contains information for each spot (e.g., Protein ID, protein name, foreground intensities, background intensities), was generated for each slide.

The tau peptide microarray was designed using 15-mer peptides with four amino acid overhangs, spanning the full sequence of PNS-tau (P10636-9) for a total of 187 tau peptides. Empty spots were used as negative controls. The arrays were printed on single microscope slides in triplicate (Jenrin Peptide Technologies, Berlin, Germany). Binding was tested per manufacturer’s protocol using plasma, diluted 1:100 in binding buffer (PBS/0.1% Tween/2% bovine serum albumin) from animals treated with either Wt-tau or P301L tau protein. Binding was detected using a 1:500 titer of HiLyte Fluor 555-labeled goat anti-mouse IgG antibody (Anaspec, Fremont, CA, USA) in binding buffer. Arrays were scanned at a fluorescence emission of 532 nm using a GenePix 4100A Microarray Scanner (Molecular Devices, Sunnyvale, CA, USA) and raw intensities adjusted by background subtraction (morphological closed followed by opening) using the scanner software (GenePix Pro 7, Molecular Devices, Sunnyvale, CA, USA). Binding was defined as peptides with fluorescence intensity greater than 3 standard errors above the mean of the slide intensities. In order to identify false positives, a microarray was incubated with the antibody alone. No signal was observed, so all peptides were included in the analysis.

Data was processed using GenePix^® Pro7 software (Molecular Devices). Each sample was corrected for background by subtracting the raw spot intensity of negative control protein from every sample spot. The results were normalized relative to corresponding concentration of Ig isotype controls in each subarray to minimize the variations that occur during sera and array processing. The mean fluorescence intensity ± standard deviation (SD) was calculated from the replicates for each antigen concentration.
Results were plotted using GraphPad Prism software version 8.4.3. Nonlinear regression model was used for dilution-response curves of serum samples. The bar plots and heatmap were used to display antibody levels of multiple samples against antigen targets, CPT time points or Ig isotypes. The data was also represented as the ratio of antigen specific antibody levels.

Scanning was performed on a confocal laser microarray scanner (Genepix 4300A, Molecular Devices), and the signal intensity (SI) was quantified using image analysis software (Genepix Pro 7, Molecular Devices) and transformed and normalised using the vsn statistical package (http://www.r-project.org). Finally the data were re-transformed (inverse log₂) to a normalised SI [21 (link)-23 ].
Antigens were considered positively recognised by an ASC-probe sample when the SI was greater than two standard deviations above the mean of NI rat sera. Amino acid sequences of the identified antigens were then analysed for features of potentially important vaccine candidates i.e. developmental expression based on EST Profile Viewer (http://www.ncbi.nlm.nih.gov/ unigene), presence of a signal peptide using SignalP -4.1 (http://www.cbs.dtu.dk/services/SignalP), and a predicted transmembrane domain with TMpred (http://www.ch.embnet.org/software/TMPRED_form.html). Also, literature searches were performed to determine the novelty of each antigen, which were considered novel when there were no reports of vaccine efficacy against S. japonicum or significant characterisation.

Humidified chamber comprising a Labnet Mini incubator (Labnet International, I5110A), containing a glass bowl with 25 ml water.
GenePix 4300A microarray scanner with integrated GenePix Pro 7 image analysis software (Molecular Devices).

For fluorescence imaging, the beads were washed 6 x with 500 μL of PBS containing 0.1% (v/v) Tween 20 (PBS-T), and twice with 500 μL of water for salts’ removal. Afterwards the beads were spread on a cleaned and polished microscope slide and dried for 30 min at 40°C. An Axon GenePix 4300 A fluorescence microarray scanner (Molecular Devices) was used for fluorescence imaging, using a resolution of 5 μm per dot. Excitation laser (488 nm or 635 nm) intensity and photomultiplier gain were adjusted to yield the maximum signal-to-background ratio. Median fluorescence intensity was calculated for a population of 50 particles using the Software GenePix Pro7 (Molecular Devices).

Sengenics KREX technology (i-OME Discovery array in combination with dual color IgG/IgA detection) was used to profile seroreactivity against 1616 self-antigens with preserved native conformation.[38 (link),39 (link)] Controls for this platform included 22 individuals from the above described control cohort as well as a commercially available pooled normal plasma (Sigma) from 50 – 70 individuals without clinical evidence of autoimmunity. Plasma was diluted 1:200 for the assay. Autoantibody binding to antigens was measured using an open format microarray laser scanner (Agilent) at 10 µm resolution, with quantification of intensities using GenePix Pro 7 software (Molecular Devices). Net fluorescence intensities were calculated by subtracting background signaling intensities, followed by log2 transformation, Loess normalization[40 (link)] using the normalizeCyclicLoess() function[41 (link)] from R package limma[42 (link)]. Batch-correction was performed using the ComBat() function[43 (link)] from R package sva.[44 (link)] To avoid potential confounding from intravenous immunoglobulin (IVIG), we excluded autoantibodies with significantly increased intensities in patients who received IVIG within 4 weeks of study enrollment compared to patients who did not receive IVIG.