Introduction
Oligosaccharides coupled to proteins form conjugates which become versatile reagents for high throughput characterisation of recognition capabilities of monoclonal antibodies, carbohydrate active enzymes, carbohydrate binding modules and other oligosaccharide binding proteins. These microarrays can be successfully printed using Arrayjet non-contact printers onto a variety of substrates. An array library of well characterised plant oligosaccharides has been developed by the Department of Plant Biology and Biotechnology at The University of Copenhagen using Arrayjet robust microarrayers (Pedersen et al., 2012).
Experimental Design
Sample Preparation
Oligosaccharide samples, (1→4)-β-D-mannohexaose and (1→5)-α-L-arabinopentaose, were prepared by chemical synthesis or hydrolysis of source polysaccharides, followed by conjugation to BSA. The samples were dissolved in a printing buffer containing 55.2% glycerol, 44% water, and 0.8% Triton X-100.
Substrates
A range of substrates were used for oligosaccharide printing, including Nexterion® NC, H, P, and E slides, as well as glass slides coated with nitrocellulose (FAST) and nitrocellulose membrane.
Inkjet Printing
Each drop dispensed by an Arrayjet microarrayer is 100 pL. For this study, 600 pL spots were printed onto the nitrocellulose membrane, while 200 pL spots were printed onto all glass slide types.
Microarray Probing
Following the blocking procedures described by Pedersen et al. (2012), nitrocellulose membranes and glass slides were incubated with anti-mannan monoclonal antibody LM21 or anti-arabinan monoclonal antibody LM6 for 2 hours (1:10 antibody dilution) in PBS and PBS containing 0.05% Tween 20, respectively. All array types were then washed with PBS and incubated for 2 hours with anti-rat or anti-mouse secondary antibodies (1:5000 dilution). Nitrocellulose microarrays were developed using a substrate containing 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT) in BCIP/NBT solution.
Image Acquisition and Analysis
Nitrocellulose membrane arrays were scanned using a flatbed scanner (Canon 8800, Søborg, Denmark). The slides were scanned using a slide scanner (GenePix 4100, Molecular Devices, Sunnyvale, USA). The output images were analysed using ImaGene 6.0 software (BioDiscovery, El Segundo, CA, USA).
Results
The images shown in Figure 1A–F were obtained using BSA conjugated to (1→4)-β-D-mannohexaose and (1→5)-α-L-arabinopentaose, printed in six replicates at a range of concentrations from 2 mg/mL to 0.5 μg/mL (Figure 1A–E) and from 2 mg/mL to 30.5 ng/mL (Figure 1F). The most sensitive detection was observed at 2 μg/mL for (1→4)-β-D-mannohexaose using the Nexterion® E slide (Figure 1D) and the FAST slide (Figure 1E). The least sensitive detection was observed at 125 μg/mL for (1→4)-β-D-mannohexaose using the Nexterion® P slide (Figure 1C).
In addition, arrays printed on the FAST slide (Figure 1E) produced a consistent spot size across all concentrations and were superior in quality compared to arrays printed on the Nexterion® E slide. Nitrocellulose membrane arrays demonstrated sensitive detection at 122.1 ng/mL (Figure 1F). For reproducibility assessment, twelve copies of arrays probed with mAb LM16 and mAb LM21 were printed onto nitrocellulose membrane and FAST slides. Representative replicate arrays are shown in Figure 2A and B. The mean spot signal from three replicate arrays was analysed and the data extracted.
Data Analysis
The data sets from the mean spot signals of arrays (Figure 2A and 2B) were plotted against each other, and r2r2 values were calculated. A low level of variability was observed between the array sets, with r2r2 values greater than 0.9 in all cases.
Advantages of Arrayjet Microarrayers
Arrayjet non-contact printers dispense samples by a highly reproducible piezo-actuation process producing quality spots that show constant reproducibility over long print runs and batch-to-batch. This bears an advantage over pin-based contact spotters, where the array quality decreases with longer print runs due to the inevitable wear of the pins with repeated usage (Pedersen et al., 2012). Arrayjet microarrayers provide flexibility in printing a large number of probes on the same slide with greater speed. This not only increases the throughput but avoids buffer loss through a concomitant concentration of samples during longer print runs (Pedersen et al., 2012).
Conclusion
The results of Pedersen et al., 2012 demonstrate that Arrayjet microarrayers can successfully print versatile, high-resolution plant oligosaccharides onto a variety of substrates at a fast speed. Consistent spot morphology, spot reproducibility, and signal intensity can be achieved using Arrayjet non-contact printers. This ‘on-the-fly‘ technology is ideal for printing carbohydrate microarrays and related applications.
Acknowledgements
This work was performed in the Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark. We extend our thanks to Professor William George Tycho Willats and Dr. Jonatan Ulrik Fangel for supporting and contributing to this application note.
References
McWilliam, I., Chong Kwan, M., and Hall, D. (2011). Inkjet Printing for the Production of Protein Microarrays. In: Protein Microarrays: Methods and Protocols (U. Korf, ed). Humana Press, New York.
Pedersen, H.L., Fangel, J.U., McCleary, B., Ruzanski, C., GroRydahl, M.G., Ralet, M.-C., Farkas, V., Schantz, L., Marcus, S.E., Andersen, M.C.F., Field, R., Ohlin, M., Knox, J.P., Clausen, M.H., and Willats, W.G.T. (2012). Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research. Journal of Biological Chemistry, 287, 39429–39438.
