Abstract
The mouse monoclonal antibody (mAb) technology still represents a key source of reagents for research and clinical diagnosis, although it is relatively inefficient and expensive and therefore unsuitable for high-throughput production against a vast repertoire of antigens. In this article, we describe a protocol that combines the immunization of individual mice with complex mixtures of influenza virus strains and a microarray-based immunoassay procedure to perform a parallel screening against the viral antigens. The protocol involves testing the supernatants of somatic cell hybrids against a capture substratum containing an array of different antigens. For each fusion experiment, we carried out more than 25,000 antigen-antibody reactivity tests in less than a week, a throughput that is two orders of magnitude higher than that of traditional antibody detection assays such as enzyme-linked immunosorbent assays and immunofluorescence. Using a limited number of mice, we can develop a vast repertoire of mAbs directed against nuclear and surface proteins of several human and avian influenza virus strains.
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Acknowledgements
We thank I. Donatelli for providing the influenza viruses and antigens used in all studies. This work was supported by a grant from EU FP & Health FLUARRAY (GA n 201960) and by FIRB grants RBIP064CRT and RBLA03C9F4.
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M.D. designed the experiments, analyzed data, supervised the project and wrote the paper; L.N., M.A.G. and B.C. conducted the experiments and analyzed the data; L.D., G.M., F.B. and R.S. intellectually contributed to this work; A.C. inspired and supervised the project, and wrote and approved the final paper.
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Supplementary information
Supplementary Figure 1
Comparison of array reactivity using slides from different batches. The images show arrays processed using the same supernatant (MAb 2A12 in panel a and MAb 7G11 in panel b, see Supplementary Table 1) but printed at different times onto different batches of slides. (TIFF 3319 kb)
Supplementary Figure 2
Images of whole slides containing several identical arrays. Each slide contains four replicas of the same array processed with the supernatants of the hybridomas 1H11, 11D7, 22C12, 17G11 (see Supplementary Table 1). The same reactivity patter observed in all array replicas on one slide demonstrates the very low variability among intra-slide spots. (TIFF 4457 kb)
Supplementary Figure 3
Layout of samples in the 384 well plate and array design. a) Table reporting the order of samples in the wells of the 384 microplate. The spotting buffer used to print each sample is reported in the right column. b) Window of the MicroGrid II software displaying the layout of the 384 microplate. A schematic representation of the 384 microplate is shown on the bottom of the panel b. The twenty-eight wells loaded with the spotting samples have been numbered. c) This screen enables the desired pin array pattern to be entered. The array pattern is designed by entering the number of the 384 microplate well in the editor. d) This screen enables one to define the position of the tool-array. By adjusting horizontal (top and bottom) and vertical (left and right) margins the pin tool placement area can be altered and the arrays that fit are generated automatically. (TIFF 4073 kb)
Supplementary Figure 4
MAb reacting to glycosyl residues. Microarray (left panel) and immunoblot (right panel) reactivity profile of the MAb 1H11 reacting against distantly related influenza virus types. The immunoblot reactivity of the MAb 1H11 was analysed against both glycosylated and de-glycosylated (Deg) virus preparations. Removal of glycosyl residues was performed using the Enzymatic Protein De-glycosylation Kit (Sigma). (TIFF 3314 kb)
Supplementary Table 1
Microarray reactivity profile against array influenza antigens of the selected MAbs. (XLS 40 kb)
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Di Cristina, M., Nunziangeli, L., Giubilei, M. et al. An antigen microarray immunoassay for multiplex screening of mouse monoclonal antibodies. Nat Protoc 5, 1932–1944 (2010). https://doi.org/10.1038/nprot.2010.161
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DOI: https://doi.org/10.1038/nprot.2010.161
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