title: Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides.
abstract: The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10μg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs.
authors: Tethis, via Russoli 3, 20143 Milano, Italy.
authors: Marzia L
authors: ML
authors: De Marni
authors: Ana
authors: A
authors: Monegal
authors: Samuele
authors: S
authors: Venturini
authors: Simone
authors: S
authors: Vinati
authors: Roberta
authors: R
authors: Carbone
authors: Ario
authors: A
authors: de Marco
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000915:Antibody Affinity; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000737:chemistry; D000941:Antigens / Q000032:analysis / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D005822:Genetic Vectors / Q000737:chemistry; D007118:Immunoassay / Q000379:methods; D057230:Limit of Detection; D049329:Nanostructures / Q000737:chemistry*; D036103:Nanotechnology; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012475:Salmonella / Q000737:chemistry; D059629:Signal-To-Noise Ratio; D057127:Single-Chain Antibodies / Q000737:chemistry; D013997:Time Factors; D014025:Titanium / Q000737:chemistry*
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D000941:Antigens; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C009495:titanium dioxide; D014025:Titanium; D051496:Receptor, Fibroblast Growth Factor, Type 1