Journal: Immunology and Cell Biology

Article Title: FLU‐LISA ( fluorescence‐linked immunosorbent assay ) : high‐throughput antibody profiling using antigen microarrays

doi: 10.1111/imcb.12618

Figure Lengend Snippet: FLU‐LISA: a rapid high‐throughput antibody binding assay. FLU‐LISA is an antigen microarray‐based binding assay that can simultaneously quantify responses to multiple antigens using minimal sample volumes. The assay can be performed on multiple sample types from multiple species including humans, mice, chickens and wild birds. Two alternative approaches to perform FLU‐LISA are available: (1) traditional FLU‐LISA, an ELISA‐like assay, performed using multiple sample dilutions that are incubated on separate arrays, in which each antigen is spotted at a single concentration; (2) one‐shot FLU‐LISA, in which each sample is incubated once at a single dilution and antigens are spotted at multiple concentrations on each array. Both alternatives provide semiquantitative characterization of the antibody repertoire to multiple antigens simultaneously. FLU‐LISA, fluorescence‐linked immunosorbent assay; MFI, Median Fluorescent Intensity.

Article Snippet: Following a 2‐h incubation, the slides were dried by centrifugation at RT for 5 min at speed 800 relative centrifugal force in a slide holder padded with Kim wipes, loaded on divided incubation trays (PEPperCHIP; PEPperPRINT, Germany) and then the samples were added and incubated with the arrays for 2 h at RT on a shaker.

Techniques: High Throughput Screening Assay, Binding Assay, Microarray, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Fluorescence

Journal: Immunology and Cell Biology

Article Title: FLU‐LISA ( fluorescence‐linked immunosorbent assay ) : high‐throughput antibody profiling using antigen microarrays

doi: 10.1111/imcb.12618

Figure Lengend Snippet: Comparison of one‐shot FLU‐LISA and traditional FLU‐LISA binding curves. (a) Comparison of the binding curves of an anti‐H3N2 Brisbane 2007 monoclonal antibody (mAb) using one‐shot FLU‐LISA. The microarray included 11 serial twofold dilutions of the A/Brisbane/10/2007 hemagglutinin antigen. The mAb was run at four different dilutions, and each dilution was incubated on a separate microarray. (b) Comparison of one‐shot FLU‐LISA with traditional FLU‐LISA. The same anti‐H3N2 mAb was run at 15 multiple dilutions on FLU‐LISA microarrays in which the antigen was spotted at 11 dilutions. Binding curves were generated across antigen dilutions ( y ‐axis) and across mAb dilutions ( x ‐axis). FLU‐LISA, fluorescence‐linked immunosorbent assay.

Article Snippet: Following a 2‐h incubation, the slides were dried by centrifugation at RT for 5 min at speed 800 relative centrifugal force in a slide holder padded with Kim wipes, loaded on divided incubation trays (PEPperCHIP; PEPperPRINT, Germany) and then the samples were added and incubated with the arrays for 2 h at RT on a shaker.

Techniques: Comparison, Binding Assay, Microarray, Incubation, Generated, Fluorescence

Journal: Immunology and Cell Biology

Article Title: FLU‐LISA ( fluorescence‐linked immunosorbent assay ) : high‐throughput antibody profiling using antigen microarrays

doi: 10.1111/imcb.12618

Figure Lengend Snippet: Pearson correlations between ELISA and one‐shot FLU‐LISA for four influenza antigens. We compared the correlations between the immunoglobulin (Ig) G area under the curve (AUC) for serum samples of 10 healthy adult individuals against four influenza strains: A/Wisconsin/67/2005 (H3N2), A/Brisbane/10/2007 (H3N2), A/California/07/2009 (H1N1) and the A/Shanghai/1/2013 (H7N9). ELISA was run using 15 serial dilutions (1:25–1:409 600) of each serum and a single antigen concentration, thus ELISA AUC was calculated across serum dilutions. FLU‐LISA microarrays were incubated once with each serum sample diluted 1:3200, and AUC was calculated across antigen dilutions. Pairwise correlations between FLU‐LISA AUC values with the different antigens are shown in red, and correlations between ELISA AUC values are shown in blue. In each of the four panels the correlation values between a single strain (Wisconsin, Shanghai, California or Brisbane) with all four strains (including itself) are visualized for each assay separately. We found that overall correlations between ELISA AUC values were higher. FLU‐LISA, fluorescence‐linked immunosorbent assay.

Article Snippet: Following a 2‐h incubation, the slides were dried by centrifugation at RT for 5 min at speed 800 relative centrifugal force in a slide holder padded with Kim wipes, loaded on divided incubation trays (PEPperCHIP; PEPperPRINT, Germany) and then the samples were added and incubated with the arrays for 2 h at RT on a shaker.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Fluorescence