Blood samples were collected from six mice in each group on day 14 after the second dose. Antibody immune responses to influenza virus were assessed by hemagglutination inhibition assay (HAI) and enzyme-linked immunosorbent assay (ELISA) as previously described (Isakova-Sivak et al., 2014 (link)). For HAI, the sera were treated with receptor destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to remove non-specific inhibitors and quantitated against four HA units of H7N9 LAIV virus. The ELISA was performed by using H7N9 LAIV (50 ng per well of sucrose purified virus) as a coating antigen on high-sorbent 96-well plates (Greiner, Germany). Ten-fold dilutions of the sera were prepared starting from 1:10 and added to the coated wells, followed by incubation with HRP-conjugated goat anti-mouse IgG antibody (Sigma). The detection of antibody binding was performed with 3,3’, 5,5’;-tetramethylbenzidine substrate (1-Step Ultra TMB–ELISA Substrate Solution, Thermo). The antibody levels were presented as the mean optical density (OD450) values for each serum dilution.
High sorbent 96 well plates
Manufactured by Greiner
Sourced in Germany
High-sorbent 96-well plates are a type of laboratory equipment designed for various applications that require high sorption capacity. These plates feature a specialized surface treatment that enhances their ability to absorb and retain a wide range of substances. The core function of these plates is to provide a reliable and efficient platform for sample preparation, purification, and analysis in various scientific and research settings.
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2 protocols using high sorbent 96 well plates
1
Antibody Responses to Influenza Vaccine in Mice
2
Ferret Serum IgG Antibody Titers
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Serum IgG antibody titers in ferret sera were determined on day 21 after each vaccine dose using enzyme-linked immunosorbent assay (ELISA). High-sorbent 96-well plates (Greiner Bio-One, Frickenhausen, Germany) were coated with 50 ng/well of either S.A. H1N1 or H3N2 LAIV sucrose-purified whole virus or recombinant 3M2e protein in a carbonate-bicarbonate buffer, in a volume of 50 µL per well at 4 °C overnight.
Two-fold dilutions of sera were prepared starting from 1:20 (for M2e antigen) or 1:200 (for H3N2 LAIV and S.A. H1N1 antigens) and added to the coated wells and incubated for 1 h at 37 °C, followed by incubation with anti-ferret IgG conjugated to horseradish peroxidase (Sigma). Antibody binding was detected with 1-Step Ultra TMB-ELISA Substrate Solution after incubation for 15 min at room temperature (Thermo Fisher Scientific). Optical density was measured at 450 nm using an xMark Microplate Spectrophotometer (BioRad). The area under the curve (AUC) of the OD450 values for all dilutions of each individual serum sample was calculated using the trapezoidal rule and expressed in arbitrary units.
Two-fold dilutions of sera were prepared starting from 1:20 (for M2e antigen) or 1:200 (for H3N2 LAIV and S.A. H1N1 antigens) and added to the coated wells and incubated for 1 h at 37 °C, followed by incubation with anti-ferret IgG conjugated to horseradish peroxidase (Sigma). Antibody binding was detected with 1-Step Ultra TMB-ELISA Substrate Solution after incubation for 15 min at room temperature (Thermo Fisher Scientific). Optical density was measured at 450 nm using an xMark Microplate Spectrophotometer (BioRad). The area under the curve (AUC) of the OD450 values for all dilutions of each individual serum sample was calculated using the trapezoidal rule and expressed in arbitrary units.
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