Immunospot plate reader

We required each site to send the plates utilized for each EP, post-completion, to the EOL for centralized reading. These readings were not utilized for the purpose of generating grading criteria. We implemented this procedure to verify the In-house reading and determine if there were issues related to the In-house ELISpot reader. The centralized reading was performed with the automated ImmunoSpot plate reader (Cellular Technologies, Cleveland Ohio). The maintenance of the instrument and the reading procedure were performed under oversight of the Quality Assurance Unit according to established standard operating procedures (see Todd et al. in this issue)

ELISpot assays to detect IFN-γ were performed as previously described (39 (link)). In brief, 96-well filter plates (Millipore) were coated with anti-mouse IFN-γ Ab overnight at 4°C (eBioscience, clone AN-18). Wells were washed and blocked with complete media. Splenocytes were freshly isolated as above and plated at 1 × 10⁶ cells per well with or without 10μM human insulin (Sigma-Aldrich) in complete RPMI 1640 [10% FBS (HyClone), non-essential amino acids, HEPES, sodium pyruvate, penicillin/streptomycin, L-Glutamine, 2 × 10⁵ M 2-ME (Invitrogen)]. Cells were cultured for 72 hours at 37°C in 5% CO₂. After incubation, plates were washed and incubated with biotinylated anti-mouse IFN-γ Ab (eBioscience, clone R4–6A2). Wells were washed and incubated with horseradish peroxidase (HRP)-conjugated streptavidin (BD Biosciences). After washing, 3-Amino-9-ethylcarbazole (AEC) substrate solution (BD Biosciences) was added and wells were monitored for spot development for 4–10 min. Cold, deionized water was used to stop substrate reaction, and plates were dried overnight. Spots were counted using an ImmunoSpot plate reader (Cellular Technology Limited).

For PRN assays, the Chicago-1 strain of MeV was mixed with serially diluted plasma and assayed for plaque formation on Vero cells in triplicate. The dilution of plasma that resulted in 50% plaque reduction was calculated. MeV-specific IgG and IgA and H-specific IgG in plasma were measured by EIAs using MaxiSorp plates (Nunc) coated with lysates from MeV-infected Vero cells (1.16 μg of protein per well; Advanced Biotechnologies) or MeV H-expressing L cells (118 (link)) as previously described (15 (link)). Avidity of MeV-specific IgG was determined by adding increasing concentrations of ammonium thiocyanate (NH₄SCN; 0.5 to 3 M) to the EIA assay for 15 min. The avidity index was calculated as the concentration of NH₄SCN at which 50% of the bound antibody was eluted.
To measure antibody-secreting cells in the bone marrow, cells isolated from bone marrow aspirates by density gradient centrifugation using Lympholyte Mammal (Cedarlane Laboratories) were incubated for 6 hours with Multiscreen ELISpot plates (MilliporeSigma) coated with MeV-infected Vero cell lysate, H-expressing L cell lysate, or baculovirus-expressed N. Bound immunoglobulin was detected with horseradish peroxidase (HRP)–conjugated goat anti-monkey IgG (Nordic), developed with stable diaminobenzidine (DAB) solution and read on an ImmunoSpot plate reader (Cellular Technology).