Synergy neo alpha plate reader

For analysis of T-cell dependent antibody production, mice were immunized intraperitoneally with 100μg NP-CGG or NP-OVA or NP-KLH, ratio 16–32, in alum (1:1). Serum from each animal was collected before immunization, and on the stated days after immunization. Titers of low and high-affinity isotype-specific antibodies to NP were measured in plates coated with NP₂₈-BSA or NP₈-BSA, respectively, using the SBA Clonotyping System (5300–05; Southern Biotechnology; Birmingham, AL, USA), according to the manufacturer’s protocol. Results were assessed by spectrophotometric measurement of absorbance at 405nm using a Biotek Synergy Neo Alpha Plate Reader (BioTek; Winooski, VT, USA). Background readings of absorbance in negative control wells were A₄₅₀<0.050.

For analysis of T cell–dependent antibody production, mice were immunized intraperitoneally with 100 μg NP-CGG ratio 20–25 in alum (1:1). On day 35 after immunization, serum was collected and titers of isotype-specific antibodies to NP were measured in plates coated with NP₂₆-BSA or NP₇-BSA with the SBA Clonotyping System, according to the manufacturer’s protocol (5300–05; Southern Biotechnology). Results were assessed by spectrophotometric measurement of absorbance at 405nm using a Biotek Synergy Neo Alpha Plate Reader (BioTek). Background readings of absorbance in negative control wells were <0.050. For enzyme-linked immunospot (ELISPOT) assay, we collected bone marrow cells on day 60 after immunization. Cells were incubated for 20 h at 37°C on NP₂₆-BSA-coated or NP₄-BSA-coated 96-well MultiScreen-HA filter plates (Millipore). NP-specific spots were visualized with goat antibody to mouse IgG1 (1034–05) or IgM (1021–05) conjugated to horseradish peroxidase (Southern Biotechnology), and color was visualized by the addition of 3,3′,5,5′-tetramethylbenzidine (Southern Biotechnology). Plates were evaluated using an automated Zeiss Elispot reader system (ZellNet Consulting, Inc).