Elx808 absorbance plate reader

The subcultured cells from 0G to 9G and 17G were spread on M9 medium containing d-Fru (0.05%) and d-Tag (0.45%). A single colony was isolated from the solid medium and inoculated into 96-well plates with M9 liquid medium supplemented with d-Fru (0.05%), d-Tag (0.45%), 0.2 mM IPTG, and 50 μg mL⁻¹ Kan in a total volume of 200 μL. Each generation of 96-well plates was incubated at 37°C for 12 h, and optical cell density was measured using a BioTek ELx808 absorbance plate reader. Three genetic regions, fruK, the Cra binding site, and T7RNAP^Pro, were amplified from 96-well plates by PCR, and PCR fragments were analyzed by DNA sequencing.

Blood samples were obtained from infants at 1 and 15 weeks and from Nigerian mothers at 1 week postpartum. All blood samples were collected in heparinized tubes and transported to the lab within 6 hours for sample processing. Plasma was removed prior to cell isolation using Ficoll density-gradient separation medium (Sigma Aldrich) and stored at −80°C until analysis. Plasma anti-tetanus IgG was measured by enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s protocol (TECAN, IBL International GmbH). The optical density at 450 nm was measured using an ELISA microplate reader (BioTek ELx808 absorbance plate reader), and a standard curve was generated using the readings from the calibrators included on each plate and used to calculate the individual titers (IU/mL). To validate each assay, we considered only calibration curves for each plate with a coefficient of determination (r²) above 0.95. Calibration curves that had an r² below 0.95 were repeated. The manufacturer provided the intra-assay and inter-assay coefficient of variation (CV%) as 6.9 and 10.4, respectively. Samples on each plate were run in duplicate and averaged. Previously tested positive samples were incorporated into subsequent runs as in-house controls.

Human IgG antibodies against TT in plasma were measured by enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s protocol (TECAN, IBL International GmbH) on samples obtained from infants at 1 and 15 weeks and a subset of mothers from the Nigerian site at 1 week postpartum. The optical density at 450 nm was measured by an ELISA microplate reader (BioTek ELx808 absorbance plate reader), and a standard curve was generated using the readings from the calibrators included on each plate and used to calculate the individual titers (IU/ml). Samples on each plate were run in duplicate, and the average of both titers was used as the final titer. Previously tested positive samples were incorporated into subsequent runs as in-house controls.