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About eight to ten-week-old Trianni mice (HHKK) (Trianni, San Francisco, USA) (2018)." href="/articles/s42003-022-04129-7#ref-CR42" id="ref-link-section-d21906725e2166"42 were used in all studies. All experiments were approved and conducted according to the guidelines of Sanofi Genzyme Institutional Animal Care and Use Committee. Mice were immunized with 50 µg of antigen in adjuvant (Sigma, catalog no. S6322) by intraperitoneal injection, and booster immunizations were performed for four to six times in two-week intervals. Sera were collected after third and last immunization to assess the antibody titers. The mice were rested for three weeks and boosted three days before harvesting secondary lymphoid tissues for processing B cells for antigen selection./p>95% on the day of transfection. Cell suspension (0.5 ml) was prepared to a final density of 3 × 106 cells/ml with Fresh Expi293 media into each well of 96-deep well culture plate (Fisher Scientific, catalog no. 07-000-873). Paired IgG1 heavy and kappa chain linear expression cassettes (LECs) were mixed (500 ng each) in 50 µl of Opti-MEM I medium. Separately, 5 µl ExpiFectamine 293 Reagent (Fisher Scientific, catalog no. A14524) was diluted in 50 µl of Opti-MEM (Fisher Scientific, catalog no. 31985062). The diluted ExpiFectamine 293 Reagent was then mixed with the DNA mix and incubated for 20 min at room temperature. This transfection mix was added to the Expi293F cell suspension in each well of the culture plate and mixed gently. The culture plate was sealed with porous Aeraseal film (Sigma Aldrich, catalog no. A9224-50EA) and incubated at 37 °C, 8% CO2, 900 rpm in a humidified (≥80%) incubator. After 18 h, a cocktail of Enhancer I and II (comes with the ExpiFectamine 293 Reagent kit; 5 µl of Enhancer 1 and 50 µl of Enhancer 2) was added to each well. The cells were incubated for another 120 h in 37 °C, 8% CO2, 900 rpm in a humidified (≥80%) incubator. Cell culture supernatant was harvested by centrifuging the culture plates at 1000 g for 30 min and the supernatants were transferred to Matrix 96-well storage tubes (Fisher Scientific, catalog no. 50-823-846). Antibody concentration in the culture supernatant was measured by Octet (ForteBio) and specific binding to the target antigen was tested by ELISA./p>

Binding with the target antigen was also tested through a cell-based assay, wherein, phage supernatants (50 µl) from individual colonies as used for ELISA, were incubated with wild type CHO cells and CHO cells expressing antigen D on the cell surface (30,000 cells/well) for 1 hr at RT. Cells were then washed three times with PBS-BSA-1% and further incubated with 50 µl/well of pre-conjugated mouse anti-M13 antibody (1:500 dilution) and Alexa-647 labelled anti-mouse antibody (1:200 dilution) (Invitrogen, catalog no. A28181) in dark at RT for 1 hr. Cells were then washed thrice with PBS-BSA-1%, suspended in 100 µl of PBS-BSA-1% and acquired in an iQue flow cytometry system (Sartorius) to evaluate binding of individual samples. The data was analyzed using Forcyte software (2020)." href="/articles/s42003-022-04129-7#ref-CR43" id="ref-link-section-d21906725e3135"43. Irrelevant Fab expressing negative control phage particles were also used as controls./p> (August 17, 2020)./p> (2018)./p> (2020)./p>