Hybridomas were generated using a standard technique (Köhler and Milstein, 1975 (
link)). Briefly, the spleen was excised and processed into a single-cell suspension, followed by lysis of erythrocytes with
1X red blood cell lysis buffer (Invitrogen, Waltham, MA, United States). The remaining 4 × 10
7 splenocytes were fused with 8 × 10
7 murine myeloma cells (P3X63Ag8.653) using 1 ml of 50% polyethylene glycol solution (Sigma-Aldrich, St. Louis, MO, United States). Fused cells were resuspended in 20% fetal bovine serum complete Dulbecco’s modified Eagle medium with hypoxanthine-aminopterin-thymidine selection supplement (Sigma-Aldrich, St. Louis, MO, United States) and plated in 96-well plates at 5 × 10
4 splenocytes per well. Plates were incubated for 10 days at 37°C in a 5% CO
2 incubator. Culture supernatant was tested for antibodies to MCF by indirect ELISA. Cells positive by ELISA were subcloned by limiting dilution and screened using the same method after 10 days. Positive subclones were expanded and supernatant was purified by
protein A/G chromatography (Thermo Scientific, Waltham, MA, United States). Purified mAbs were evaluated for specificity by Western blotting as described below. One mAb, 4H2, bound to a known antigen in MCF, chitinase 1 (CTS1), and was pursued for gene rescue analyses.
Jugler C., Grill F.J., Eidenberger L., Karr T.L., Grys T.E., Steinkellner H., Lake D.F, & Chen Q. (2022). Humanization and expression of IgG and IgM antibodies in plants as potential diagnostic reagents for Valley Fever. Frontiers in Plant Science, 13, 925008.
