Mouse monoclonal igg

Freshly cut TMA sections were immuno-stained on one day and in one experiment. Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121 °C in pH 2.0, in target retrieval solution (Biogenex, San Ramon, CA, USA). Primary antibodies specific for Tn antigen/CD175 (mouse monoconal IgG, dilution 1:150, Thermo Fisher Scientific, Waltham, MA, USA, cat. number MA1-80055) and STn antigen/CD175s (mouse monoclonal IgG, dilution 1:150, Thermo Fisher Scientific, Waltham, MA, USA, cat. number MA1-90577) was applied at 37 °C and pH 7.8 for 60 min. Bound antibody was then visualized using the EnVision Kit (Dako, Glostrup, Denmark) according to the manufacturer’s directions. CD175 and CD175s staining was found in the cytoplasm and the apical membrane of positive cells. Tumors with complete absence of staining were scored as “negative”. Tumor samples with ≥10% positivity, and with membranous accentuation, displaying a homogenous staining of the complete core were accordingly scored as “positive”.

Saccharomyces cerevisiae yeast display strain EBY100 (Leu-, Try-phenotype) has genomic insertion of a GAL1-AGA1: URA3 ura3-52trp1 leu2Δ1 his Δ 200 prp4:HIS2prb1Δ1.6R can1GAL was maintained in YPD medium [25 (link)]. The yeast cells (EBY100) harboring display vector pDNL-6 [24 ] were grown in SD-CAA minus Trp and Ura medium. For yeast display, the scFv containing yeast cells were grown for 20 h at 18–20 °C in SG/R-CAA medium (identical to SD-CAA medium except glucose was replaced by galactose and raffinose) supplemented with antibiotics [26 (link)]. The bacterial strain DH5α E. coli DH5α E. coli (F-80dlacZ M15 (lacZYA-argF) U169 recA1 endA1hsdR17 (rk−, mk+) phoAsupE44 -thi-1 gyrA96 relA1) was used for cloning and propagation of plasmid DNA. The bacterial strain BL21 (DE3) F⁻ompThsdS_B (r_B⁻m_B⁻) gal dcm (DE3) was used for the expression of soluble scFv protein under the T7 promoter. The displayed scFv protein was detected by flow cytometry (FACS), using mouse monoclonal IgG that recognizes the c myc tag (9E10) (Thermofisher Scientific, Waltham, MA, USA, CatLog: 980). Phycoerythrin-labeled-goat anti-mouse (H+L) IgG (BioLegend Way, San Diego, CA, USA, CatLog: 405307 and Alexa-633 streptavidin (Thermofisher Scientific, Waltham, MA, USA, CatLog: S21375) were also employed in the FACS analysis.