cd160 by55  (Miltenyi Biotec)

Journal: Scientific Reports

Article Title: A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

doi: 10.1038/s41598-020-76069-3

Figure Lengend Snippet: Biopanning of the scFv phage display library on Streptactin magnetic beads. ( a ) Schematic representation of the biopanning process employing the StreptagII-Streptactin system. CD160 fused with Twin streptag was capture directly from cell supernatant on the surface of Streptactin magnetic beads. Phages were incubated with the antigen’ coated beads and bound phages were subsequently recovered by eluting with a 30 mM biotin solution and used to amplify the library for the next biopanning round. ( b ) The process of coating and elution of the beads throughout the biopanning process was assessed by flow cytometry analysis. These were stained during the several steps of the procedure (coating, blocking with 3%BSA and elution) using commercial anti CD160-PE. ( c ) Enrichment of the phage library for CD160 was determined after three rounds of biopanning on beads. The bacterial library from the different rounds of selection was induced to express the soluble scFv fragment in the supernatant. This was used to stain SupT1 cells overexpressing human CD160, due to the presence of a myc-tag at the C-terminus of productive scFvs we were able to detect binding using anti myc antibody. 2xTY media + anti myc-tag-DL549 and the commercial anti-CD160-PE Ab were used as controls. ( d ) The 15 individual bacterial colonies identified in the third round of biopanning carrying unique combination of CDR1,2 and 3 were screened by against SupT1 CD160 positive and negative cells (NT).

Article Snippet: Anti CD160 antibody (BY55-PE, BD Pharmigen) was used as positive control and either induced bacteria supernatant from previous round of biopanning or 2xTY medium as negative control for the staining.

Techniques: Magnetic Beads, Incubation, Flow Cytometry, Staining, Blocking Assay, Selection, Binding Assay

Journal: Scientific Reports

Article Title: A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

doi: 10.1038/s41598-020-76069-3

Figure Lengend Snippet: Characterization of the monoclonal scFv identified in the biopanning. ( a ) Alignment to the IMGT database of Rattus norvegicus germline variable sequence showed rearrange sequences originated from family 2 and 5 of the VH genes. We identified five different HCDR3 in which three were represented in multiple clones and three occurring only once. ( b ) Flow cytometry analysis of the five selected HCDR3 expressed secreted as chimeric scFv fused with mIgG2aFc. Supernatant from transfected HEK293T cells was used to stain SupT1 negative and SupT1 CD160 positive cells; the binding was detected with a secondary anti-mouseIgGFc-PE and anti CD160-PE (BY55) was used as positive control. ( c ) The affinity of these five scFv was determined using Biacore T200 technology. Individual scFvs were captured on a CM5 chip and CD160 was injected as analyte at 5 different concentration (3.70 nM, 11.11 nM, 33.33, nM, 100 nM and 300 nM) in a single-cycle kinetic study. The double-reference subtracted sensorgrams fitted with the 1:1 Langmuir binding model; kinetic and model fitting data are reported in the table.

Article Snippet: Anti CD160 antibody (BY55-PE, BD Pharmigen) was used as positive control and either induced bacteria supernatant from previous round of biopanning or 2xTY medium as negative control for the staining.

Techniques: Sequencing, Clone Assay, Flow Cytometry, Transfection, Staining, Binding Assay, Positive Control, Injection, Concentration Assay

Journal: Scientific Reports

Article Title: A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

doi: 10.1038/s41598-020-76069-3

Figure Lengend Snippet: Functional assays as chimeric antigen receptor. Four different healthy donors PBMCs were transduced with lentivirus to express the five different scFvs fused in a third generation (CD28-OX40ζ) CAR structure and tested in a cytotoxic assay. T cells were incubated with SupT1 CD160 positive and negative cells (NT) at different effector to target ratio (1:2, 1:4 and 1:8). The residual number of target cells for each donor was normalized against non-engineered PBMCs and all the five CARs demonstrated excellent killing at both 24 h and 72 h ( a ). Total cytokine production was assessed at 72 h and showed high levels of interferon-gamma (IFN-γ) and IL-2 ( b ) with no background on target-negative cells. The mean from each donor was plotted individually and analysed using two-way ANOVA with Dunnett’s post-test for comparison between CARs within the same target group. ***p < 0.0001.

Article Snippet: Anti CD160 antibody (BY55-PE, BD Pharmigen) was used as positive control and either induced bacteria supernatant from previous round of biopanning or 2xTY medium as negative control for the staining.

Techniques: Functional Assay, Transduction, Incubation