Journal: Nature Communications

Article Title: Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment

doi: 10.1038/ncomms11804

Figure Lengend Snippet: ( a ) ELISA results from twenty independent hybridomas presented as per cent maximum absorbance compared to anti-IA g7 mouse hybridoma clone 10–2.16 as positive control. Plates were coated with p63:IA g7 monomer and compared to InsB 9–23 :IA g7 monomer-coated plates. Supernatant was added and secondary antibody was used to measure binding by ELISA. Media alone was used for a negative control (dashed line). ( b ) CFSE proliferation assay in response to p63 peptide was performed using splenocytes from BDC2.5 mice. The per cent of divided CD4 + T cells is shown with maximum division with peptide alone (87.5%) and inhibition with each hybridoma supernatant screened. Data are representative from two independent experiments. ( c ) CFSE-labelled BDC2.5 T cells were cultured for 4 days in the presence of p63 or p31 peptide with varied concentrations of FS1 MAb or isotype control IgG1 antibody (1.72 μM). The per cent divided CD4 + T cells is shown for each concentration of blocking FS1 MAb compared with maximum proliferation with no antibody (no Ab) or isotype Ab. FS1 MAb effects on IFNγ cytokine production from the cultured cells (middle panel) and IL-17A cytokine production (bottom panel). Data are representative from two independent experiments in duplicate for each antigen concentration. ( d ) In vitro antibody staining on antigen-presenting cells following peptide pulse with p63 or OVA 141–160 using purified clone FS1 MAb to detect p63 loaded cDCs (CD8α + CD11c + MHCII + , CD3e − , F4/80 − ), and B cells (B220 + ,MHCII + ,CD11c − ,CD3ɛ − ,F4/80 − ) but not CD4 + or CD8 + T cells (CD3ɛ + , CD11c − , CD11b − , B220 − , F4/80 − ) compared with no p63 peptide negative control. Data are representative from three independent experiments. ( e ) In vivo staining of antigen-presenting cells with FS1 MAb following footpad immunization. NOD mice received p63 or OVA 141–160 peptide and 1.5 h following injection the popliteal lymph node was collected and stained for antigen-specific presentation using biotinylated FS1 MAb. FS1 MAb staining was detected on DCs and B cells, but not T cells using fluorochrome-linked streptavidin. Statistical significance was calculated using a two-tailed Student's t -test. Data are representative from two independent experiments with 2–4 mice per group.

Article Snippet: Peptides used for in vivo immunization and in vitro stimulation, and peptide pulsing include p63 (RTRPLWVRME), p31 (YVRPLWVRME), OVA 141–160 (CARELINSWVESQTNGIIRN) (Genemed Synthesis), 2W (EAWGALANWAVDSA) (Genscript).

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control, Binding Assay, Negative Control, Proliferation Assay, Inhibition, Cell Culture, Concentration Assay, Blocking Assay, In Vitro, Staining, Purification, In Vivo, Injection, Two Tailed Test