mouse ifn-γ/il- 2 double- color enzymatic elispot assay kit  (Cellular Technology Ltd)

Journal: Molecular Therapy Oncology

Article Title: Preclinical development and clinical safety assessment of a synthetic peptide conjugate enabling endogenous antibody binding to promote innate receptor engagement

doi: 10.1016/j.omton.2025.200954

Figure Lengend Snippet: IFNγ and TNFα-release by memory T cells in response to LUR1-6 conjugates Peptides and LUR1-6 conjugates were incubated in human whole blood from prostate cancer patients and healthy donors, pre and post a DTP vaccination, (A) and (B), or with and without a mouse anti-MTTE IgG2a antibody, (C) and (D), in a circulating blood loop assay. T cell surface markers and intracellular IFN-γ and TNF-α were analyzed with flow cytometry. The conjugates tested were LUR1-6, LUG1-6 (see ), and SLP1-6, which are the free synthetic long peptides of respective conjugates. The [MTTE] 3 -CMV conjugate contains the HLA-A∗0201-restricted epitope pp65(NLV) from CMV and CMV lysates were used as positive controls. MTTE3-irrelevant (MTTE-irrel.) contains a scrambled SLP sequence (DGLQGLLLGLRQRIETLEGK) without any know human T cell epitopes. Representative flow cytometry dot plots of the three responding donors from (A) and (C) are displayed in (B) and (D). The cells were gated as CD45RO + CD3+CD4-CD8+ and the % of IFN-γ+ and TNF-α+ cells are displayed. (E) SLP-specific IFN-γ producing T cells upon LUG2 vaccination in seropositive HLA-DR4 humanized transgenic mice were measured in a recall-ELISpot assay. Result shown as mean ± SD. Significance was tested with Mann Whitney-test ∗ p < 0.05.

Article Snippet: The immunogenicity of the SLP contained in LUG2 construct with and without the TAP sequence: ARWWLLHETDSAVAAARQIYVAAFTVQAAAE (UV02) and LLHETDSAVAAARQIYVAAFTVQAAAE (UV08) were determined using the splenocytes harvested from immunized mice in an ex vivo IFN-γ ELISpot assay (ELISpot kit for mouse IFN-γ/3321-2A, Mabtech, Stockholm, Sweden) according to the instructions from the manufacturer.

Techniques: Incubation, Flow Cytometry, Sequencing, Transgenic Assay, Enzyme-linked Immunospot, MANN-WHITNEY