The TCR cloning and expression protocol10 (link) is described in the Supplementary Information . We used TCR-deficient Jurkat cells (JurkatΔαβ)35 (link) transduced to express CD4 and CD8 as reporter cells. To assess antigen specificity of the cloned TCRs, we pulsed dendritic cells derived from patient PBMCs with candidate peptides (10 μg ml−1 unless specified otherwise) for 2 h in complete RPMI. Reporter cells stably expressing TCR were co-cultured in 96-well round bottom plate with pulsed dendritic cells, in a ratio of 20:1 (0.5 × 106 Jurkat-expressing TCR cells to 2.5 × 104 dendritic cells) in complete RPMI. After 24 h at 37 °C and 5% CO2, the supernatant was collected and diluted 1:2 with ELISA Assay Diluent (Biolegend) and IL-2 production was measured using the Human IL-2 ELISA MAX Standard Kit (Biolegend) according to the manufacturer’s instructions. OVA peptide was used as a negative control and PMA/ionomycin was used as a positive control.
Human il 2 elisa max standard kit
Manufactured by BioLegend
The Human IL-2 ELISA MAX Standard Kit is a laboratory equipment product designed for the quantitative measurement of human interleukin-2 (IL-2) in biological samples. It employs the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify the presence of IL-2 in the sample.
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Lab products found in correlation
2 protocols using human il 2 elisa max standard kit
1
TCR Cloning and Antigen Specificity Assay
2
Antibody Production and Immune Modulation
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Example 11
The four expression vectors, pHuPRO2-IgG1.AA, pHuPRO5-IgG1.AA, pBS839 and pBS841, are stably transfected into CHO-K1 as described above. HuPRO2-IgG1AA, HuPRO5-IgG1.AA, BS839 and BS841 are purified from culture supernatants of their respective CHO-K1 transfectants with a protein A affinity column as described above. SDS-PAGE analysis under reducing conditions is carried out to examine the size of heavy and light chains of these four antibodies.
The biological activity of BS839 and BS841 to enhance immune responses is analyzed by measuring the expression level of IL-2 in human T cells. Human PBMC are grown in RPMI-1640 containing 10% FBS and 10 μg/ml of PHA-L for three days (3-day old PBMC) as described above. Approximately one hundred thousand cells of the 3-day old PBMC are incubated in a well of a 96-well plate, which is precoated with 1 μg/ml of mouse anti-human CD3 monoclonal antibody OKT3, in the presence of (i) no additional antibody, (ii) 1 μg/ml of HuPRO2-IgG1.AA, (iii) 1 μg/ml of HuPRO5-IgG1.AA, (iv) 1 μg/ml of HuOHX14DS-IgG1.AA, (v) 1 μg/ml of HuPRO2-IgG1.AA and HuOHX14DS-IgG1.AA, (vi) 1 μg/ml of HuPRO5-IgG1.AA and HuOHX14DS-IgG1.AA, (vii) 1 μg/ml of BS841, and (viii) 1 μg/ml of BS839 for one day at 37° C. in a 7.5% CO2 incubator. IL-2 concentration in culture supernatants is measured using the Human IL-2 ELISA MAX Standard Kit (BioLegend).
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