biotinylated mouse anti human ifn  (Mabtech Inc)

Journal: PLoS ONE

Article Title: Impairment of IFN-Gamma Response to Synthetic Peptides of Mycobacterium tuberculosis in a 7-Day Whole Blood Assay

doi: 10.1371/journal.pone.0071351

Figure Lengend Snippet: The solid line represents the median. IFN-gamma response to peptide pools (covering ESAT-6, CFP-10, and Rv2654) and recombinant ESAT-6 and CFP-10 in the 7 day WBA (n = 29, Panel B). “PP” indicates peptide pool and “r” indicates the recombinant proteins. Recombinant proteins induced significantly higher responses than the peptide pools (p<0.0001 for both ESAT-6 and CFP-10). IFN-gamma concentrations are shown as pg/ml. Lines indicate median response.

Article Snippet: Detection of IFN-gamma by ELISA was performed as previously described , using the mouse anti-human IFN-gamma monoclonal capture antibody (BD Pharmingen 551221) and the biotinylated mouse anti-human IFN-gamma detection antibody (BD Pharmingen 554550).

Techniques: Recombinant

Journal: PLoS ONE

Article Title: Impairment of IFN-Gamma Response to Synthetic Peptides of Mycobacterium tuberculosis in a 7-Day Whole Blood Assay

doi: 10.1371/journal.pone.0071351

Figure Lengend Snippet: Correlation between QFT-GIT and IFN-gamma response in the 7-day whole blood assay.

Article Snippet: Detection of IFN-gamma by ELISA was performed as previously described , using the mouse anti-human IFN-gamma monoclonal capture antibody (BD Pharmingen 551221) and the biotinylated mouse anti-human IFN-gamma detection antibody (BD Pharmingen 554550).

Techniques:

Journal: PLoS ONE

Article Title: Impairment of IFN-Gamma Response to Synthetic Peptides of Mycobacterium tuberculosis in a 7-Day Whole Blood Assay

doi: 10.1371/journal.pone.0071351

Figure Lengend Snippet: PBMC of two donors were set up with either peptide pool or recombinant ESAT-6 and cultured for 1,3,5,7 days. IFN-gamma was measured in the culture supernatant by ELISA. Filled symbols represent peptide stimulation, open symbols represent stimulation with recombinant protein. Donor 1 is represented by circles, and Donor 2 by squares.

Article Snippet: Detection of IFN-gamma by ELISA was performed as previously described , using the mouse anti-human IFN-gamma monoclonal capture antibody (BD Pharmingen 551221) and the biotinylated mouse anti-human IFN-gamma detection antibody (BD Pharmingen 554550).

Techniques: Recombinant, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

doi: 10.1186/1479-5876-2-9

Figure Lengend Snippet: Summary of assay conditions that were tested and selected for the autologous tumor IFN-γ ELISPOT assay

Article Snippet: 50 μl/well of 2 μg/ml mouse anti-human IFN-γ-biotinylated mAb (BD Pharmingen, San Diego, Ca) in DPBS/1%BSA/0.05% Tween was added and the plates were incubated for 2 h at room temperature.

Techniques: Enzyme-linked Immunospot, Cell Culture, Incubation

Journal: Journal of Translational Medicine

Article Title: A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

doi: 10.1186/1479-5876-2-9

Figure Lengend Snippet: Optimization of PBMC preparation for use in the tumor IFN-γ ELISPOT assay . Cryoperserved PBMC were thawed and rested in culture medium either overnight (O/N) or for 2 h and assessed for optimal reactivity to tumor cells. Pre- (white columns) and postvaccine (grey columns) PBMC (1 × 10 5 cells/well) were co-cultured with sCD40Lt activated autologous tumor cells (2 × 10 5 cells/well) for 48 h in the ELISPOT assay. Significantly (p < 0.05) more IFN-γ production by responding T cells was observed in post- compared to prevaccine PBMC when PBMC were rested overnight. Data is presented as average number of spots per well ± SD and is representative of four experiments with similar results.

Article Snippet: 50 μl/well of 2 μg/ml mouse anti-human IFN-γ-biotinylated mAb (BD Pharmingen, San Diego, Ca) in DPBS/1%BSA/0.05% Tween was added and the plates were incubated for 2 h at room temperature.

Techniques: Enzyme-linked Immunospot, Cell Culture

Journal: Journal of Translational Medicine

Article Title: A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

doi: 10.1186/1479-5876-2-9

Figure Lengend Snippet: Optimization of effector to target cell ratio for use in the tumor IFN-γ ELISPOT assay . Various concentrations of pre- (white columns) and postvaccine (grey columns) PBMC (1 × 10 5 , 2 × 10 5 or 3 × 10 5 cells/well) were co-cultured with different concentrations of sCD40Lt activated autologous tumor cells (1 × 10 5 , 2 × 10 5 or 3 × 10 5 cells/well) for 48 h in the ELISPOT assay. Cryoperserved PBMC were thawed and cultured overnight. Data for PBMC concentrations of 3 × 10 5 and 2 × 10 5 cells/well are not shown because the resulting spots were too numerous and/or diffuse to count accurately. Significantly (p < 0.01) more IFN-γ production by responding T cells was observed in post- compared to prevaccine PBMC samples regardless of the effector to target ratio. Moreover, significantly (p < 0.01) more tumor specific T cells were enumerated at the 100:200 effector to target ratio compared to the other ratios tested. Data is presented as average number of spots per well ± SD and is representative of four experiments with similar results.

Article Snippet: 50 μl/well of 2 μg/ml mouse anti-human IFN-γ-biotinylated mAb (BD Pharmingen, San Diego, Ca) in DPBS/1%BSA/0.05% Tween was added and the plates were incubated for 2 h at room temperature.

Techniques: Enzyme-linked Immunospot, Cell Culture

Journal: Journal of Translational Medicine

Article Title: A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

doi: 10.1186/1479-5876-2-9

Figure Lengend Snippet: Incubation of PBMC and tumor cells for 48 h enhanced the sensitivity of the tumor IFN-γ ELISPOT assay . Pre- (white columns) and postvaccine (grey columns) PBMC (1 × 10 5 cells/well) were co-cultured with sCD40Lt activated autologous tumor cells (2 × 10 5 cells/well) for 24, 48 and 72 h in the ELISPOT assay. Significantly (p < 0.05) more IFN-γ production by responding T cells was observed in post- compared to prevaccine PBMC samples regardless of the length of incubation. Moreover, a significantly higher number of tumor-reactive T cells were observed at the 48 h in the postvaccine PBMC (p < 0.01). Data is represented as average number of spots per well ± SE (n = 3). Data for 72 h is not shown because the resulting spots were too numerous and/or diffuse to count accurately.

Article Snippet: 50 μl/well of 2 μg/ml mouse anti-human IFN-γ-biotinylated mAb (BD Pharmingen, San Diego, Ca) in DPBS/1%BSA/0.05% Tween was added and the plates were incubated for 2 h at room temperature.

Techniques: Incubation, Enzyme-linked Immunospot, Cell Culture

Journal: Journal of Translational Medicine

Article Title: A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

doi: 10.1186/1479-5876-2-9

Figure Lengend Snippet: Medium containing HuAB serum enhanced the ability to measure tumor-reactive T cells . Pre- (white columns) and postvaccine (grey columns) PBMC (1 × 10 5 cells/well) were co-cultured with sCD40Lt activated autologous tumor cells (2 × 10 5 cells/well) for 48 h. The optimal media for the tumor IFN-γ ELISPOT assay was evaluated by comparing HuAB and FBS as the serum source. Although both HuAB and FBS containing media yielded significant (p < 0.001 and p < 0.05, respectively) differences between pre- and postvaccine T cell reactivity to tumors, medium containing HuAB yielded significantly (p < 0.05) more IFN-γ production.

Article Snippet: 50 μl/well of 2 μg/ml mouse anti-human IFN-γ-biotinylated mAb (BD Pharmingen, San Diego, Ca) in DPBS/1%BSA/0.05% Tween was added and the plates were incubated for 2 h at room temperature.

Techniques: Cell Culture, Enzyme-linked Immunospot

Journal: Journal of Translational Medicine

Article Title: A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

doi: 10.1186/1479-5876-2-9

Figure Lengend Snippet: Comparison of three different substrate detection systems for the tumor IFN-γ ELISPOT assay . The AEC, BCIP and TrueBlue™ substrate detection systems were evaluated for spot quality and signal to noise (stimulated to unstimulated) ratio using flu matrix peptide (FMP)-reactive PBMC from normal donors. PBMC (3 × 10 5 cells/well) alone and stimulated with FMP (5 μg/ml) were run in the ELISPOT assay for 24 hr. Spots were visualized using AEC, BCIP/NBT and TrueBlue™ substrate detection systems according to the manufacturer's instructions. Images from the plate scan generated by the ImmunoSpot analyzing system are shown. Data is represented as average number of spots per well ± SD.

Article Snippet: 50 μl/well of 2 μg/ml mouse anti-human IFN-γ-biotinylated mAb (BD Pharmingen, San Diego, Ca) in DPBS/1%BSA/0.05% Tween was added and the plates were incubated for 2 h at room temperature.

Techniques: Enzyme-linked Immunospot, Generated

Journal: Journal of Translational Medicine

Article Title: A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

doi: 10.1186/1479-5876-2-9

Figure Lengend Snippet: Representative wells of a tumor IFN-γ ELISPOT assay . Pre- and postvaccine PBMC (1 × 10 5 cells/well) were co-cultured with sCD40Lt activated autologous tumor cells (2 × 10 5 cells/well) for 48 h as described in Materials and Methods. Wells containing PBMC alone and tumor cells alone served as controls. Samples were tested blinded in triplicates. Images from the plate scan generated by the ImmunoSpot analyzing system are shown. Average numbers of spots per well are indicated. Data is from a representative experiment of nine with similar results.

Article Snippet: 50 μl/well of 2 μg/ml mouse anti-human IFN-γ-biotinylated mAb (BD Pharmingen, San Diego, Ca) in DPBS/1%BSA/0.05% Tween was added and the plates were incubated for 2 h at room temperature.

Techniques: Enzyme-linked Immunospot, Cell Culture, Generated

Journal: Journal of Translational Medicine

Article Title: A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

doi: 10.1186/1479-5876-2-9

Figure Lengend Snippet: Reproducibility of the tumor IFN-γ ELISPOT assay . Pre- and postvaccine PBMC from three patients (P 1–3) were co-cultured with autologous sCD40Lt activated tumor cells for 48 h as described in Materials and Methods. All samples were tested blinded in three independent experiments by three technicians. The numbers of spots per 1 × 10 5 effectors were significantly (p ≤ 0.001) higher in the postvaccine as compared to the prevaccine samples for all three patients. For each patient the data is presented as spots per 1 × 10 5 effector cells. The correlation of the results between technicians was highly significant (R 2 above 0.9 for all experiments).

Article Snippet: 50 μl/well of 2 μg/ml mouse anti-human IFN-γ-biotinylated mAb (BD Pharmingen, San Diego, Ca) in DPBS/1%BSA/0.05% Tween was added and the plates were incubated for 2 h at room temperature.

Techniques: Enzyme-linked Immunospot, Cell Culture