hm2252  (Hycult Biotech)

Journal: Clinical and Developmental Immunology

Article Title: Soluble Triggering Receptor Expressed on Myeloid Cells 1 Is Released in Patients with Stable Chronic Obstructive Pulmonary Disease

doi: 10.1155/2007/52040

Figure Lengend Snippet: ELISA for sTREM-1. (a) Titration of recombinant human TREM-1::IgG1 either in the absence (filled symbols) or presence of normal human serum (open symbols). (b) Serial dilution of serum from a septic patient. The r 2 values are the correlation coefficients of the depicted data.

Article Snippet: For the detection of soluble TREM-1 (sTREM-1), anti-TREM-1 (6B1) mAb was coated at 0.5 μ g/mL in PBS, then blocked by addition of 100 μ L of 15% BSA for 2 hours at 37 ° C and washed.

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Recombinant, Serial Dilution

Journal: JCI Insight

Article Title: A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A*02:01 complex

doi: 10.1172/jci.insight.151624

Figure Lengend Snippet: ( A ) Binding of 6B1 to T2 cells pulsed with or without peptides. pIRS2, WT-IRS2, pCDC25b, or WT1-RMF peptide at a concentration of 20 μg/mL was pulsed onto T2 cells overnight. Cells were washed and stained with 6B1 mAb at the concentration of 3 μg/mL, followed by secondary mAb staining. The staining included secondary mAb or isotype control human IgG1. ( B ) In parallel, HLA-A2 expression was determined by staining the cells with anti–HLA-A2 mAb BB7 clone T2. Uns, unstained. ( C ) 6B1 titration was performed on T2 cells pulsed with indicated peptides and stained with indirect staining with 6B1 at concentrations ranging from 10 μg/mL to 0.1 μg/mL. ( D ) Binding kinetics of 6B1 was measured by biolayer interferometry (BLI) as indicated in Methods. The pIRS2 peptide sequence was substituted with alanine at positions 1, 2, 4, 5, 6, 7, 8, or 9 or with glycine (G3) indicated as A1–A9, and G3 and the binding of 6B1 (3 μg/mL) was determined by indirect staining and flow cytometric analysis. ( E ) T2 cells alone or pulsed with HPV peptide were the negative controls. ( F ) The same cells were simultaneously stained with anti–HLA-A2 mAb, clone BB7.2, to measure the relative binding of the peptides to HLA-A2 molecule. ( G ) Similarly, threonine substituted peptide with (pT-4) or without phosphate (WT-T4) at the position 4 was pulsed onto T2 cells and the binding of 6B1 mAb was determined by flow cytometry. ( H ) The same cells were simultaneously stained with anti–HLA-A2 mAb, clone BB7.2, to measure the relative binding of the peptides to HLA-A2 molecule.

Article Snippet: The OctetRed system (ForteBio, Pall LLC) was used to determine the binding properties of mAb 6B1.

Techniques: Binding Assay, Concentration Assay, Staining, Expressing, Titration, Sequencing, Flow Cytometry

Journal: JCI Insight

Article Title: A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A*02:01 complex

doi: 10.1172/jci.insight.151624

Figure Lengend Snippet: 6B1 recognizes arginine at position 1 and phosphate at Ser-4 in a panel of HLA-A2-bindng phosphopeptides

Article Snippet: The OctetRed system (ForteBio, Pall LLC) was used to determine the binding properties of mAb 6B1.

Techniques:

Journal: JCI Insight

Article Title: A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A*02:01 complex

doi: 10.1172/jci.insight.151624

Figure Lengend Snippet: ( A – F ) Recognition of the naturally presented pIRS2/A2 complex on the tumor cell surface by 6B1 in a pIRS2/HLA-A2–restricted manner was determined by flow cytometric analysis. Human leukemia cell lines BV173, SET2, ovarian cancer cell line SKOV-3 and Burkitt’s lymphoma cell line Jeko, and HLA-A2 – T leukemia cell line Jurkat were stained with 6B1 conjugated to APC at 10 μg/mL, followed by flow cytometric analysis. T2 cells pulse with pIRS2 was used as a positive control. Unstained cells and isotype hIgG1 were used as negative controls. Data are representative of 3 experiments. ( G – J ) Similarly, normal human cardiomyocytes, cardiac fibroblasts and thymic fibroblasts ( H – J ) and AML cell line AML-14 ( G ) were stained with 6B1 or isotype control (3 μg/mL) and followed by goat anti–human IgG Fab2 conjugated to FITC. ( K – M ) HLA-A2 expression ( K ) was simultaneously measured by anti HLA-A2 mAb (clone BB7.2) conjugated to APC. ( L and M ) Whole blood from HLA-A2 + ( L ) or HLA-A2 – ( M ) healthy donor was stained with the 6B1 or isotype control (3 µg/mL) and mAbs to CD15, CD33, and CD45RA; red blood cells were lysed; washed; and run on flow cytometry. The data represent staining from 5 separated experiments with multiple donors.

Article Snippet: The OctetRed system (ForteBio, Pall LLC) was used to determine the binding properties of mAb 6B1.

Techniques: Staining, Positive Control, Expressing, Flow Cytometry

Journal: JCI Insight

Article Title: A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A*02:01 complex

doi: 10.1172/jci.insight.151624

Figure Lengend Snippet: ( A ) Schematic of 6B1 BisAb panel. Five different BisAb formats engaging anti-CD3 mAb 2LK are shown as indicated. Binding of 6B1 BisAbs to T2 cells. ( B – E ) T2 cells ( B ) were pulsed with pIRS2 ( D ), WT-IRS2 ( E ), or HPV ( C ) peptide at a concentration of 20 μg/mL and were stained with 6B1 BisAbs at the concentration of 1 or 0.1 μg/mL, followed by secondary anti-His-tag mAb staining. The staining included secondary mAb (2Ab) or isotype control human IgG1 (hiso).

Article Snippet: The OctetRed system (ForteBio, Pall LLC) was used to determine the binding properties of mAb 6B1.

Techniques: Binding Assay, Concentration Assay, Staining

Journal: JCI Insight

Article Title: A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A*02:01 complex

doi: 10.1172/jci.insight.151624

Figure Lengend Snippet: ( A – D ) T2 cells alone ( A ) or pulsed with pIRS2 ( B ), WT-IRS2 ( C ), or irrelevant EW ( D ) peptides at 50 μg/mL were incubated with PBMCs at an E: T of 30:1 and mAbs at indicated concentrations, and the cytotoxicity was measured by a 5-hour 51 Cr-release assay. Each data point was the average of triplicate cultures ± SD; data are representative of 2 similar experiments. ( E – H ) Similarly, T cell–mediated cytotoxicity against tumor cell lines MDA-MB-231 ( E ), SKOV-3 ( F ), TPC-1 ( G ), or A375 ( H ) was measure by LDH-release assay using EBV-T cells at an E:T ratio of 20:1, with BisAbs at the indicated concentrations. Each data point was the average of triplicate cultures ± SD; data are representative of 3 similar experiments. ( I and J ) AML PDXs were stained with mAbs to CD33 and HLA-A2. The cells were labeled with CFSE and were incubated with activated T cells at an E:T ratio of 6:1, in the presence of 6B1 (1+1) or isotype control at 10 μg/mL. After overnight culture, cells were harvested, washed, and stained with mAb to CD33. Percentage reduction of total CFSE + cells was determined as killing of the cells. ( K ) The percentage lysis of 6B1 (1+1) group was plotted over isotype control.

Article Snippet: The OctetRed system (ForteBio, Pall LLC) was used to determine the binding properties of mAb 6B1.

Techniques: Incubation, Release Assay, Lactate Dehydrogenase Assay, Staining, Labeling, Lysis

Journal: JCI Insight

Article Title: A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2/HLA-A*02:01 complex

doi: 10.1172/jci.insight.151624

Figure Lengend Snippet: ( A ) Linear regression of binding interface energy in Rosetta energy units (REU) with in vitro binding of 6B1 to the indicated phosphopeptide complexes. Regression line passes through mean of top 10 scoring models for each phosphopeptide, shaded regions represent 95% CI, and R value represents Pearson correlation coefficient and respective 2-tailed P value. ( B ) Representative model of 6B1 in complex with pIRS2/HLA-A2. 6B1 VH, VL, HLA-A2, and phosphopeptide shown in dark green, light green, mauve, and orange, respectively. Phosphoserine residue is colored red. ( C ) 6B1-pIRS2/HLA-A2 complex at different angles showing contacts of CDRH3 tyrosines (green) in contact with phosphopeptide (orange), with phosphoserine (SEP) distinguished in red. Phosphopeptide residues are labeled at their approximate locations. ( D ) 6B1-pKMD/HLA-A2 complex viewed at the same angles as in C .

Article Snippet: The OctetRed system (ForteBio, Pall LLC) was used to determine the binding properties of mAb 6B1.

Techniques: Binding Assay, In Vitro, Labeling