Clone:
REA661
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC
Alternative names:
B7, B7-1, BB1

Extended validation for CD80 Antibody, anti-human, REAfinity™

Specificity

Epitope competition
In order to compare the epitope specificity of an antibody, the clone being used is compared with other known clones recognizing the same antigen in a competition assay.
Other clonesOverlap in epitope recognition with REA661
2D10++
QA18A16++
W17149D++
L307.4+
Cells were incubated with an excess of purified unconjugated CD80 (REA661) antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker. Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.
Knockout validation
To ensure antibody specificity, the target gene is knocked out in a suitable cell line using the CRISPR/Cas9 system and the knockout is confirmed by sequencing of the target locus. The antibody is considered to bind specifically to the intended epitope if no antibody binding to the knockout cells can be detected. The antibody staining is controlled by fluorescence microscopy and/or flow cytometry.
WT
KO
View details
Fluorescence microscopy image of CD80 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD80-PE (REA661, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Fluorescence microscopy image of CD80 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD80-PE (REA661, red) and counterstained with DRAQ5 (blue) as DNA stain.
Fluorescence microscopy image of CD80 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD80-PE (REA661, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Overlay histogram showing flow cytometric analysis of CD80 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD80-PE, clone (REA661). Flow cytometry was performed with the MACSQuant
®
Analyzer. Cell debris, dead cells and cell doublets were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
Overlay histogram showing flow cytometric analysis of CD80 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD80-PE, clone (REA661). Flow cytometry was performed with the MACSQuant
®
Analyzer. Cell debris, dead cells and cell doublets were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Sensitivity

Performance comparison
Selected fluorochrome conjugated antibodies from Miltenyi Biotec were compared to commercially available hybridoma clones in flow cytometry analysis.
View details
Flow cytometric comparison of different clones for CD80. Peritoneal macrophages from C57BL/6 mice were stained with CD80 antibodies and with a suitable counterstaining. As a control, CD80 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for CD80. Peritoneal macrophages from C57BL/6 mice were stained with CD80 antibodies and with a suitable counterstaining. As a control, CD80 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for CD80. Peritoneal macrophages from C57BL/6 mice were stained with CD80 antibodies and with a suitable counterstaining. As a control, CD80 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for CD80. Peritoneal macrophages from C57BL/6 mice were stained with CD80 antibodies and with a suitable counterstaining. As a control, CD80 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for CD80. Peritoneal macrophages from C57BL/6 mice were stained with CD80 antibodies and with a suitable counterstaining. As a control, CD80 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
Flow cytometric comparison of different clones for CD80. Peritoneal macrophages from C57BL/6 mice were stained with CD80 antibodies and with a suitable counterstaining. As a control, CD80 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
Fixation data
To provide an indication on how an antibody performs after fixation of cells, in-house data on staining results before and after fixation with 3.7% formaldehyde using Miltenyi Biotec antibodies are provided. Different experimental settings may lead to different results.
No fixation
Post fixation
View details
Comparison of staining pattern on non-fixed and fixed cells using CD80 (REA661). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.
View details
Comparison of staining pattern on non-fixed and fixed cells using CD80 (REA661). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.
Comparison of staining pattern on non-fixed and fixed cells using CD80 (REA661). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.

Specifications for CD80 Antibody, anti-human, REAfinity™

Overview

Clone REA661 recognizes the human CD80 antigen, also known as B7-1. CD80 is a 262 amino acid long 60 kDa molecule and a member of the immunoglobulin superfamily. Together with CD86 (B7-2) it belongs to the B7 family of costimulatory molecules. CD80 is expressed on activated B cells, dendritic cells, and monocytes/ macrophages. The interaction of CD80 with its ligands CD28 and CD152 (CTLA-4) plays a critical role in induction and regulation of immune responses. Binding of CD80 to CD28, which is constitutively expressed on T cells, provides a costimulatory signal and induces T cell proliferation and cytokine production. In contrast, binding to CTLA-4 strongly inhibits proliferation and IL-2 secretion by T cells.
Additional information: Clone REA661 displays negligible binding to Fc receptors.

Alternative names

B7, B7-1, BB1

Detailed product information

Technical specifications

CloneREA661
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman, non-human primate
Cross-reactivity
rhesus monkey (
Macaca mulatta
)
AntigenCD80
Alternative names of antigenB7, B7-1, BB1
Molecular mass of antigen [kDa]29
Distribution of antigenT cells, B cells, dendritic cells, Langerhans cells, macrophages, monocytes
Entrez Gene ID941
RRIDAB_2751414, AB_2802032, AB_2802016, AB_2659256, AB_2659257, AB_2751432

Resources for CD80 Antibody, anti-human, REAfinity™

Certificates

Please follow this
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to search for Certificates of Analysis (CoA) by lot number.

Reviews for CD80 Antibody, anti-human, REAfinity™

Staining CD80 in THP1 Cells

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CD80-Biotin, human (130-110-269)

The aim was to detect CD80 on PMA activated THP1 cells. This antibody did its work.

References for CD80 Antibody, anti-human, REAfinity™

Publications

  1. Freeman, G. J. et al. (1989) B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells. J. Immunol. 143(8): 2714-2722
  2. Lanier, L. L. et al. (1995) CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL. J. Immunol. 154(1): 97-105
  3. Krummel, M. F. and Allison, J. P. (1995) CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182(2): 459-465
  4. Linsley, P. S. et al. (1993) The role of the CD28 receptor during T cell responses to antigen. Annu. Rev. Immunol. 11: 191-212

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