Clone:
REA675
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC, MICS, IF, IHC, MC
Alternative names:
B4, Leu-12

Extended validation for CD19 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 REA675
SJ25C1++
HIB19++
LT19++
J3-119++
Cells were incubated with an excess of purified unconjugated CD19 (REA675) 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 CD19 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD19-PE (REA675, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Fluorescence microscopy image of CD19 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD19-PE (REA675, red) and counterstained with DRAQ5 (blue) as DNA stain.
Fluorescence microscopy image of CD19 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD19-PE (REA675, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Overlay histogram showing flow cytometric analysis of CD19 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD19-PE, clone (REA675). 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 CD19 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD19-PE, clone (REA675). 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 CD19. Human peripheral blood mononuclear cells (PBMCs) were stained with CD19 antibodies and with a suitable counterstaining. As a control, CD19 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 CD19. Human peripheral blood mononuclear cells (PBMCs) were stained with CD19 antibodies and with a suitable counterstaining. As a control, CD19 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 CD19. Human peripheral blood mononuclear cells (PBMCs) were stained with CD19 antibodies and with a suitable counterstaining. As a control, CD19 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 CD19. Human peripheral blood mononuclear cells (PBMCs) were stained with CD19 antibodies and with a suitable counterstaining. As a control, CD19 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 CD19. Human peripheral blood mononuclear cells (PBMCs) were stained with CD19 antibodies and with a suitable counterstaining. As a control, CD19 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 CD19. Human peripheral blood mononuclear cells (PBMCs) were stained with CD19 antibodies and with a suitable counterstaining. As a control, CD19 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 CD19 (REA675). 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 CD19 (REA675). 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 CD19 (REA675). 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 CD19 Antibody, anti-human, REAfinity™

Overview

REA675 recognizes the human CD19 antigen, a type I transmembrane glycoprotein of 95 kDa that belongs to the immunglobulin superfamily. CD19 is expressed on B cells throughout most stages of B cell differentiation, though its expression is down-regulated during their terminal differentiation to plasma cells. Expression of CD19 is also found in the majority of B cell–derived malignancies. CD19 is further present on follicular dendritic cells. On B cells, CD19 associates with CD21, CD81, and CD225 (Leu-13) forming a signal transduction complex.
Additional information: Clone REA675 displays negligible binding to Fc receptors.

Alternative names

B4, Leu-12

Detailed product information

Technical specifications

CloneREA675
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman
AntigenCD19
Alternative names of antigenB4, Leu-12
Distribution of antigenB cells, dendritic cells
RRIDAB_2726198, AB_2726476, AB_2726199, AB_2726472, AB_2726195, AB_2784031, AB_2784030, AB_2726479, AB_2726202, AB_2751240, AB_2751237, AB_2726477, AB_2726200, AB_2726473, AB_2726196, AB_2726478, AB_2726201, AB_2726480, AB_2726203, AB_2726474, AB_2726197, AB_2801882, AB_2726475

References for CD19 Antibody, anti-human, REAfinity™

Publications

  1. Nadler, L. M. et al. (1983) B4, a human B lymphocyte-associated antigen expressed on normal, mitogen-activated, and malignant B lymphocytes. J. Immunol. 131(1): 244-250
  2. Fujimoto, M. et al. (2000) CD19 regulates intrinsic B lymphocyte signal transduction and activation through a novel mechanism of processive amplification. Immunol. Res. 22(2-3): 281-298
  3. Poe, J. C. et al. (2001) CD19, CD21, and CD22: multifaceted response regulators of B lymphocyte signal transduction. Int. Rev. Immunol. 20(6): 739-762
  4. Tedder, T. F. et al. (2002) CD19-CD21 complex regulates an intrinsic Src family kinase amplification loop that links innate immunity with B-lymphocyte intracellular calcium responses. Biochem. Soc. Trans. 30(4): 807-811

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