CD19 Antibody, anti-human, REAfinity™

Name: CD19 Antibody, anti-human, REAfinity™
Availability: InStock

CD19 Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA675

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC, MC

Alternative names:

B4, Leu-12

Flow cytometry applications forCD19 Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 615
PE-Vio 770
PerCP-Vio 700
Vio Bright B515
Vio Bright R720
Vio Bright V423
VioBlue
VioGreen

Conjugate:

APC

Recommended dilution:

1:50

Remark:

Cells should be stained prior to fixation, if formaldehyde is used as a fixative.

Tested:

QC tested

Products used:

130-114-168, 130-113-642

Protocols:

Cell surface flow cytometry staining protocol (PBS/EDTA/BSA 1:50)

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with CD19 antibodies or with the corresponding REA Control (S) antibodies (left images) as well as with CD20 antibodies. Flow cytometry was performed using the MACSQuant^® Analyzer. The Tandem Signal Enhancer has been used to increase binding specificity of tandem-dye–conjugated antibodies. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of tandem conjugates.

MICS (MACSima Imaging Cyclic Staining) applications forCD19 Antibody, anti-human, REAfinity™

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

human

Products used:

130-114-168, 130-113-642

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Mass cytometry applications forCD19 Antibody, anti-human, REAfinity™

pure

Conjugate:

pure

Reported:

32676612,

Products used:

130-122-301

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 clones	Overlap 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

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.

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.

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.

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.

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

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

Clone	REA675
Clonality	monoclonal
Isotype	recombinant human IgG1
Isotype control	REA Control Antibody (S), human IgG1
Host	human cell line
Type of antibody	Primary antibodies, Recombinant antibodies
Species	human
Antigen	CD19
Alternative names of antigen	B4, Leu-12
Distribution of antigen	B cells, dendritic cells
Entrez Gene ID	930
RRID	AB_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_2921775, AB_2921776, AB_2801882, AB_2904805, AB_2904804, AB_2726475

Resources for CD19 Antibody, anti-human, REAfinity™

Documents and Protocols

Scientific posters

Increased sensitivity of flow cytometry for detection of minimal residual disease and circulating tumor cells in B Non-Hodgkin Lymphoma

Certificates

Please follow this

link

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

References for CD19 Antibody, anti-human, REAfinity™

Publications

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
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
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
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

Applications

Extended validation