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Monoclonal Antibodies

Anti-PLA2R [12-6-5]

Invented by Paul Brenchley from University of Manchester
Invented at University of Manchester
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Antigen: PLA2R N-terminus fragment (Alanine20- Proline663)
1µg of recombinant full length extracellular domain of human PLA2R (NC8) and a N-terminus fragment of PLA2R (NC3) were subjected to SDS PAGE followed by western blot using Ms anti-PLAR (Cl12-6-5) at dilution 1:10000.
Immunofluorescence image of PLA2R over-expressing podocytes revealing cell surface expression of the PLA2R receptor using Ms anti-PLAR (Cl12-6-5) at dilution 1:400 (under 40x lens).
Overlay flow cytometry histograms of wild type (dark blue line) and over-expressing PLA2R podocytes (low expressor, green line; high expressor, red line) stained with Ms Cl12-6-5) at dilution 1:200. Negative controls were unlabelled cells (turquoise line) and mouse IgG (grey line).
Normal human kidney showing glomerular specific staining. IHC Protocol Info: HIER pH8 (CC1) for 64 minutes, 36 minute primary antibody (PLA2R Mouse 12-6-5) incubation at room temperature using a dilution of 1:5000, using Ventana Ultraview DAB detection system.
Normal human kidney showing podocyte specific staining. IHC Protocol Info: HIER pH8 (CC1) for 64 minutes, 36 minute primary antibody (PLA2R Mouse 12-6-5) incubation at room temperature using a dilution of 1:5000, using Ventana Ultraview DAB detection system.
Membranous nephropathy kidney showing staining of PLA2R in the capillary wall of the glomerular basement membrane. IHC Protocol Info: HIER pH8 (CC1) for 64 minutes, 36 minute primary antibody (PLA2R Mouse 12-6-5) incubation at room temperature using a dilution of 1:5000, using Ventana Ultraview DAB detection system.
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Catalogue Number 152570
Applications FACS IHC IF WB
Antigen/Gene or Protein Targets Phospholipase A2 receptor 1
Reactivity Human
Relevance Monoclonal antibody which detects PLA2R, the major antigen for idiopathic membranous nephropathy.

Background and Research Application
PLA2R is the major auto-antigen in the autoimmune kidney disease, membranous nephropathy. Detection of PLA2R antigen by IHC in the immune complexes present in the glomerular basement membrane is used as a diagnostic marker for the disease. PLA2R is also involved in clearance followed by suppression of their potent enzymatic activities of secretory phospholipase A2 (sPLA2) from the blood, including group sPLA2-X and a snake venom sPLA2. PLA2R acts a tumour suppressor protein and it is associated with cell senescence and differentiated cells.
Host Mouse
Immunogen Human PLA2R extracellular sequence N-C3 (containing domains N-terminal Cysteine rich-FibII-CTLD1-CTLD2-CTLD3). Genbank acc no. U17033; amino acids 20-663 Recombinant protein expressed in HEK 293-EBNA-1 cells
Immunogen UniProt ID Q13018
Subclass IgG2b
Myeloma Used Sp2/0-Ag14
Notes Production Details
Purified using multi-step affinity chromatography with protein A.

Storage Conditions
Store at -20 degrees frozen. Avoid repeated freeze/thaw cycles.

Points of Interest
Recommended dilution:
WB: 1:5000-1:10000
FC: 1:200
IF: 1:400
IHC: 1:5000-1:20000

Affinity:
KD 5.4x10-10 M (determined by surface plasmon resonance between recombinant human PLA2R and increasing concentration of Ms Clone 12-6-5 antibody).

IHC protocol

System: Automated Ventana Benchmark ULTRA
Detection Kit: UltraView DAB Detection System
Antigen Retrieval: 64 minute heat in CC1 (Tris based pH8.4 buffer)
Antibody concentration: 1+5000
Antibody Incubation: 36 minute at room temperature.
Counterstain: Haematoxylin 12 min, Bluing Reagent 4 min.
Positive Tissue Control: Normal kidney

Extended information including detection system reagents: The automated Ventana BenchMark ULTRA IHC ⁄ ISH Staining Module (Ventana Co., Tucson, AZ, USA) was used together with the Ultraview 3, 3’ diaminobenzidine (DAB) version 3 detection system (Ventana Co.). Tissue sections (4 µm) were deparaffinized and incubated in EZPrep Volume Adjust (Ventana Co.). At intervals between steps the slides were washed with a TRIS-based Reaction Buffer, pH 7.6. A heat-induced antigen retrieval protocol set for 64 min was carried out using a TRIS– ethylenediamine tetracetic acid (EDTA)–boric acid pH 8.4 buffer (Cell Conditioner 1). The sections were incubated with ultraviolet inhibitor blocking solution for 4 min, then with antibody to PLA2R for a set time of 36 min at room temperature. This was followed by incubation with horseradish peroxidase-linked secondary antibody (8 min.), followed by DAB chromogen and substrate (8 min.), and copper enhancer for 4 min. Counterstain (haematoxylin II) was applied for 12 min before an incubation of 4 min with bluing reagent.

Concentration
1mg/ml as standard
Research Area Cardiovascular, Cell Signaling & Signal Transduction, Metabolism

References

There are 3 reference entries for this reagent.

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References: 3 entries

Fresquet et al. 2017. Sci Rep. 7(1):6876. PMID: 28761153.

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PLA2R binds to the annexin A2-S100A10 complex in human podocytes.

Europe PMC ID: 28761153

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Obrisca et al. 2015. Biomed Res Int. 2015:249740. PMID: 26576418.

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References: 3 entries

Fresquet et al. 2017. Sci Rep. 7(1):6876. PMID: 28761153.

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PLA2R binds to the annexin A2-S100A10 complex in human podocytes.

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Obrisca et al. 2015. Biomed Res Int. 2015:249740. PMID: 26576418.

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Inventor Information