United Kingdom (UK)
Recombinant monoclonal antibody to HSP70. Manufactured using AbAb’s Recombinant Platform with variable regions (i.e. specificity) from the hybridoma cmhsp70.1 (C92F3B1).
UniProt Accession Number of Target Protein: P0DMV8
Alternative Name(s) of Target: HSP70-1; HSP70.1; HSPA1A; HSX70; HSP72; mHsp70; Heat shock 70 kDa protein 1A; Heat shock 70 kDa protein 1
Immunogen: The original antibody was generated by grafting the CDRs of the mouse antibody on human framework regions. The parental mouse antibody was generated by immunizing mice with an SBD-derived synthetic peptide TKDNNLLGRFELSG (TKD peptide; aa 450–463) with complete Freud’s adjuvants.
Specificity: This antibody binds a region including amino acids 473-504 (TKDNNLLGRFELSG) of the C-terminal substrate-binding domain (SBD) of membrane bound Hsp70 (mHsp70). It binds a 14-mer peptide termed”. This protein is located intracellularly and plays an important part in various cellular processes, including protection of the proteome from stress, folding and transport of newly synthesized polypeptides, activation of proteolysis of misfolded proteins and the formation and dissociation of protein complexes. It plays a pivotal role in the protein quality control system, ensuring the correct folding of proteins, the re-folding of misfolded proteins and controlling the targeting of proteins for subsequent degradation. It maintains protein homeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. Membrane bound Hsp70s is frequently over-expressed in patients affected by leukemia and has been identified as a potential target for cancer therapies.
Application Notes: The IgG1 version of this antibody binds the C-terminal substrate-binding domain (SBD) of the human membrane heat shock protein mHsp70 with an affinity of 3nM. The binding characterization of this antibody was done using ELISA. This antibody can detect aa 473–504) fused to domain III of HSA in a western blot assay (PMID: 20556545). The Fab version of this antibody binds mHsp70 with an affinity of 35 nM. The fab version can successfully identify membrane-associated Hsp70 on tumor cell lines both in immunofluorescence staining and flow cytometry (PMID: 20123884). The original mouse antibody cmHsp70.1 can selectively induce antibody-dependent cellular cytotoxicity (ADCC) of membrane Hsp70+ mouse tumor cells by unstimulated mouse spleen cells. Tumor killing could be further enhanced by activating the effector cells with TKD and IL-2 (PMID: 21187371). Cy5.5-conjugated cmHsp70.1 was capable of being endocytosed and accumulated in the tumor and was used for near-infrared fluorescence imaging of tumors. The tumor-selective internalization of mAb cmHsp70.1 might enable targeted uptake of toxins or radionuclides into Hsp70 membrane-positive tumors (PMID: 20406322). cmHsp70.1 antibody-conjugated gold nanoparticles were used in the real time imaging of small tumors (PMID: 26392771). This antibody was also used in the immunohistochemical and flow cytometric analysis of mHsp70 expressed on glioblastoma multiforme tumors (PMID: 29177668).
Antibody first published in:
Zettlitz et al. Humanization of a mouse monoclonal antibody directed against a cell surface-exposed epitope of membrane-associated heat shock protein 70 (Hsp70). Mol Biotechnol. 2010 Nov;46(3):265-78. PMID:20556545
Note on publication:
Describes the humanization of a mouse antibody against membrane-associated heat shock protein 70 (Hsp70).
Flow cytometry using anti-HSP70 antibody cmhsp70.1 (C92F3B1) (Ab03197). Paraformaldehyde fixed HeLa cells permeabilized with 0.5% Triton were stained with the anti-unknown specificity antibody (Ab00178-23.0; isotype control, black line) or the rabbit IgG version of cmhsp70.1 (C92F3B1) (Ab03197-23.0, blue line) at a dilution of 1:100 for 1h at RT. After washing, the bound antibody was detected using a goat anti-rabbit IgG AlexaFluor® 488 antibody at a dilution of 1:1000, and the cells were analyzed using a FACSCanto flow-cytometer.
Western blot using anti-HSP70 antibody cmhsp70.1 (C92F3B1) (Ab03197). HEK293 (A) (0.0001µg/ml), A431 (B) (0.0001µg/ml), HeLa (C) (0.0001µg/ml), MCF7 (D) (0.0001µg/ml), A549 (E) (0.0001µg/ml), and Jurkat (F) (0.001µg/ml) cells lysate (35µg protein in RIPA buffer) were resolved on an SDS-PAGE gel, and blots were probed with the chimeric rabbit version of cmhsp70.1 (C92F3B1) (Ab03197-23.0) at the mentioned respective concentrations before detection using an anti-rabbit secondary antibody. A primary incubation of 1 hour was used, and protein was detected by chemiluminescence.
Immunofluorescence staining of HeLa cells with anti-HSP70 antibody cmhsp70.1 (C92F3B1) (Ab03197). Immunofluorescence analysis of paraformaldehyde fixed HeLa cells, permeabilized with 0.15% Triton, on Shi-fix™ coverslips stained with the chimeric rabbit IgG version of cmhsp70.1 (C92F3B1) (Ab03197-23.0) (1:100 dilution) for 1h followed by Alexa Fluor® 488 secondary antibody (1:1000 dilution), showing cytoplasmic and some nuclear staining. The nuclear stain is DAPI (blue). Panels show, from left-right, top-bottom, Ab03197-23.0, DAPI, merged channels, and an isotype control. The isotype control was an unknown specificity antibody (Ab00178-23.0) followed by staining with Alexa Fluor® 488 secondary antibody.