zap-conjugates

Rothenberg ME, Nusse Y, Kalisky T, Lee JJ, Dalerba P, Scheeren F, Lobo N, Kulkarni S, Sim S, Qian D, Beachy PA, Pasricha PJ, Quake SR, Clarke MF (2012) Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice. Gastroenterology 142:1195-1205.e1196. doi: 10.1053/j.gastro.2012.02.006

Objective: To investigate the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5 stem cells.

Summary: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5+ stem cells.

Usage: For saporin experiments, small intestinal organoids with crypt buds and visible Paneth cells were passaged as single cells. Before embedding in Matrigel, dissociated cells were incubated with Anti-cKit-SAP (biotinylated-2B8 mixed with Streptavidin-ZAP). Treatment led to a marked decrease in organoid formation.

Related Products: Streptavidin-ZAP (Cat. #IT-27)

Selbo PK, Weyergang A, Eng MS, Bostad M, Maelandsmo GM, Hogset A, Berg K (2012) Strongly amphiphilic photosensitizers are not substrates of the cancer stem cell marker ABCG2 and provides specific and efficient light-triggered drug delivery of an EGFR-targeted cytotoxic drug. J Control Release 159(2):197-203. doi: 10.1016/j.jconrel.2012.02.003

Summary: Many anti-cancer drugs are substrates of the ATP-binding cassette transporter ABCG2. Unfortunately ABCG2 is also thought to play an important role in multi-drug resistance and the protection of cancer stem cells against chemotherapeutics and photodynamic therapy. This paper examined whether photosensitizers used in photochemical internalization (PCI) are substrates for ABCG2. Streptavidin-ZAP (Cat. #IT-27) was combined with biotinylated EGF and applied to cells in culture; saporin (Cat. #PR-01) was used as a control. The data show that PCI with the EGF-saporin toxin did not utilize ABCG2 to enter cells.

Related Products: Streptavidin-ZAP (Cat. #IT-27), Saporin (Cat. #PR-01)

Minami SS, Sun B, Popat K, Kauppinen T, Pleiss M, Zhou Y, Ward ME, Floreanig P, Mucke L, Desai T, Gan L ( 2012 ) Selective targeting of microglia by quantum dots. J Neuroinflammation 9(1):22 . doi: 10.1186/1742-2094-9-22

Related Products: MonoBiotin-ZAP (Cat. #BT-ZAP)

Q: I have a mouse monoclonal antibody and a rabbit polyclonal antibody that I would like to test using your secondary conjugate system. Which products do I need to order? 

A: For mouse monoclonals, you can use: Mab-ZAP (Cat. #IT-04) – Cells that internalize your mouse monoclonal antibody will be eliminated. Or, Fab-ZAP mouse (Cat. #IT-48) – Cells that internalize your mouse monoclonal IgG antibody will be eliminated.

For rabbit polyclonals, you can use: Rab-ZAP (Cat. #IT-05) – Cells that internalize your rabbit polyclonal antibody will be eliminated. Or, Fab-ZAP rabbit (Cat. #IT-57) – Cells that internalize your rabbit IgG antibody will be eliminated.

The difference between Fab-ZAP products and other secondary conjugates is that Fab-ZAP is made with a monovalent secondary antibody which eliminates the possibility of cap formation as cross-linking of the Fab-ZAP molecules cannot occur. The Fab-ZAP products will still recognize the heavy and light chains of antibodies, and should be used in the same way and at the same molar concentrations as the original secondary conjugates (such as Mab-ZAP and Rab-ZAP). Cytotoxicity assays using Fab-ZAP (mouse) have demonstrated an improved EC50 when directly compared to Mab-ZAP.

Any of our secondary conjugates offer a very cost-effective diagnostic method for screening primary antibodies for in vitro or in vivo use.

Related Products: ZAP Conjugates

Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang L-P, Gimotty PA, Gilks CB, Lal P, Zhang L, Coukos G (2011) Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells. Nature 475:226-230. doi: 10.1038/nature10169 PMID: 21753853

Objective: To investigate whether a direct link between tumor hypoxia and tolerance occurs through the recruitment of regulatory cells.

Summary: Tumor hypoxia promotes the recruitment of regulatory T (Treg) cells through induction of expression of the chemokine CC-chemokine ligand 28 (CCL28), which, in turn, promotes tumor tolerance and angiogenesis.

Usage: In vivo depletion of CD4+ CD25+ cells was achieved by intraperitoneal administration of anti-CD25 or an immunotoxin consisting of anti-mouse CCR10 or anti-mouse CCR3 antibody conjugated at an equimolar ratio to Streptavidin–ZAP. Anti-CCR10–SAP depleted 90% of CCR101 or CCR31 cells. Anti-CCR10–SAP suppressed tumour growth and abrogated the effects of CCL28 overexpression, whereas anti-CCR3–SAP had no effect on tumor growth.

Related Products: Streptavidin-ZAP (Cat. #IT-27)

Q: We’ve been using Mab-ZAP to test our primary mouse monoclonal antibody in cytotoxicity assay. Relative to the Primary mAb-Mab-ZAP complex, the Mab-ZAP alone has quite a bit of activity (40-60% growth inhibition) at the recommended concentrations used (45 or 100 ng/well) or even half that dose. Even though we get a dose response, the higher concentrations of primary antibody give me less cytotoxic activity than the lower concentrations. Can you give your thoughts on this matter?

A: The effect you are seeing is something that is actually typical and indicates that you are using the material correctly. Described in Kohls et al., unbound primary antibody will compete with primary antibody bound to a secondary conjugate may reduce cytotoxicity through competitive inhibition of the primary antibody-Mab-ZAP complex. This is especially noticeable with our Fab-ZAP line of secondary conjugates.

We still recommend that our customers try a 10 nM dose as a starting point, but always recommend adjusting the concentration to better accommodate their experiments. As a reference, our data sheets show a cytotoxicity curve with a starting concentration of 10 nM and an ending concentration of 1 fM.

Related Product: Mab-ZAP (Cat. #IT-04)

References

1. Kohls MD et al. Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 28(1):162-165, 2000.

Q: I would like to know if the secondary antibody used to prepare Mab-ZAP (Cat. #IT-04) reagent binds to heavy chain of mIgG’s (only) or if it recognizes light chains as well?

A: Mab-ZAP will recognize whole IgG and will bind to both the heavy and light chain.

Related Product: Mab-ZAP (Cat. #IT-04)

Mitra N, Banda K, Altheide TK, Schaffer L, Johnson-Pais TL, Beuten J, Leach RJ, Angata T, Varki N, Varki A (2011) SIGLEC12, a human-specific segregating (pseudo)gene, encodes a signaling molecule expressed in prostate carcinomas. J Biol Chem 286(26):23003-23011. doi: 10.1074/jbc.M111.244152

Summary: Siglec 12 (sialic acid-binding immunoglobulin-like lectin 12) is a sugar molecule that has mutated in humans to be inactive, but is active in other primates. The human version is found on some macrophages, various epithelial cell surfaces, and some human carcinoma cell lines. Using Mab-ZAP (Cat. #IT-04) and monoclonal antibodies against Siglec 12, the researchers demonstrated binding and internalization in a prostate cancer cell line, indicating that Siglec 12 may be a target for some cancer therapies.

Related Products: Mab-ZAP (Cat. #IT-04)

Q: Using Mab-ZAP (Cat. #IT-04) in a cytotoxicity assay, I obtained a nice dose-response curve up to around 10-9 M of antibody and then lost progressively the toxic effect of Mab-ZAP. What should I do to improve in my assay?

A: If the highest dose for which you got a good response was 10 nM and you lost effect when the primary concentration was increased beyond that, then that is the result we would expect. Often we have seen in cytotoxicity assays that when the primary antibody concentration is raised beyond a certain level (10-100 nM frequently being that level) there is so much free primary antibody that it competes with the Mab-ZAP-bound antibody for binding sites, thereby reducing the toxic effect. We recommend that you pre-incubate your primary with Mab-ZAP before adding the solution to the wells.

Related: Mab-ZAP (Cat. #IT-04)

Chung JS, Shiue LH, Duvic M, Pandya A, Cruz PDJ, Ariizumi K (2011) Sezary syndrome cells overexpress syndecan-4 bearing distinct heparan sulfate moieties that suppress T-cell activation by binding DC-HIL and trapping TGF-beta on the cell surface. Blood 117(12):3382-3390. doi: 10.1182/blood-2010-08-302034

Summary: Syndecan-4 (SD-4) is a transmembrane heparan sulfate proteoglycan. The Sézary syndrome (SS) subset of cutaneous T-cell lymphoma overexpresses distinct heparan sulfate moieties, giving the authors a specific target for these cells. Biotinylated DC-HIL- Fc (the extracelluar domain of dendritic cell- associated heparan sulfate proteoglycan- integrin ligand fused to Fc of mouse IgG) was combined at a 1:1 molar ratio with streptavidin-ZAP (Cat. #IT-27). In vitro, this targeted toxin eliminated SS cells, preventing their proliferation and suggesting a method for SS treatment.

Related Products: Streptavidin-ZAP (Cat. #IT-27)