zap-conjugates

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)

Sawada R, Sun SM, Wu X, Hong F, Ragupathi G, Livingston PO, Scholz WW (2011) Human monoclonal antibodies to Sialyl-Lewisa (CA19.9) with potent CDC, ADCC, and antitumor activity. Clin Cancer Res 17(5):1024-1032. doi: 10.1158/1078-0432.CCR-10-2640

Summary: In this work the authors investigated the use of a carbohydrate antigen, sialyl-Lewisa (CA19.9), as a target for cancer therapeutics. Human monoclonal antibodies were generated against CA19.9 and characterized using ELISA and flow cytometry. To assess internalization one antibody, 5B1, was combined with Hum-ZAP (Cat. #IT-22) and applied to CA19.9-expressing BxPC3 cells. The cytotoxicity of the 5B1-Hum- ZAP complex indicates that CA19.9 may be a target for cancer therapy.

Related Products: Hum-ZAP (Cat. #IT-22)

Q: When we contacted you to find out more about having a custom saporin conjugation performed with our primary antibody, you recommended that we use the ATS secondary conjugate system to determine that our antibody was specific to the population we want to eliminate. We looked more at the website, and it seems that we are supposed to start with Anti-M-ZAP, Cat. #IT-30 (our primary Ab is a mouse IgM), and use the Mouse IgM-SAP, Cat. #IT-41 for control. Is this correct?

A: If your primary antibody is a mouse IgM, then you are correct that Anti-M-ZAP (Cat# IT-30) is the appropriate secondary conjugate to use. As for control conjugates, the best control would be a secondary conjugate using an IgM isotype control mixed with Anti-M-ZAP. An alternative would be to use Goat IgG-SAP (Cat# IT-19) made with normal goat IgG that mimics Anti-M-ZAP without the specific affinity for mouse IgM.

Once you determine you need a direct conjugate made between your mouse IgM primary antibody and saporin, then you would want to use the Mouse IgM-SAP (Cat# IT-41) as a control toxin just as you use your direct conjugate.

Related: ZAP Conjugates, Control Conjugates, Custom Conjugates

Yang MY, Chaudhary A, Seaman S, Dunty J, Stevens J, Elzarrad MK, Frankel AE, St Croix B (2011) The cell surface structure of tumor endothelial marker 8 (TEM8) is regulated by the actin cytoskeleton. Biochim Biophys Acta 1813(1):39-49. doi: 10.1016/j.bbamcr.2010.11.013

Summary: Tumor endothelial marker 8 (TEM8) is a cell surface protein that is up-regulated on tumor blood vessels. Overexpression of this protein, however, produces a form that is not recognized by the SB5 monoclonal antibody used to bind TEM8. While cells expressing normal levels of TEM8 were killed by an application of biotinylated SB5 plus either 1 nM or 10 nM streptavidin-ZAP (Cat. #IT-27), cells overexpressing the protein did not bind the immunotoxin. Understanding the structural differences between the two forms of TEM8 will help in the design of therapeutic antibodies against these tumor cells.

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

Quadros EV, Nakayama Y, Sequeira JM (2010) Targeted delivery of saporin toxin by monoclonal antibody to the transcobalamin receptor, TCblR/CD320. Mol Cancer Ther 9(11):3033-3040. doi: 10.1158/1535-7163.MCT-10-0513

Summary: Vitamin B12 is necessary for cell proliferation. Cancer cells display an increased expression of TCb1R, the receptor that facilitates the intake of B12. In order to evaluate the potential of using immunotoxins to eliminate cancer cells expressing TCb1R the authors performed a series of in vitro experiments using their monoclonal antibodies plus Mab-ZAP (Cat. #IT-04). The results indicate that this is a viable therapeutic model that causes minimal peripheral damage.

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

Kuroda K, Liu H, Kim S, Guo M, Navarro V, Bander NH (2010) Saporin toxin-conjugated monoclonal antibody targeting prostate-specific membrane antigen has potent anticancer activity. Prostate 70(12):1286-1294. doi: 10.1002/pros.21164

Summary: Current treatments for prostate cancer are only moderately effective. In this work the authors examined the cytotoxic efficacy of an anti-prostate-specific membrane antigen (PMSA) antibody conjugated to saporin on PMSA-positive cell lines. hJ591, a humanized anti-PMSA antibody, was biotinylated and combined with streptavidin-ZAP (Cat. #IT-27). The hJ591-streptavidin-ZAP complex was specifically cytotoxic to PMSA-positive cell lines, and had anti-cancer activity in a xenograft model. This work demonstrates the anti-cancer potential of targeting PMSA.

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

Hovinga KE, Shimizu F, Wang R, Panagiotakos G, Van Der Heijden M, Moayedpardazi H, Correia AS, Soulet D, Major T, Menon J, Tabar V (2010) Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate. Stem Cells 28:1019-1029. doi: 10.1002/stem.429 PMID: 20506127

Summary: The antibody CD105 (1:1,000) was incubated with Mab-ZAP, to allow binding and formation of a CD105 antibody-saporin complex, which was added to explants or to a human cerebral microvessel endothelial cell line as control.  CD105 antibody was also incubated with a Goat-IgG-SAP for control  that does not bind CD105.  The conjugates were injected into the explant under a dissecting microscope after gently incising the explant surface for better access.

Related Products: Mab-ZAP (Cat. #IT-04), Goat IgG-SAP (Cat. #IT-19)