zap-conjugates

Bostad M, Kausberg M, Weyergang A, Olsen C, Berg K, Høgset A, Selbo P (2014) Light-triggered, efficient cytosolic release of IM7-saporin targeting the putative cancer stem cell marker CD44 by photochemical internalization. Mol Pharm 11:2764-2776. doi: 10.1021/mp500129t

Summary: CD44 is known as a common cancer stem cell (CSC) marker. Given that CSC’s seem to have the ability to resist many therapeutic agents, the authors investigated the use of photochemical internalization (PCI) while targeting CD44-expressing CSC’s. An immunotoxin was constructed by biotinylating a pan CD44 antibody and combining it with Streptavidin-ZAP (Cat. #IT-27) at a 4:1 biotinylated antibody to Streptavidin-ZAP molar ratio. Various cancer cell lines were incubated with the toxin at a concentration of 0.825 nM. The toxin showed specific cytotoxicity to CD44-expressing cell lines, demonstrating the efficacy of PCI in conjunction with targeted toxins to treat some cancers

Related Products: Streptavidin-ZAP (Cat. #IT-27), Anti-CD44-SAP (Cat. #IT-72)

Q: I ordered the Streptavidin-ZAP (Cat. #IT-27) and had my antibody biotinylated a couple of months ago. I am ready to begin the first round of experiments to determine the concentration needed for the secondary. How much of the biotinylated antibody should I put to combine with the streptavidin for intravitreal injections? Can you please send me a protocol for how to determine the ratio of primary to secondary?

A: Streptavidin-ZAP should be mixed with the biotinylated material at an equimolar concentration. The Streptavidin-ZAP you ordered should have included a data sheet which gives the protein concentration and molecular weight, which you would use to determine the molar concentration. We have a calculator page on our website which can help with this if needed.

Listed below is a publication using Streptavidin-ZAP combined with a biotinylated antibody being used in intravitreal injections. The reference describes in detail the quantities they tried. You can also browse references on our site to see how scientists use ATS products to accomplish their research goals and publish in respected journals.

References

1. Ren C et al. Direct retino-raphe projection alters serotonergic tone and affective behavior. Neuropsychopharmacology 38(7):1163-1175, 2013.

Alonso M, Gregorio J, Davidson M, Gonzalez J, Engleman E (2014) Depletion of inflammatory dendritic cells with anti-CD209 conjugated to saporin toxin. Immunol Res 58:374-377. doi: 10.1007/s12026-014-8511-6

Objective: To investigate a strategy that avoids monocyte intermediates to deplete inflammatory dendritic cells (DCs). Mice with an abundance of inflammatory DCs as a consequence of lipopolysaccharide exposure were treated with anti-CD209 antibody conjugated to saporin.

Summary: The results demonstrate depletion of CD209+ DCs. This strategy could prove useful for the targeted reduction of inflammatory DCs in disease.

Usage: Streptavidin-ZAP mixed with biotinylated Anti-CD209 was delivered i.v. to mice. Inflammatory DCs were markedly depleted.

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

Lund K, Bostad M, Skarpen E, Braunagel M, Krauss S, Duncan A, Hogset A, Selbo P (2014) The novel EpCAM-targeting monoclonal antibody 3-17I linked to saporin is highly cytotoxic after photochemical internalization in breast, pancreas and colon cancer cell lines. MAbs 6(4):1038-1050. doi: 10.4161/mabs.28207

Summary: The epithelial cell adhesion molecule (EpCAM) is an attractive diagnostic and therapeutic target for a wide range of human carcinomas. It has also been found on cancer stem cells, increasing the interest in targeting and eliminating cells that express it. The authors have created a monoclonal antibody that binds EpCAM, and use several assays to demonstrate the antibody’s potential as an oncology tool. In one series of assays the biotinylated antibody was combined with streptavidin-ZAP (Cat. #IT-27), and in conjunction with photochemical internalization was shown to have specific cytotoxicity on several different cancer cell lines over a range of concentrations.

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

Leoni G, Rattray M, Fulton D, Rivera A, Butt AM (2014) Immunoablation of cells expressing the NG2 chondroitin sulphate proteoglycan. J Anat 224(2):216-227. doi: 10.1111/joa.12141

Summary: In this work the authors use an antibody against the NG2-glia marker chondroitin sulphate proteoglycan (CSPG) along with Mab-ZAP (Cat. #IT-04) on cell lines and brain slices to eliminate cells expressing CSPG. The results demonstrate selective and effective killing, providing a method to study the function of these cells.

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

Hess SM, Young EF, Miller KR, Vincent BG, Buntzman AS, Collins EJ, Frelinger JA, Hess PR (2013) Deletion of naive T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance. Transpl Immunol 29(1-4):138-145. doi: 10.1016/j.trim.2013.10.005

Summary: The authors utilized biotinylated peptide-MHC class I tetramers with Streptavidin-ZAP (Cat. #IT-27) to selectively delete a specific population of alloreactive T cells in mice. Animals received iv 33-pmol injections of the toxic tetramer, and the data indicate that these toxic tetramers can prevent the induction of donor-specific responses that result in organ rejection.

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

Q: Some suppliers sell their streptavidin conjugates in amounts given as streptavidin equivalents. Is that also the case for your product, Streptavidin-ZAP? What is important to me is to know what the molar concentration of streptavidin conjugate is, and the volume of your preparation.

A: The molar concentration of Streptavidin-ZAP (Cat. #IT-27) will depend on the lot; the accompanying data sheet will contain the molecular weight. We recommend that you mix Streptavidin-ZAP and your biotinylated material at equimolar concentrations. For our in-house in vitro quality control assays, we make a stock vial containing both 1 mM of biotinylated material and 1 mM of Streptavidin-ZAP diluted in media in 150 ml total volume. From this stock vial, we add 10 ml to each well of a plate containing cells in 90 µl volumes, which then dilutes the stock material to its correct concentration of 100 nM.

Check out the calculators on our website:

• Calculate Volume Required to Dilute a Solution
• Calculate Molarity of a Solution
• Calculate Volume of a Solution
• Calculate Mass of a Solution
• Convert Between Moles and Grams
• Convert Molar Units
• Convert Liter Units

Q: So, each mole of streptavidin will bind 4 moles of biotin?

A: Streptavidin-ZAP was created for use as an initial diagnostic step with biotinylated targeting agents, before moving on to a direct linkage between the optimal targeting agent and Saporin. The biotin-streptavidin interaction should be considered a linker; the major players are the targeting agent and Saporin. The targeting agent to Saporin ratio is kept at 1:2 M. When pre-mixing the biotinylated moiety with Streptavidin-ZAP in an equimolar ratio the ability of Streptavidin-ZAP to bind up to 4 biotins ensures that most of the biotinylated moiety will have Streptavidin-ZAP attached (no free biotinylated moiety). Streptavidin equivalents would not be appropriate as the Saporin moiety in Streptavidin-ZAP is the primary focus of the technology.

Bostad M, Berg K, Hogset A, Skarpen E, Stenmark H, Selbo PK (2013) Photochemical internalization (PCI) of immunotoxins targeting CD133 is specific and highly potent at femtomolar levels in cells with cancer stem cell properties. J Control Release 168(3):317-326. doi: 10.1016/j.jconrel.2013.03.023

Summary: Targeted therapies for cancer can be trapped in the lysosome and compartmentalized away from the target. Photochemical internalization is a method to increase the efficacy of these compounds by releasing the therapeutic portion of the molecule from the endocytic vesicles to the cytosol by the use of light. The authors demonstrate this method on cells expressing the cancer stem cell marker CD133. Biotinylated antibodies against CD133 were combined with Streptavidin-ZAP (Cat. #IT-27) and applied to cell lines.

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

Burgos-Ojeda D, McLean K, Bai S, Pulaski H, Gong Y, Silva I, Skorecki K, Tzukerman M, Buckanovich RJ (2013) A novel model for evaluating therapies targeting human tumor vasculature and human cancer stem-like cells. Cancer Res 73(12):3555-3565. doi: 10.1158/0008-5472.CAN-12-2845

Objective: To evaluate tumor vascular markers (TVM) expression in a human embryonic stem cell–derived teratoma (hESCT) tumor model previously shown to have human vessels.

Summary: The model tested represents a useful tool to test anti-human TVM therapy and evaluate in vivo human CSC tumor biology.

Usage: In vitro – Anti-THY1-SAP (biotinylated Anti-THY1 mixed equimolar with Streptavidin-ZAP) was incubated with mesenchymal stem cells (MSC); resulting in statistically significant MSC death. In vivo – Anti-THY1-SAP or control (Rat IgG-SAP) was administered intravenously. Treated ovarian tumors showed delayed growth and significant reduction in central tumor viability.

Related Products: Streptavidin-ZAP (Cat. #IT-27), Rat IgG-SAP (Cat. #IT-17)

Yamazaki CM, Nakase I, Endo H, Kishimoto S, Mashiyama Y, Masuda R, Futaki S, Koide T (2013) Collagen-like cell-penetrating peptides. Angew Chem Int Ed Engl 52(21):5497-5500. doi: 10.1002/anie.201301266

Objective: To overcome drawbacks of cell-penetrating peptide (CPP)-conjugated forms, namely instability against attack by proteases, and binding to serum proteins that lowers the availability of the CPP to target cells.

Summary: Complexes were prepared by combining biotinylated CPPs with Streptavidin-ZAP to evaluate the impact of utilizing a rigid collagen-like triple-helical scaffold to improve CPP performance. There was low adsorption onto serum proteins and triple-helical CPPs are extremely stable in animal serum. Such unique properties are expected to be advantageous to long-term applications in cell culture systems and to in vivo drug delivery.

Usage: Biotinylated CPPs were interacted with Streptavidin-ZAP (SA-ZAP); IT-27: Streptavidin-ZAP

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