Q: Concerning Hum-ZAP (Cat. #IT-22) and Rat-ZAP (Cat. #IT-26), are they monovalent or bivalent to their target immunoglobulins?
A: The secondary conjugates Hum-ZAP and Rat-ZAP are, in fact, bivalent and so do have the theoretical possibility of causing internalization when the primary would not – a false positive. In fact, we have never heard of this happening, mainly because the theoretical situation is difficult to put into practice – probably things get a little bulky on the cell surface.
Our idea is that the secondary conjugates are meant for large-scale screening in a very cost-effective manner, and upon identification of a positive, that primary antibody can be biotinylated and tested in vivo with streptavidin-ZAP (Cat. #IT-27). Streptavidin-ZAP can also cause oligomerization, but it’s used at equimolar amounts to the primary antibody, so that may not happen to an appreciable amount. However, the best method is to have a primary immunotoxin constructed through custom synthesis, in which saporin is directly coupled to the targeting agent.
Penaloza-MacMaster P, Masopust D, Ahmed R (2009) T-cell reconstitution without T-cell immunopathology in two models of T-cell-mediated tissue destruction. Immunology 128:164-171. doi: 10.1111/j.1365-2567.2009.03080.x
Summary: Although antigen-specific T cells are vital to adaptive immune responses, they also contribute to a variety of diseases. In this work the authors examined the possibility of selectively removing epitope-specific T cells while preserving immune function. Biotinylated MHC class I tetramers were combined with streptavidin-ZAP (Cat. #IT-27) and used in a mouse transferable T-cell-dependent neurological disease model. This technique resulted in a dramatic reduction in targeted antigen specific T cells with no observable bystander toxicity.
Q: We were wondering how an IgM primary antibody might work in a Mab-ZAP assay. I realize that the conjugated antibody is an anti-IgG whole molecule antibody. However there may well be aspects/epitopes shared in common between IgG and IgM that might render an IgM primary useful with the Mab-ZAP reagent… or not? Has anyone looked at this with your products?
A: We do believe, but have not confirmed, that you will see a cross-reactivity, but at a lower level. We do sell a second immunotoxin for IgM’s, Anti-M-ZAP (Cat. #IT-30) which is made from a goat anti-murine IgM.
Hess PR, Buntzman AS, Murray SL, Young EF, Frelinger JA (2009) Featured Article: Selective deletion of CD8+ T cells by saporin-coupled MHC class I tetramers. Targeting Trends 10(1)
Rouleau C, Curiel M, Weber W, Smale R, Kurtzberg L, Mascarello J, Berger C, Wallar G, Bagley R, Honma N, Hasegawa K, Ishida I, Kataoka S, Thurberg BL, Mehraein K, Horten B, Miller G, Teicher BA (2008) Endosialin protein expression and therapeutic target potential in human solid tumors: sarcoma versus carcinoma. Clin Cancer Res 14:7223-7236. doi: 10.1158/1078-0432.CCR-08-0499
Summary: Endosialin is an antigen expressed in many human cancer cell lines. As part of a wide-ranging study investigating clinical specimens, cell culture, and animal models, this group used Hum-ZAP (Cat. #IT-22) combined with a humanized anti-endosialin antibody in cell proliferation assays. Mouse IgG-SAP (Cat. #IT-18) was used as a control. The anti-endosialin antibody and Hum-ZAP were incubated together in equimolar concentrations then applied to cells in culture. Various cancers, including synovial sarcoma, fibrosarcoma, and osteosarcoma among others, were found to express endosialin.
Zhao XY, Liu HL, Liu B, Willuda J, Siemeister G, Mahmoudi M, Dinter H (2008) Tomoregulin internalization confers selective cytotoxicity of immunotoxins on prostate cancer cells. Transl Oncol 1:102-109. doi: 10.1593/tlo.08124
Summary: Tomoregulin is a type 1 transmembrane protein with a short cytoplasmic tail, and is found in the brain and prostate. After confirming cell surface localization by flow cytometry, and determining expression levels by whole-cell binding assays, the authors evaluated the use of tomoregulin as a target for immunotoxin therapy. Cells transfected with tomoregulin were treated with anti-tomoregulin + Mab-ZAP (IC50 = 160 pM; Cat. #IT-04) in vitro. The results demonstrate the potential for tomoregulin in prostate cancer treatment.
Siva AC, Wild MA, Kirkland RE, Nolan MJ, Lin B, Maruyama T, Yantiri-Wernimont F, Frederickson S, Bowdish KS, Xin H (2008) Targeting CUB domain-containing protein 1 with a monoclonal antibody inhibits metastasis in a prostate cancer model. Cancer Res 68:3759-3766. doi: 10.1158/0008-5472.CAN-07-1657
Summary: CUB domain-containing protein 1 (CDCP1) is an antigen expressed on several metastatic cancers, as well as on CD34+ and CD133+ myeloid leukemic blast cells. After demonstrating in vitro activity of the monoclonal antibody 25A11 with Mab-ZAP (Cat. #IT-04) and Hum-ZAP (Cat. #IT-22) the authors had a custom conjugation of 25A11 and saporin made for testing in mice. Goat-IgG-SAP (Cat. #IT-19) was used as a control for in vivo experiments, and saporin (Cat. #PR-01) was the control in vitro. The direct conjugate significantly inhibited tumor growth as well as metastasis in vivo.
One of the tests you can use to test your targeting agent for internalization is the in vitro Cytotoxicity Assay. Protocols to assist in preparing for, executing and interpreting results are now posted on our website.
There are several protocols available.
Preparing for a Cytotoxicity Assay using Secondary Conjugates. This protocol will be helpful when using our secondary antibody-saporin conjugates with your primary antibody. These include Anti-M-ZAP (Cat. #IT-30), Goat-ZAP (Cat. #IT-36), Hum-ZAP (Cat. #IT-22), Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), and Rat-ZAP (Cat. #IT-26).
Preparing for a Cytotoxicity Assay using Streptavidin-ZAP. This protocol will be helpful when using our streptavidin-saporin conjugate (Streptavidin-ZAP, Cat. #IT-27) with your biotinylated targeting agent (peptide, ligand, cytokine, growth factor, antibody, etc.).
Concentration Calculation: Convert molarity to mg/ml and mg/ml to molarity. This protocol will help in determining the correct amount of material to use in your assay. There is also a link to an Online Calculator.
Cytotoxicity Assay for Targeted Toxins in vitro. This protocol includes photos of what your plates should look like during the assay process. It takes five days to complete this assay. Start on a Monday and develop on Friday. There are many factors that go into a successful cytotoxicity assay. This protocol should help you design and execute appropriately.
Preparing Cytotoxicity Data. This protocol will give an example of how to process the data from a Cytotoxicity Assay. ATS uses SOFTMax Pro software connected to a plate reader to determine the A490 value. Then we import this data into Prism software (GraphPad) to conduct further data analysis. Here is a figure generated with Prism.
We hope these protocols will be helpful to you in your research. If there are additional protocols or tutorials we can provide, please do not hesitate to ask.
This graph gives important information about how the potency of your targeted toxin. The ED50 is the Median Effective Dose (produces desired effect in 50% percent of population). The lower this number is, the more potent the targeted toxin.
Q: I’m using your secondary conjugate Mab-ZAP (Cat. #IT-04) and it’s not killing my cells. I’m not following the protocol on the data sheet. I’m doing flow cytometry. I have 70,000 cells per well. I mix Mab-ZAP with my primary antibody and add it. When I count the cells, there is no decrease. My cells grow very slowly. I didn’t see anything after 72 hours.
A: The protocol on the data sheet is described in detail in the article by Kohls et al.
70,000 cells per well is a lot of cells per well. We use between 500 and 2500 over a 72-hour period and then develop with MTS.
If your cells are slow-growing, you may want to wait a little longer to develop the assay, because the whole metabolism process is slowed. This is a weakness of the MTS system — you have to have a certain number of cells in the end in the control cells to get a decent reading on your plate reader.
In this case, you might want to try a more sensitive assay such as protein or DNA synthesis inhibition with incorporation of radiolabeled leucine or thymidine.
Q: I purchased your secondary conjugate, Mab-ZAP (Cat. #IT-04). I am preparing to do a cytotoxicity assay and I’m wondering if my primary antibody should be sterilized prior to combining with Mab-ZAP?
A: Depending on the conditions of your lab in which you are using your antibody, it is possible that within a 72-hour period, you may see bacterial growth in your plates if the antibody was accidentally exposed to bacteria. It is recommended that, if you feel comfortable with the antibody, you can just go ahead and try it without sterilizing it, and if you do see bacterial growth, you can certainly filter sterilize the material through a 0.2 micron filter before using.
