Q: For in vitro cytotoxicity assays, could you tell me: 1) whether you incubate primary with your Saporin secondary for a specific amount of time prior to cell addition, and 2) do you use a single concentration of secondary per well or a primary:secondary ratio — like 1:2 or 1:4?
A: The primary antibody should be incubated with the ZAP product for 20 min prior to addition to the cells. Internalization often happens so quickly that you would lose some efficacy due to the antibody being bound and internalized prior to the ZAP product complexing with the primary. We do recommend maintaining a constant 5 nM (~ 45 ng/well) concentration of the ZAP product in the well and titrating your primary only. This way the EC50 you generate will be the EC50 of the primary antibody with all else held constant. The best starting concentration for your primary antibody is 10-100 nM in the well.
Q: I ordered a targeted toxin. Will it come in powder form? How do I re-dissolve it?
A: Our Saporin conjugate products are all provided in sterile PBS solution within a concentration range of 0.5 – 3 mg/ml. Saporin is an extremely safe ‘toxin’ to handle in standard laboratory environments when in solution for several reasons. Solutions in general are easier to corral and keep contained than powders and consequently are less likely to accidentally end up on an individual’s skin, tongue, or in one’s eyes. As a lyophilized product, Saporin would also be present at an extremely high concentration such that there is cause for concern should it contact the body of the user in any way. Lastly, our Saporin conjugates have historically required dilution prior to use for both in vitro and in vivo procedures. As such, it is much easier to ensure the amount of material you, as a customer, are receiving and the subsequent dilution is accurately adjusted to your desired concentration when providing these products already in solution. If upon receiving a Saporin conjugate you believe the product to be lyophilized or in a powder form, please contact us immediately, prior to opening the vial.
Q: I’m interested in your anti-DBH-saporin toxin for lesioning central catecholaminergic neurons. I see from the product description that the antibody used is a mouse monoclonal – designed to specifically target rat DBH. My interest is to produce targeted lesions in mouse transgenic. Will this product still work specifically? Thanks.
A: Unfortunately, we do not have really good data to support the use of our Anti-DBH-SAP (Cat. #IT-03) in mice. There is significant homology between mouse and rat DBH, however the actual antigen for both the mouse monoclonal we use in the immunotoxin and an alternate unpurified rabbit polyclonal, is native bovine DBH enzyme. For further background information there are two references where our product was used in mice. The references are listed below.
An early sympathetic nervous system influence exacerbates collagen-induced arthritis via CD4+ / CD25+ cells.[1] The sympathetic nervous system can play conflicting roles in collagen-induced arthritis (CIA). CD4+CD25+ T cells can play an immunoregulatory effect in this system depending on the expression of the FoxP3 transcription factor. Mice received 5-µg intraperitoneal injections of anti-DBH-SAP to induce an early sympathectomy. The results indicate that the sympathetic nervous system increases disease severity in CIA by stimulating some of the proinflammatory aspects of CD4+CD25+ T cells.
An opposing time-dependent immune-modulating effect of the sympathetic nervous system conferred by altering the cytokine profile in the local lymph nodes and spleen of mice with type II collagen-induced arthritis.[2] In this work the authors examined the role of the sympathetic nervous system (SNS) in late stages of chronic arthritis. 5 µg intraperitoneal injections of anti-DBH-SAP in mice were used to confirm that previous 6-OHDA injections caused a sympathectomy. The results demonstrate that the SNS supports inflammation during the asymptomatic phase of arthritis, but inhibits inflammation during the chronic symptomatic phase.
Q: Our lab is getting ready to begin a project using one of your targeted toxins. We already did a preliminary experiment to try out the material, but we have a couple of questions before we start the larger project. First, do you have any protocols or references for injecting intrathecally?
A: Thank you for your inquiry. We appreciate the opportunity to get involved in projects before they begin. At Advanced Targeting Systems, we do not do any in vivo work, just in vitro, however we have collaborated with many fine laboratories that have good experience with intrathecal injections. If you search PubMed with the keywords ‘saporin’ and ‘intrathecal’ you will be able to view references that will give you good information on techniques and protocols. Prior to beginning your project you will want to submit your animal care guidelines to your IACUC committee. Turner et al. published an article that will be helpful regarding intrathecal injections. [1]
Q: The second question is in two parts: 1) how do we determine the appropriate dose, and 2) how do we know saporin is not killing indiscriminately at that dose?
A: You should always use a control when determining the appropriate dose. A basic premise of the ATS targeting technology is that if a control (saporin alone or a control conjugate) evokes a response, then the dose is too high. Whenever a new shipment of targeted toxin is received, the proper working dilution should be ascertained before beginning a project. The targeted toxin data sheet states:
“There may be lot-to-lot variation in material; working dilutions must be determined by end user. If this is a new lot, assess the proper working dilution before beginning a full experimental protocol.”
If you search on the ATS website for the species and route of administration you plan to use, you can look through the publication summaries and see the dose that was used for that particular study. That will give you a ballpark range in which to start your dose titration. Just keep in mind: if the control kills cells, the dose is too high.
Q: I have been using your ZAP Antibody Internalization Kit. It is working well for me, but I can only test one antibody at a time. Do you offer the ZAP kit in larger sizes?
A: We do offer kits with sufficient components to test multiple antibody candidates. We offer “Z4” and “Z10” sizes of kits that include all of the same consumable components of the original ZAP kit in quantities sufficient to test 4 or 10 antibodies, respectively. While the included recommended protocol is identical to the original ZAP kit, the added materials provide an opportunity for the experienced researcher to streamline their experiment by testing multiple antibody candidates at one time.
Q: I ordered a control conjugate to use alongside my targeted conjugate, but the two products are at different concentrations. How much control conjugate should I use?
A: Conjugate products are often of differing protein concentrations, meaning dilution of one is usually necessary to ensure comparable amounts of control conjugate and targeted conjugate are used. This adjustment can be done on a molar basis or a protein concentration basis. The data sheet shipped with each Advanced Targeting System conjugate specifies the molecular weight of the product. There are various calculators available on the ATS website.
By using these tools, calculations can be done that will ensure the same number of molecules of both control and targeted conjugate are used in your experiment. Alternatively, if the molecular weights of the two products are similar, calculations can be done to use the same amount of control protein as targeted conjugate protein in your experiment.
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.
Q: How long does it take to see the cell death occurring from the use of targeted toxins using saporin? Is there a time course of hours or days?
A: The figure below illustrates the time course of cell death very effectively. Internalization and cytotoxicity of SP-SAP in primary cultures of neonatal spinal cord neurons. Confocal image of neurons where the Substance P receptor; NK1R (SPR) immunofluorescence (A, C, D) appears red, areas of concentrated SPR immunofluorescence appear yellow. (A, C, and D) SPR immunofluorescence in neurons 2 hours, 1 day, and 4 days, respectively, after treatment with SP-SAP. (B) Confocal image showing SAP immunofluorescence (yellow) 2 hours after SP-SAP treatment.
These images were projected from 14 optical sections acquired at 0.8-mm intervals with a 603 lens. Bar, 25 mm.
It is recommended that you wait for two weeks to allow for all debris to be cleared and the animal to regain normal eating and sleeping habits.
Q: We have a publication in review and put this statement in the paper, “The mouse monoclonal antibody to the low affinity nerve growth factor receptor (p75NTR; Advanced Targeting Systems) was derived from immunization of mice with WM245 melanoma cells and recognizes p75NTR in human, primate, rabbit, sheep, dog, cat, and pig. According to the manufacturer’s information, the antibody was tested by flow cytometry.” One of the reviewers wants to know more about the flow cytometry used to characterize this antibody (Cat. #AB-N07). Can you help, please?
A: This antibody is routinely tested by flow cytometry. The quality control flow data can be found on the data sheet on our website. HS294T cells, human metastatic melanoma cells, were used in flow cytometry with Anti-p75NTR (ME20.4, Cat. #AB-N07). Cells were treated with 4 µg of AB-N07 and subsequently with Anti-murine IgG-FITC (Cat. #FL-07). This assay shows the binding affinity of AB-N07 to cells known to express p75NTR.
