- Home
- Knowledge Base
- FAQ
FAQ
Anti-DBH-SAP specificity
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.
Related Products: Anti-DBH-SAP (Cat. #IT-03)
References
- Harle P et al. An early sympathetic nervous system influence exacerbates collagen-induced arthritis via CD4+CD25+ cells. Arthritis Rheum 58:2347-2355, 2008.
- Harle P et al. 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. Arthritis Rheum 52:1305-1313, 2005.
Intrathecal Injections and Dosage
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.
References
Conjugate Calculations
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.
ZAP Kit Sizes
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.
Related Products: ZAP Conjugates
Streptavidin-ZAP concentration ratio
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
Time Course of Cell Death
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.
References
Flow cytometry for AB-N07
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.
Streptavidin-ZAP concentration
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.
Saporin Cleavage
Can you comment on the mechanism by which the SAP toxin is cleaved off from the antibody? Your website indicates that the cleavage occurs in the endosome. I just want to verify that it is not cleaved in the lysosome.
From what we have gathered, there is a great probability that something gets broken in the endosome. We know this from the peptide ligand toxins that bind to GPCRs. They would be rapidly returned to the cell surface through receptor recycling if there wasn’t some sort of cleavage. In the case of FGF-SAP (Cat. #IT-38) , e.g., we know that FGF is extensively degraded in the endosome through proteolytic degradation (Lappi et al, 1994). There is occasionally a disulfide linker between the toxin and antibody, but there is some controversy that this is cleaved: many say yes, some say no, mainly because the redox potential is not sufficient. This would ignore the presence of thiol reductase enzymes. The single chain antibody fusion protein-toxins are quite toxic. The linkage there is clearly through peptide bonds (they are fusion proteins) so the easiest response to this question is that there is proteolytic degradation. Since saporin is tremendously resistant to proteases, it can’t be stopped.
Lappi DA, Ying W, Barthelemy I, Martineau D, Prieto I, Benatti L, Soria M, Baird A (1994) Expression and activities of a recombinant basic fibroblast growth factor-saporin fusion protein. J Biol Chem 269(17):12552-12558. doi: PMID: 8175664
Targeting B Cells
How can I target B-cells? Which secondary conjugate should I use?
ATS makes secondary conjugates that use your primary antibody to target cells for elimination. Just mix your primary antibody with a secondary antibody conjugated to the ribosome-inactivating protein, Saporin, to screen your antibodies. The cells targeted by your primary antibody are eliminated.
Recognizes whole IgG
Hum-ZAP (Cat. #IT-22) is made with
a bivalent secondary antibody
that recognizes whole IgG.
B-Cells have endogenous IgGs.Fab-ZAP (Cat. #IT-51) is made with
a monovalent secondary antibody
that recognizes whole IgG.
B-Cells have endogenous IgGs.Recognizes Fc region
FabFc-ZAP (Cat. #IT-65) is made with
a monovalent secondary antibody
that recognizes ONLY the FC portion of IgG.
Antagonists vs. Targeted Toxins
Q: Recently, I attended a talk where the speaker said that a targeted toxin was able to work when an antagonist did not. Can you explain how your technology is different?
A: Certainly. It is a very interesting question and one that helps explain the Targeting Technology quite well. An antagonist is used to block a receptor on a cell to keep it from binding a target molecule and activating the cell. For example, a substance P antagonist binds to the substance P (NK-1) receptor. The hypothesis was that if the antagonist binds to the receptor, substance P cannot bind and the cell will not be activated. The reality is that there are other receptors besides the substance P receptor on that cell. If any of these other receptors bind to their target molecules, then the cell will still be activated.
It sometimes is not enough to block one particular receptor. The ATS targeting technology (molecular surgery) can use any of these cell surface receptors to target and completely eliminate the cell. That way, there are no receptors left to bind; no cell left to be activated. Importantly, molecular surgery cleanly removes one particular cell type and does not damage bystander cells. Once the debris from the targeted cell is cleared away, there is nothing remaining to interfere or affect the normal action/interaction of other cells.
Related Products: Targeted Toxins
Lethal Dose of Saporin in Mice
Q: What is the LD50 of saporin in mice? Do you have references for this?
A: Thank you for your question. It is very helpful to have this information to calculate the appropriate dose for systemic administration.
According to the work of Thorpe et al., saporin alone has an acute LD50, when delivered intravenously, of 6.8 mg/kg in mice. Histologic examination of kidneys from mice receiving near-lethal doses of saporin revealed necrosis of the convoluted tubules. Other major organs had only minor changes.
Once saporin is attached to an immunoglobulin, the LD50 drops dramatically to 1.0 mg/kg in systemic administration. Near-lethal doses of the conjugates, by contrast to saporin alone, inflicted major damage to the liver and spleen of the mice while the kidneys (and other organs) appeared normal under histologic examination.
References
Reducing Agent in Media
Q: We will be using your chick-ZAP secondary conjugate (Cat. #IT-62) and noticed that in your protocol you mention not to use a reducing agent in your media. Our normal growth media contains beta mercaptoethanol at 100 μM. Will this be a problem?
A: Officially, we would recommend allowing the cells to acclimate to media that contains NO BMe, and then proceed with your experiments. However, some of our in-house experiments use cells that are cultured in media containing 50 μM BMe, and we have not seen that concentration affect the toxin’s effectiveness, but we have not tried a concentration as high as 100 μM.
Related Product: Chick-ZAP (Cat. #IT-62)
Do you remove unconjugated antibody from Custom Conjugates
Q: We are interested in having a custom conjugation of saporin and our antibody. Do you remove unconjugated antibody from the final material you send us?
A: We do remove the unconjugated antibody and saporin from the final product that we send to you. And perhaps to answer a question you may not be asking, but may be curious about, the unconjugated material is not particularly usable, after it has been removed from the final product as it has been slightly modified in preparation for the conjugation.
See also: Custom Conjugates
Pan-neural targeting toxins
Q: I’d like to know which of your products are the pan-neural targeting toxins? I need an agent to kill all nerves in tissue preps.
A: OX7-SAP (Cat. #IT-02) should be perfect for this application. We recommend you examine your neurons with OX-7 antibody to see if they are positive. The only complication would be if you want to look at T-lymphocytes that also express Thy 1.
Related Product: OX7-SAP (Cat. #IT-02)
Staining with FITC-labeled Anti-Saporin
Q: I plan to use your Secondary Antibody Conjugates, Rab-ZAP (Cat. #IT-05), and Fab-ZAP Rabbit (Cat. #IT-57) with my primary antibody and would like to observe eliminated cells using a fluorescence microscope. The idea is to co-culture cancer cells and fibroblast cells, and kill fibroblast cells only with a specific primary antibody. Then I want to observe the eliminated fibroblast cells and take pictures with a fluorescence microscope. Can you recommend a protocol?
A: In order to stain and visualize the cells that are being eliminated, it would be best to stain for Saporin using a fluorescently-tagged antibody such as the FITC-labeled Saporin antibody (Cat. #AB-15AP-FL). By washing off the media after a day, and then staining for saporin, one would illuminate only the cells that have internalized the saporin (marking them for death). The cells that do not stain for saporin will live.
Related Product: FITC-labeled Saporin antibody (Cat. #AB-15AP-FL)
Anti-NGFr blocking antibody
Q: I have a question regarding your antibody to NGF (p75) receptor antibody (Cat. #AB- N01AP). Could you please tell me how you determined that it is a blocking antibody? Has this information been published?
A: We list on our website that one application for this antibody is for blocking the function of nerve growth factor receptor. This information was presented in an abstract at the Society for Neuroscience Meeting held in 1994.
Huber LJ, Lee K-F, Dreyfus CF, Chao MV (1994) Generation and characterization of a murine p75 receptor blocking antibody. Soc Neurosci Mtg, Miami Beach FL, Abstract #23-12.
Please check out the other references on our website for publications describing applications for this antibody.
Related Product: Anti-NGFr (Cat. #AB-N01AP)
Choosing the Correct Secondary Conjugate
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
Cytotoxicity Assay Protocols
Q: We are setting up some experiments in rat where we’d use your 192-IgG-SAP (Cat. #IT-01) in cytotoxicity assays. How much material do we need to order?
A: You can find protocols for calculating the amount of material needed for a cytotoxicity assay and protocols for the assay and interpretation of results on our website.
What charge does Bombesin-SAP and Blank-SAP have
Q: I am using your Bombesin-SAP (Cat. #IT-40) to kill GRP-receptor in mouse brain. I have a plan to inject it by iontophoresis. Do you know which charge dose Bombesin-SAP and Blank-SAP (Cat. #IT-21) have; plus charge or negative charge?
A: Both of these products will have a negative charge, though you may have to look into the literature for any needed guidance on dosing with that type of delivery.
Related Products: Bombesin-SAP (Cat. #IT-40), Blank-SAP (Cat. #IT-21)