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FAQ
Assay Parameters for Mab-ZAP
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)
Destroy Hippocampal Neurons
Q: Is there a saporin derivative available that selectively destroys all or some hippocampal neurons?
A: Assuming that you mean to eliminate cell bodies from the hippocampus, rather than just projections, a neat paper by Martin et al. describes the use of SSP-SAP (Cat #IT-11) to do this. NPY-SAP (Cat. #IT-28) could also be interesting.
Related: SSP-SAP (Cat. #IT-11), NPY-SAP (Cat. #IT-28)
References
Custom Mouse IgM Conjugate and Controls
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
Time Course for Toxins
Q: I am interested in your product GAT1-SAP (Cat. #IT-32). Before ordering, I would like to know how long it takes until the toxin produces a lesion; Is seven days enough?
A: The usual time-course for saporin toxins is that behavioral effects start appearing at four days and usually plateau at seven days. However, keep in mind that it takes some time for dead cells to be cleared out, so histology on the animal should wait until at least two weeks after administration.
Related: Targeted Toxins
Immunotoxin Tracers
Q: In a previous question, you mentioned mixing anti-DBH-SAP (Cat. #IT-03) with a tracer to monitor drug delivery. Which tracer would you recommend? We were thinking of using FluoroGold. If we do not use a tracer, we were thinking of using a neutral red solution to dilute the stock of anti-DBH-SAP in order to be able to visibly see the toxin being injected into the spinal cord. Could there be an issue of pH if we used neutral red with anti-DBH-SAP? Our concern is that the toxin is not being ejected from the pipette tip or that it is not being taken up into the pipette tip as we can not see it (it’s the same color as the mineral oil). We are confident in the targeting of the spinal area for injection as we have previously used FluoroGold only and then were able to visualize it in the area of interest.
A: Our Scientific Advisor, Dr. Ronald G. Wiley, uses Fast Green dye (0.01-0.1% w/v) in the toxin injection solutions. He originally chose Fast Green because intracellular electrophysiologists had long used it while doing intracellular recordings and shown it was non-toxic. Fast Green has more contrast than Neutral Red (easier to see) and does not affect pH significantly. He has used it with many saporin-containing toxins with success.
Dr. Wiley says, “There are two issues when you talk about using “tracers” with targeted toxins: 1) tracing the acute injection volume to be sure it goes into the animal correctly, and 2) tracing the neurons that projected to the injection site and were therefore susceptible to being killed by the toxin.
Dr. Wiley does not use separate anatomic tracers for the immunotoxins, the only agents taken up and retrogradely transported efficiently. Since ATS immunotoxins are so efficient you have to use a high efficiency tracer such as cholera toxin B (but not WGA since it may not play well with saporin).
Dr. Wiley does not favor FluoroGold (a tin compound) because he has seen some local toxicity at FluoroG injection sites which might impair uptake and/or transport of a targeted toxin, and it is not clear if it is compatible with saporin-containing toxins.
Related: Anti-DBH-SAP (Cat. #IT-03), Anti-DBH-SAP Administration
How long does it take to kill the target cell?
Q: Saporin (Cat. #PR-01) has been shown to enzymatically inhibit the function of the ribosome, which follows that protein synthesis is then inhibited. Inhibition of protein synthesis brings about “cell death” to my knowledge. To detect “cell death” usually does not take a longer time to detect than “growth inhibition,” I suppose. So what I would like to ask you is: “at least” how many hours will it take to detect “cell death” caused by saporin. In your protocol, the recommended duration of assay is 72 hours. Does that duration contain much allowance? Of course, the duration must be dependent on the speed (or efficiency) of internalization of saporin, I understand. But once saporin is internalized, how many hours (or minutes) will it take to kill the target cell?
A: 72 hours is for the great majority of cell lines, but there are a very few that require 48 hours and a very few that require 96 hours (maybe 1 of each of the 100 or so that we’ve tried). The variation in time from 72 hours is not much on the shorter side, but is only limited by the few living cells proliferating on the longer side.
It is easy to see dead cells in the microscope, so you may want to visually check your cells at different times to verify that 72 hours is correct.
How many hours will it take after internalization to kill a cell? Quite a few, because there are several processes that need to occur: the enzyme must inactivate a sufficient number of ribosomes to inhibit protein synthesis, and then the cell has to stop living because of the turnover and loss of those proteins. That takes time.
Related: Saporin (Cat. #PR-01)
Are Hum-ZAP and Rat-ZAP bivalent?
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.
Related: ZAP Conjugates
Lot-to-Lot Variation
Q: We have a question about two 192-IgG-SAP (Cat. #IT-01) lots. According to your data sheet there is an approximate 4-fold difference in ED50 between your new lot and old lot. We also observed a clear difference in behavior between animals dosed with the new batch and the old one. It is thus obvious that the new lot needs to be diluted to achieve the same results, however, we are uncertain if this can be calculated just based on the ED50 values. Do you have any experience about dose-responses with the different lots in terms of size of lesion?
A: We don’t have an exact correlation between in vitro and in vivo activity, unfortunately. We state on the data sheet to check a new batch on a small number of animals.
“There may be lot-to-lot variation in material; working dilutions must be determined by end user. If this is a new lot, you must assess the proper working dilution before beginning a full experimental protocol.”
Related: Targeted Toxins
Anti-Melanopsin Protocol
Q: We’re interested in trying out your melanopsin antibody (Cat. #AB-N38) using immunohistochemistry in mouse retina. Do you have a recommended protocol?
A: This protocol has been utilized successfully with anti-melanopsin.
Related: Anti-Melanopsin (Cat. #AB-N38), Anti-Melanopsin, affinity-purified (Cat. #AB-N39)
Antigen for HRP-labeled Antibody to p53
Q: Regarding your HRP-labeled Antibody to p53 (Cat. #AB-236), your data sheet states the antigen was 15-40 a.a. Does this a.a. count from N-terminus?
A: There was an error on the data sheet which has since been corrected. The AB-236 immunogen is a KLH-conjugated peptide corresponding to amino acids 6-45. The numbering does start from the N-terminus.