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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)
Higher concentration and less cytotoxicity
Q: We’ve been using Mab-ZAP to test our primary mouse monoclonal antibody in cytotoxicity assay. Relative to the Primary mAb-Mab-ZAP complex, the Mab-ZAP alone has quite a bit of activity (40-60% growth inhibition) at the recommended concentrations used (45 or 100 ng/well) or even half that dose. Even though we get a dose response, the higher concentrations of primary antibody give me less cytotoxic activity than the lower concentrations. Can you give your thoughts on this matter?
A: The effect you are seeing is something that is actually typical and indicates that you are using the material correctly. Described in Kohls et al., unbound primary antibody will compete with primary antibody bound to a secondary conjugate may reduce cytotoxicity through competitive inhibition of the primary antibody-Mab-ZAP complex. This is especially noticeable with our Fab-ZAP line of secondary conjugates.
We still recommend that our customers try a 10 nM dose as a starting point, but always recommend adjusting the concentration to better accommodate their experiments. As a reference, our data sheets show a cytotoxicity curve with a starting concentration of 10 nM and an ending concentration of 1 fM.
Related Product: Mab-ZAP (Cat. #IT-04)
References
Does Mab-ZAP bind heavy and light chain?
Q: I would like to know if the secondary antibody used to prepare Mab-ZAP (Cat. #IT-04) reagent binds to heavy chain of mIgG’s (only) or if it recognizes light chains as well?
A: Mab-ZAP will recognize whole IgG and will bind to both the heavy and light chain.
Related Product: Mab-ZAP (Cat. #IT-04)
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)