FAQ

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

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)

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

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

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.

Panda S. et al. 2002. Melanopsin (Opn4) requirement for normal light- induced circadian phase shifting. Science 298(5601):2213-2216.

Related: Anti-Melanopsin (Cat. #AB-N38), Anti-Melanopsin, affinity-purified (Cat. #AB-N39)

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.

Related: HRP-labeled Antibody to p53 (Cat. #AB-236)

Q: We are using SSP-SAP (Cat. #IT-11) to lesion NK-1r-bearing neurons. I have the conjugate diluted in solution and was wondering whether or not it is okay to leave it out at room temperature overnight? I would like to use an aliquot over a period of two days. Also, would it be okay to combine it the next day to a new, thawed aliquot?

A: We suggest, instead of leaving material out at room temperature, that you store at 4°C over the two days. Yes, you can combine samples.

Related: SSP-SAP (Cat. #IT-11)

Q: Could you please tell me if the Somatostatin 14 antibody (Cat. #AB-04) will also pick up the Somatostatin 28 residue?

A: Yes, it will, because they share the sequence of SS14. However, the Somatostatin-28 antibody (Cat. #AB-05) will not see Somatostatin-14.

Related: Anti-Somatostatin-14 (Cat. #AB-04), Anti-Somatostatin-28 (Cat. #AB-05)

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.

Related: Mab-ZAP (Cat. #IT-04), Anti-M-ZAP (Cat. #IT-30)

Q: I am planning an experiment to investigate the effects of ablation of spinal NK-1r-expressing cells (using intrathecal injection of SSP-SAP, Cat. #IT-11). In the first part of the experiment I want to destroy the NK-1r-expressing cells before surgical modification. I am unsure how long after injection of SSP-SAP I should carry out the surgery. I was thinking of carrying out surgery at the two-week time point as in a 2007 Neuroscience paper by Wiley et al. Their immunocytochemistry showed a large reduction in staining at this time point. Any advice you could give me would be much appreciated.

A: Two weeks is probably fine. Generally cells begin to lose function at four days, but people wait longer because there is a clean-up by microglia/macrophage that removes the markers that people use for detection/demonstration of efficacy. Mantyh et al. were conservative with a 30-day wait for saporin clearance.

Related: SSP-SAP (Cat. #IT-11)

References

1. Wiley RG et al. Anti-nociceptive effects of selectively destroying substance P receptor-expressing dorsal horn neurons using [Sar(9),Met(O(2))(11)]-substance P-saporin: Behavioral and anatomical analyses. Neuroscience 146:1333-1345, 2007.
2. Mantyh PW et al. Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278:275-279, 1997.