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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.
SSP-SAP Aliquot Temperature
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)
Somatostatin Antibodies
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)
IgM Primary Antibody Assay
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.
Saporin Clearance
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
- 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.
- Mantyh PW et al. Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278:275-279, 1997.
Apoptosis or Necrosis?
Q: Do targeted toxin-treated cells die by apoptosis?
A: There are, allegedly, two ways for cells to die: by apoptosis or necrosis. According to Fiorenzo Stirpe (the discoverer of saporin), saporin-intoxicated cells die both ways, some by one, others by the other.
There is good literature that states that cells die by apoptosis. Saporin and apoptosis gives 25 hits in PubMed. For instance:
Bergamaschi G, Perfetti V, et al. (1996). Saporin, a ribosome-inactivating protein used to prepare immunotoxins, induces cell death via apoptosis. Brit J Haemat 93:789-794.
However, Seeger et al., did not find evidence of apoptosis in an electron microscopy study with cells dying from 192-IgG-SAP and concluded they die from necrosis. Saporin and necrosis gives 11 hits in PubMed.
So, saporin-treated cells seem to die by both apoptosis and necrosis. The customer is always right.