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?
We don’t have an exact correlation between in vitro and in vivo activity, unfortunately. We always state on the data sheet to check a new batch on a small number of animals. Actually, I see that we merely suggest that; we’ll change it to be more explicit.
“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.”
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.
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?
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.
You may find this paper from long ago quite interesting: Olsnes et al., J Biol Chem. 251(13):3985-3992, 1976 Jul 10. It concerns relatives of saporin, ricin and abrin, that have their own, probably more efficient, cell binding chains and are true toxins (whereas saporin has no binding chain and therefore no real toxicity on its own). Incredibly, protein synthesis inhibition at high doses is underway after 1 hour!