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.
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.”
Q: We’re interested in trying out your melanopsin antibody (Cat. #AB-N38) using immunohistochemistry in mouse retina. Do you have a recommended protocol?
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.
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.
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.
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.
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.
One of the tests you can use to test your targeting agent for internalization is the in vitro Cytotoxicity Assay. Protocols to assist in preparing for, executing and interpreting results are now posted on our website.
There are several protocols available.
Preparing for a Cytotoxicity Assay using Secondary Conjugates. This protocol will be helpful when using our secondary antibody-saporin conjugates with your primary antibody. These include Anti-M-ZAP (Cat. #IT-30), Goat-ZAP (Cat. #IT-36), Hum-ZAP (Cat. #IT-22), Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), and Rat-ZAP (Cat. #IT-26).
Preparing for a Cytotoxicity Assay using Streptavidin-ZAP. This protocol will be helpful when using our streptavidin-saporin conjugate (Streptavidin-ZAP, Cat. #IT-27) with your biotinylated targeting agent (peptide, ligand, cytokine, growth factor, antibody, etc.).
Concentration Calculation: Convert molarity to mg/ml and mg/ml to molarity. This protocol will help in determining the correct amount of material to use in your assay. There is also a link to an Online Calculator.
Cytotoxicity Assay for Targeted Toxins in vitro. This protocol includes photos of what your plates should look like during the assay process. It takes five days to complete this assay. Start on a Monday and develop on Friday. There are many factors that go into a successful cytotoxicity assay. This protocol should help you design and execute appropriately.
Preparing Cytotoxicity Data. This protocol will give an example of how to process the data from a Cytotoxicity Assay. ATS uses SOFTMax Pro software connected to a plate reader to determine the A490 value. Then we import this data into Prism software (GraphPad) to conduct further data analysis. Here is a figure generated with Prism.
We hope these protocols will be helpful to you in your research. If there are additional protocols or tutorials we can provide, please do not hesitate to ask.
This graph gives important information about how the potency of your targeted toxin. The ED50 is the Median Effective Dose (produces desired effect in 50% percent of population). The lower this number is, the more potent the targeted toxin.
