Q: What are neuropeptide-toxins and how do they work?
A: Neuropeptide-toxin conjugates are made up of the ribosome-inactivating protein, saporin, coupled to a naturally-occurring or synthetically-modified neuropeptide such as Substance P or dermorphin. The conjugate has binding specificity similar to the native, unconjugated neuropeptide. When the neuropeptide binds to its cognate receptor, the conjugate is internalized. Once inside the target cell within an endosome, the neuropeptide and saporin separate and some of the saporin translocates into the cytoplasm where it catalytically inactivates ribosomes resulting in cell death.
Q: Are neuropeptide-toxins effective suicide transport agents?
A: The general answer to this question is not currently known. However, in the instance of intrathecally injected dermorphin-SAP (Cat. #IT-12), the evidence does NOT favor suicide transport of the neuropeptide-toxin conjugate. When supramaximal doses of dermorphin-SAP (750 ng) are injected into the lumbar subarachnoid space of adult rats, less than 1% of lumbar dorsal root ganglion cells show evidence of saporin activity. This is in spite of the fact that many of these neurons express the targeted mu opioid receptor on their central terminals in the superficial dorsal horn of the spinal cord. This assertion is based on analysis of over 16,000 neurons from dorsal root ganglia in six rats.
Q: Does Mab-ZAP (Cat. #IT-04) bind to the FC portion of mouse IgG?
A: The antibody used to create our Mab-ZAP (IT-04), will react with whole molecule mouse IgG, which includes the Fc portion and the two antigen binding Fab portions.
Q: Can your FabFc-ZAP human (Cat# IT-65) bind to the Fc portion of another species, such as mouse IgG? It looks like it binds to mouse IgG in our assay.
A: The antibody used to create our FabFc-ZAP Human (IT-65), can react with the Fc (gamma) portion of human IgG heavy chain and should not react with the Fab portion of human IgG. However, there could be minimal cross-reaction with mouse, horse, or bovine serum proteins, and it is possible to see cross-reaction with immunoglobulins from other species.
Q: We recently spoke to you about performing a custom saporin conjugation using our antibody. Is 0.09% azide in PBS in the antibody stock acceptable?
A: There are a number of dialysis steps within the conjugation protocol that will ultimately remove the azide from your antibody solution. So as long as your antibody will be happy in PBS without azide during the procedure, sending the material in 0.09% azide is fine. The final conjugate will be returned to you in PBS, sterile-filtered, without azide.
Q: In general, how many saporin molecules are incorporated per antibody? Can we test this by HPLC?
A: We aim for 2-2.5 moles of saporin per mole of antibody. You should be able to see differences in HPLC between your antibody with one vs. two vs. three saporins attached, however we will provide you with a saporin molar ratio and a product that has had free saporin and free antibody removed from the final conjugate.
Q: Is the dosage of Fab-ZAP always enough for any level of antibody concentration?
A: The 4.5 nM dosage of Fab-ZAP is the recommended concentration. We do not typically see unspecific killing at 4.5 nM on most cell lines. If the concentration of Fab-ZAP is increased, it may undergo bulk-phase endocytosis and kill cells indiscriminately. A lower concentration of Fab-ZAP may lead to antibody competition, resulting in a lack of killing of cells at the highest concentration of antibody.
Q: Each concentration is suggested to perform 6 replications, can it be adjusted more or less in practice?
A: Yes, the assay design is meant to be a thorough approach but can be adjusted by the user. We recommend 6 replications based on our 96-well plate template design. The concentration of Fab-ZAP is 4.5 nM in the suggested protocols.
Q: Can Fab-ZAP detect the targeted antibody still in supernatant?
A: As long as there is nothing in the supernatant inhibiting the reactivity of Fab-ZAP, it should work. We do not typically recommend this, but in theory it should be possible. I would be cautious of this approach based off of the presumed lack of established concentration of antibody.
Q: Instead of performing the reaction between our biotinylated peptide and Streptavidin-ZAP at the initially provided concentration of Strep-ZAP (20 µM), is it OK if the reaction is done at a 10-fold more dilute concentration? This request is to ensure we don’t have any solubility problems with our very tricky lipophilic peptide. Our protocol would be to first dilute Streptavidin-ZAP to 2 µM with PBS and then add the peptide in DMSO (10% final), and store the aliquoted resulting 1.82 µM solution?
A: In regards to your question, while keeping in mind your solubility concerns, we suggest that you:
Proceed with diluting the Streptavidin-ZAP to 2 uM with PBS as you suggest, BUT, only react the amount of Streptavidin-ZAP necessary for the next step.
Store the undiluted and unreacted Streptavidin-ZAP at -80°C until you’re ready for more conjugate.
We understand the solubility of the peptide is a concern, and rightfully so. However, we also do not want to compromise the Streptavidin-ZAP during storage, considering its value.
Q: We have your ZAP internalization kit and I have a question regarding the concentrations used in the cytotoxicity assay. The Hum-ZAP used in the assay (mentioned in the PDF protocol) is 4.5 nM and the target agent was 10 nM to 1 fM. Is there a stoichiometric relation between Hum-ZAP and the target agent concentrations?
A: To answer your question simply, yes, there is a stoichiometric relation between a secondary conjugate and the targeting agent.
Q: If I use higher concentrations of the target antigen, then should I also increase the concentration of Hum-ZAP?
A: It may be intuitive to think that using a higher dose of primary antibody induces a higher amount of cell death, but as seen in the attached figure, at the highest concentration of 192-IgG (10 nM = Log -8) there is a lessened amount of killing, at a 25-fold lower concentration, as compared to the antibody. The explanation for this is that, at the higher concentrations of primary antibody, there is more unconjugated 192-IgG and fewer 192-IgG+Fab-ZAP complexes. The free 192-IgG then out-competes the conjugates for cell surface binding sites which, in turn, decreases the amount of Saporin being internalized, hence less cell death.
