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.
Q: Your targeted toxin kits come with different controls. I’m not sure of the best way to use them; there is included unconjugated antibody, unconjugated saporin, and a control conjugate, mouse IgG-SAP. Should I use them all in the same experiment or for different purposes?
A: For mouse IgG-containing conjugates, the ideal control is Mouse IgG-SAP (Cat. # IT-18). Mouse IgG-SAP — that is, saporin conjugated to mouse IgG — that has no specific antigen for targeting is the best control. Unconjugated saporin is still considered a second good control, useful in cases where down-regulation by the antibody is a concern.
Q: What about for the peptide toxins?
A: We have produced Blank-SAP as a control for the peptide ligand toxins. Blank-SAP (Cat. #IT-21) is a peptide that has the usual common amino acids that are found in peptide neurotransmitters, but arranged in a sequence that is random and not detected in homology searches. So, it should never find an amenable receptor. This is quite an important control; the peptide ligand toxins are often delivered directly to tissue, and there are cases in which there will be no toxicity or non-specific toxicity.
As any journal reviewer will tell you, it’s very important to document the specificity, and with Blank-SAP as a control, you can definitively show that toxicity is due to proper targeting, rather than non-specific cytotoxicity. This should provide the information needed so the reviewer doesn’t have to make you go back and document specificity with further experimental work!
Q: I’ve been looking at your secondary conjugates and want to see if my targeting agent is specific to certain cells. Which secondary conjugate should I use?
A: It depends on two factors: 1) the type of assay you want to use, and 2) the kind of targeting agent you want to use.
For in vitro assays, in particular, internalization assays, you can use any of the ZAP Antibody Internalization Kits (Z-Kits that include all the materials necessary to test your targeting agent).
For the Antibody Internalization Kits, you use your primary antibody and select the appropriate secondary antibody species, depending on the isotype of your primary antibody — e.g. for a human antibody, use Hum-ZAP (Cat. #KIT-22-Z), Fab-ZAP human (Cat. #KIT-51-Z), FabFc-Human (Cat. #KIT-65-Z), Hug-M-ZAP (Cat. #KIT-43-Z), or Fab-ZAP Hug-M (Cat. #KIT-78-Z).
Or you can biotinylate your antibody and use Biotin-Z Antibody Internalization Kit (Cat. #KIT-27-Z).
For in vivo applications, it depends on the kind of targeting agent you want to use. Regardless of whether you use an antibody, peptide or ligand, you will need to biotinylate the material first. ATS offers a biotinylation service that is efficient and economical.
If you are using a biotinylated peptide, you will use a kit that includes the appropriate control — Streptavidin-ZAP Peptide Kit (Cat. #KIT-27-B).
If you are using a biotinylated antibody, you will use the Streptavidin-ZAP Antibody Kit that includes the appropriate antibody species control: BIgG-SAP Human (Cat. #KIT-27-Ahu), BIgG-SAP Mouse (Cat. #KIT-27-Amu), BIgG-SAP Rabbit (Cat. #KIT-27-Arb), BIgG-SAP Rat (Cat. #KIT-27-Art). Select the kit that matches the species of your biotinylated primary antibody.
Over the years, ATS has frequently been asked about Saporin’s safety for use in the lab as well as when used clinically. Residual awareness of alternate Ribosome-Inactivating Proteins (RIPs) and ‘toxins’ such as Ricin have caused some researchers new to the use of RIPs to question the belief that Saporin is safe. Unlike Type 2 RIPs (such as Ricin), Type I RIPs, like Saporin have no binding chain and consequently no means of entering the physiological space necessary for the protein to act as a toxin. The following is a review of safety in handling and potential toxicity within the human body for systemic events not related to normal research applications of Saporin conjugates, including Substance P-Saporin (SP-SAP), which is a therapeutic under development for the treatment of chronic pain.
The acute LD50 for saporin in mice (25 g) is 6.8 mg/kg;[1] that would translate in humans (75 kg) to 510 mg! A concentration of about 100 nM is the threshold to see even a vague hint of saporin toxicity. In human blood, that would correspond to 24 mg injected systemically into a person. The fermentation process to produce recombinant saporin has a titer of 2 mg/L meaning that the production broth itself contains no more than 67 nM concentration of saporin. Furthermore, the final protein concentrations from production batches of recombinant Saporin used in our drug are 4 mg/ml, meaning 6 mL of final material would need to accidentally end up in a human before the ‘hint of toxicity’ threshold would potentially be met.
The toxicology studies of SP-SAP contained within ATS’s IND prior to the current human Phase I clinical trial evaluated effects related to the intended method of administration, intrathecal local injection. SP-SAP is not expected to ever be a self-administered therapy, so the effects of gross off-target events, such as accidental auto-injection, swallowing, spillage, or immersion were not considered.
The table below[2] highlights antibody-saporin conjugates approved by the FDA for Phase I/II clinical trials in humans. The therapeutics listed below were administered intravenously and imply what the FDA accepted as non-toxic levels of saporin-based conjugates in these studies.
Looking more closely at the study by French et al.,[3] several milligrams of antibody conjugate were repeatedly injected into human patients under a FDA regulated clinical trial and peak serum levels tested, demonstrating rapid clearing of saporin from the system.
As a company that specializes in Saporin, our two-plus decades of experience working with the protein in research, preclinical, and clinical environments has taught us that with minimal standard laboratory precautions users are not at any real risk of toxic effects. Even our CSO, after 30+ years of working with Saporin exhibits undetectable levels of Saporin antibodies in his blood!
