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: 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: 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: For in vitro cytotoxicity assays, could you tell me: 1) whether you incubate primary with your Saporin secondary for a specific amount of time prior to cell addition, and 2) do you use a single concentration of secondary per well or a primary:secondary ratio — like 1:2 or 1:4?
A: The primary antibody should be incubated with the ZAP product for 20 min prior to addition to the cells. Internalization often happens so quickly that you would lose some efficacy due to the antibody being bound and internalized prior to the ZAP product complexing with the primary. We do recommend maintaining a constant 5 nM (~ 45 ng/well) concentration of the ZAP product in the well and titrating your primary only. This way the EC50 you generate will be the EC50 of the primary antibody with all else held constant. The best starting concentration for your primary antibody is 10-100 nM in the well.
Q: We have a publication in review and put this statement in the paper, “The mouse monoclonal antibody to the low affinity nerve growth factor receptor (p75NTR; Advanced Targeting Systems) was derived from immunization of mice with WM245 melanoma cells and recognizes p75NTR in human, primate, rabbit, sheep, dog, cat, and pig. According to the manufacturer’s information, the antibody was tested by flow cytometry.” One of the reviewers wants to know more about the flow cytometry used to characterize this antibody (Cat. #AB-N07). Can you help, please?
A: This antibody is routinely tested by flow cytometry. The quality control flow data can be found on the data sheet on our website. HS294T cells, human metastatic melanoma cells, were used in flow cytometry with Anti-p75NTR (ME20.4, Cat. #AB-N07). Cells were treated with 4 µg of AB-N07 and subsequently with Anti-murine IgG-FITC (Cat. #FL-07). This assay shows the binding affinity of AB-N07 to cells known to express p75NTR.
Q: We are setting up some experiments in rat where we’d use your 192-IgG-SAP (Cat. #IT-01) in cytotoxicity assays. How much material do we need to order?
A: You can find protocols for calculating the amount of material needed for a cytotoxicity assay and protocols for the assay and interpretation of results on our website.
