Today’s topic is time-course for our saporin conjugates. Common questions we get are (1) how long does it takes to see cell death, (2) how long should I wait before performing histology on an animal, or (3) how long before I see behavioral changes in an animal?
In this image from Mantyh et. al. (1997), we are looking at confocal imagery of the binding and internalization of our peptide conjugate SP-SAP to the NK1r receptor in primary cultures of neonatal spinal cord neurons.
As you can see, conjugate binding occurs immediately where within hours, the SP-SAP has recognized and bound to the NK1 receptor. Here we see the areas of concentrated NK1R expression marked by yellow immunofluorescence.
But how long until you see cell death? Here is a cytotoxicity graph of in vitro data of our antibody conjugate 192-IgG-SAP. These are typical data after cells have been treated for 3 days, which is standard protocol.
Waite et al. (1994) used 192-IgG-SAP and showed the appearance of behavioral effects associated with neuronal loss at day four and plateauing at day 7.
This coincides with the time course seen in vitro. At this point, microglia will infiltrate, but stop at 7 days, which is probably the peak day for infiltration. Once there is complete removal of the detritus, microglia down-regulate and at 14 days all debris is cleared and the animal begins to regain normal eating and sleeping habits. This is the idea behind waiting about 2 weeks before performing histology.
ZAP conjugates allow you to screen targeting agents in a quick and cost-efficient way, looking for specificity, functional binding, internalization, and EC50 determination.
We are finishing off our topic of ZAP conjugates with a closer look at the typical in vitro data you can expect to see from a cytotoxicity assay.
In this example, you are looking at the cytotoxicity curves using various secondary conjugates when compared to the direct conjugate, 192-IgG-SAP.
Mab-ZAP, our bivalent secondary antibody that recognizes whole IgG, reacted with primary antibody, produces a similar potency to the directly linked conjugate of saporin to the same antibody.
Interestingly, in this example Fab-ZAP and FabFc-ZAP, which both use a monovalent secondary antibody, reacted with primary antibody produced a cytotoxic effect greater than 12-fold over the direct conjugate.
In this example, you will also see an interesting phenomenon with ZAP secondary conjugates. 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 example, at the highest concentration of 192-IgG (10 nM = Log (-8)) there is a lessened amount of killing, at a 25-fold lower concentration when compared to the antibody.
The explanation is that at the higher concentrations of primary antibody, there are more unconjugated 192-IgG and fewer 192-IgG plus Fab-ZAP complexes. So, this free 192-IgG can then out-compete the conjugates for cell surface binding sites, which, in turn, decreases the amount of saporin being internalized, hence less cell death.
Our publication in the Journal of Toxins, provides a nice review of this phenomenon.
We are continuing to highlight our ZAP antibody internalization kits and our line of secondary antibody saporin conjugates.
ZAP conjugates allow you to screen targeting agents in a quick and cost-efficient way, looking for specificity, functional binding, internalization, and EC50 determination.
We also highlighted a publication that did a great job in showcasing how they can be used to screen antibodies for the best candidate, and then proceed with having us perform a custom conjugation of that antibody directly to saporin.
Here is information about the chemistry and why we offer conjugates that use whole, bivalent, IgG as well as monovalent Fab IgG.
Our first release of ZAP conjugates utilized whole-molecule IgG, bivalent secondary antibodies that recognized both heavy and light chains of primary antibodies. In theory though, this presented a possible limitation if the bivalent nature of an antibody suggested that cross-linking could occur on the cell surface and contribute to a phenomenon known as ‘cap formation’. The ‘cap’ could potentially induce some level of endocytosis that would lead to cytotoxicity and produce a false positive for internalization of a primary antibody.
We designed new secondary conjugates (given the moniker Fab-ZAP) which used monovalent antibodies that would continue to recognize whole IgG, but lacked components that would contribute to capping.
We now offer 3 main categories: (1) Conjugates made with bivalent antibodies that recognize whole IgG, (2) conjugates made with monovalent antibodies that recognize whole IgG, and (3) conjugates made with monovalent antibodies that recognize only the Fc region of IgG.
We are highlighting our ZAP antibody internalization kits, as well as our line of secondary antibody saporin conjugates. These products are given the moniker “ZAP”in place of saporin.
The ZAP conjugates produced by ATS consist of a variety of secondary antibodies that allow a large number of targeting agents to be screened quickly and cost-efficiently for specificity, functional binding, internalization, and EC50 determination.
The conjugates are constructed using either species-specific secondary antibodies, or streptavidin (for use with biotinylated Targeting Agents), and they are chemically attached to Saporin, a potent plant ribosome-inactivating protein.
Use of a ZAP conjugate eliminates the time-consuming and expensive step of conjugating each targeting-agent-candidate to the payload. The ZAP conjugate can simply be reacted with a targeting agent in a single step and added to cells in culture conditions.
Once the reacted conjugate has been administered, the Targeting Agent seeks out and binds its receptor and takes the saporin payload inside the cells of interest, where it is released within the cytosol to inactivate the ribosomes. Cells that don’t express the target cell surface marker don’t bind or internalize the ZAP-targeting agent complex, and won’t be affected. Saporin has no binding chain, and no means of getting into cells on its own.
Our line of pHast conjugates are one of our fastest tools to quantitatively test your primary antibody’s specificity, binding, and internalization, providing results in 1 day.
The pHast conjugate uses a secondary antibody that is cross-linked to a pH-dependent fluorescent reporter and will piggy-back onto your primary antibody.
This fluorescent reporter will increase intensity as the pH of its surroundings becomes more acidic, such as you would see on the inside of a cell.
There are various options available in our pHast Conjugates line.
Our pHast conjugates can recognize and bind to various species, such as human, mouse, rat, rabbit, goat, guinea pig, chicken, and the newest arrivals are the conjugates that can bind to VHH domain, typically seen in heavy chain in alpaca IgG.
We also offer streptavidin conjugated to our pHast dyes which opens up the choice to use a biotinylated targeting agent.
Not only do we offer various species for targeting, but we also offer various pH dependent fluorescent dyes, allowing you to multiplex in the same experiment.
We’re highlighting one of our antibodies, a rabbit polyclonal against Melanopsin. This antibody is offered as both serum (AB-N38) and affinity purified (AB-N39). Intrinsically photosensitive retinal ganglion cells (ipRGCs) are cells that express melanopsin. These ipRGCs, with their long processes, are involved in the perception of light and dark and are circadian rhythm determinants. Our Melanopsin antibody recognizes a portion of the N-terminal region of the mouse melanopsin extracellular domain and does not cross-react with melanopsins of other species, so it’s specific to mouse.
Here is how other researchers are using them in recent publications. This Melanopsin antibody may be the antibody your lab needs.
Kim et. al. used (AB-N38) at a 1:2,000 dilution to quantify ipRGCs in the retina and confirm that Per1-deficiency did not affect melanopsin-positive cell abundance.
And finally, Zhu et. al. using affinity purified (AB-N39) at a 1:2000 dilution to identify and quantify ipRGCs in retinal whole-mounts following ischemic injury.
I am using your Fab-ZAP Human (IT-51) and I want to know how many antibody molecules can a secondary antibody conjugate theoretically bind to?
Answer
A whole IgG (bivalent) secondary antibody in theory is capable of binding two antigen molecules. Since our Fab-ZAP conjugates actually use a monovalent secondary antibody, in theory they would be able to each bind one antigen molecule. Our Fab-ZAP human conjugates use polyclonal monovalent secondary antibodies raised against both heavy and light chain of IgG and can cross-react across immunoglobulin classes and subclasses of the same species since they share the same light chain. In theory it would be possible that your primary antibody could have several secondary conjugates attached.
Question
When exploring the usage concentration for Fab-ZAP Human, what is the recommended concentration range (for example, is it a few times the initial concentration of the analyte antibody / the highest test concentration)?
Answer
Our standard protocol (in vitro only) for our Fab-ZAP secondary conjugates is to maintain a constant concentration of 4.5 nM of the Fab-ZAP while titrating the primary antibody, usually in a range starting at 10 nM with 1:5 to 1:10 serial dilutions. The goal would be to have the primary antibody as a ‘variable’ and the Fab-ZAP as the ‘constant’. Also, it’s important to saturate the primary antibody with Fab-ZAP secondary conjugate, as any ‘free unreacted primary antibody’ would compete with ‘antibody+Fab-ZAP conjugate’ for binding sites on the cell. I have attached a publication that discusses this topic.
How is a targeted toxin able to inhibit a cellular process when an antagonist did not?
An antagonist is used to block a receptor on a cell to keep it from binding a target molecule and activating the cell.
For example, a substance P antagonist binds to the substance P (NK-1) receptor. The hypothesis was that if the antagonist binds to the receptor, substance P can’t bind and the cell won’t be activated.
The reality is that there are other receptors besides the substance P receptor on that cell. If any of these other receptors bind to their target molecules, then the cell will still be activated.
The Targeted Toxins with our targeting technology can use any of these cell surface receptors to target and completely eliminate the entire cell. Since there are no receptors left to bind; no cell left to be activated.
Importantly, our conjugates cleanly remove one particular cell type and don’t damage bystander cells.
Once the debris from the targeted cell is cleared away, there is nothing remaining to interfere or affect the normal action/interaction of other cells.
Have you been wondering if ordering in bulk is the better move for your upcoming project? Wondering if there is pricing incentive when buying larger quantities? The quick answer is yes!
The best cost per microgram is larger 1 mg size, where we typically give the biggest price break and seeing upwards of a 20% discount when comparing four 250 ug vials and the 1 mg vial.
Another benefit of ordering in bulk is maintaining lot consistency for your project. This allows you to develop a consistent protocol and not have to be concerned with variability in batch-to-batch protein concentrations.
We understand that typically the upfront cost of buying in bulk can be the limiting factor, so I want to take a second and let you know that we also offer plans where you can reserve 1-2 mg over a 12-month period for a specific lot, and just pay for the amount that you draw down.
ATS offers a custom biotinylation service to biotinylate antibodies and peptides, especially when customers plan to use them with our Streptavidin-ZAP.
We have been seeing a trend of variability with commercially available antibodies, where customers will see over or under derivatization of material and this can be an issue if you plan to use it with Streptavidin-ZAP.
There are a few fundamentals to look for when choosing/creating your biotinylated targeting agent:
(1) the linker doesn’t impair target-binding affinity,
(2) the linker should be stable and
(3) the linker should be able to release payload efficiently.
Important details to know about your biotinylated targeting agent:
(1) the ratio of biotin to the protein,
(2) unreacted biotin is purified away
(3) the length of linker because long linkers could interfere with binding and
(4) location of where biotin is attached such that important protein side-chains weren’t impacted.
If any of these parameters are unfamiliar to you or you weren’t given these details, then I strongly encourage you to talk to us about a custom biotinylation service.
