FAQ

Q: I have a question about what solutions might be incompatible with the conjugated saporins. We have done an experiment where we injected a mixture of saporin conjugate (same batch we’ve used in previous studies here) and a cocktail of 5,7-dihydroxytryptamine and 6-hydroxydopamine (in 0.1% ascorbic acid) to try to deplete multiple neurotransmitters. 

The way we did this was to prepare both solutions at double strength and to mix them immediately before loading the syringe and placing the injections. So the final solution has 0.05% ascorbate, 0.01 mg/ml saporin conjugate, and I think 6 µg/µl 5,7-DHT and 4 mg/ml 6-OHDA.

Anyway, we are doing the histology now and the cholinergic lesion didn’t work. I’m wondering whether the ascorbic acid might have either damaged the conjugation of the saporin to the antibody, or have inactivated the saporin molecule itself somehow.

A: You have well-described what the problem is. A reducing agent will inactivate the toxin, and of course, ascorbic acid is a potent reducing agent. Because of your experience, we have added a line in the data sheet to caution people. This is the first report of this happening in nearly fourteen years of business, so it just had not been an issue.

Related: Targeted Toxins

Q: I’m using your secondary conjugate Mab-ZAP (Cat. #IT-04) and it’s not killing my cells. I’m not following the protocol on the data sheet. I’m doing flow cytometry. I have 70,000 cells per well. I mix Mab-ZAP with my primary antibody and add it. When I count the cells, there is no decrease. My cells grow very slowly.  I didn’t see anything after 72 hours.

A: The protocol on the data sheet is described in detail in the article by Kohls et al.

70,000 cells per well is a lot of cells per well. We use between 500 and 2500 over a 72-hour period and then develop with MTS.

If your cells are slow-growing, you may want to wait a little longer to develop the assay, because the whole metabolism process is slowed. This is a weakness of the MTS system — you have to have a certain number of cells in the end in the control cells to get a decent reading on your plate reader. 

In this case, you might want to try a more sensitive assay such as protein or DNA synthesis inhibition with incorporation of radiolabeled leucine or thymidine.

Related: ZAP Conjugates

References

1. Kohls MD et al. Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 28(1):162-165, 2000.

Q: Can you tell me how I would calculate the amount of a SAP-type targeted toxin that I might want to buy based on a desire to bilaterally kill cells of interest within a typical diffusion zone around an injection site in a rodent brain? Use CRF-SAP (Cat. #IT-13) as an example. Are we talking a few hundred dollars to run 40 animals or thousands of dollars?

A: For intraparenchymal application, usually 100 nanograms per injection site is too much; on the order of 5-10 nanograms is appropriate (for example: Lappi et al.)

So for CRF-SAP: at 10 nanograms per site, 20 nanograms per animal times 30 animals is 600 nanograms. Actual purchase price may vary. However, the average cost for this experiment using one vial of CRF-SAP in the 25-microgram size will run $8-$10 to treat all 30 animals. The CRF-SAP kit includes unconjugated saporin and a control immunotoxin; if you purchase that, the cost for the experiment described with 30 animals will be $12-$15.

Your question about how many injections are needed depends on the diffusion at the site of injection of the agent. Usually, peptide ligand toxins do not diffuse very far and in some cases (such as SSP-SAP, Cat. #IT-11) more than one injection is required. For the antibody targeted toxins, the antibody targeting agent is more resistant to tissue proteases and so there is a greater diffusion, though many researchers like to use several injections to completely cover an area. We’re not sure how big of an area you need or want to deplete; that would, of course, require a dosing regimen to determine.

Q: Will one injection permanently and reliably accomplish the deed?

A: One injection might or might not deplete the entire area that you are interested in; again, it depends on diffusion distance of the agent. However, those cells that are eliminated are eliminated permanently and will not come back. Plasticity to replace them is usually not seen.

Q: What are the typical criticisms (e.g., perhaps related to non-specific effects or interpretive difficulties) that the use of such products engender?

A: The typical reviewer requests demonstration of specificity and making a determination that, in fact, the proper cell type is hit and non-target cells are left intact is important.

Related: Targeted Toxins

References

1. Lappi DA et al. Entering through the doors of perception: characterization of a highly selective Substance P receptor-targeted toxin. Neuropeptides 34(5):323-328, 2000.

Q: I purchased your secondary conjugate, Mab-ZAP (Cat. #IT-04). I am preparing to do a cytotoxicity assay and I’m wondering if my primary antibody should be sterilized prior to combining with Mab-ZAP?

A: Depending on the conditions of your lab in which you are using your antibody, it is possible that within a 72-hour period, you may see bacterial growth in your plates if the antibody was accidentally exposed to bacteria. It is recommended that, if you feel comfortable with the antibody, you can just go ahead and try it without sterilizing it, and if you do see bacterial growth, you can certainly filter sterilize the material through a 0.2 micron filter before using.

Related: Mab-ZAP (Cat. #IT-04)

Q: I have a few questions about the Alexa488-labeled affinity-purified NGFr antibody (Cat. #AB-N43-FLA). Is it specific to extracellular p75? Can you use it on live cells? Does it work on fixed cells? Does it cause activation of the p75 receptor (i.e., result in apoptosis or changes in axon outgrowth in neuronal cells)?

A: This product does recognize extracellular p75 in both live and fixed cells. As for the activation, that’s an interesting question. There is no evidence of 192-IgG either causing apoptosis or neurite outgrowth as far as I can see. Chandler et al. (1984) report that the antibody “partially inhibits the regeneration of neurites from primed PC12 cells,” and it enhances NGF binding. But that’s about it, despite several studies being done with PC12 cells and in vivo. We assume all this holds upon treatment with 192-IgG-SAP (Cat. #IT-01) — until the cell dies from saporin poisoning.

Chandler CE, Parsons LM, Hosang M, Shooter EM (1984) A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells. J Biol Chem 259(11):6882-6889.

Related: NGFR (192-IgG, p75) Mouse Monoclonal, Alexa488-labeled (Cat. #AB-N43-FLA)

Q: Is there is any indication that intrastriatal administration of 192-IgG-SAP (Cat. #IT-01) will lesion the cholinergic neurons of the striatum. My sense from reviewing the literature is that these cholinergic neurons are not susceptible to the toxin, but I thought I’d ask to see if you had any information / experience regarding this point.

A: No, I don’t think it will work because the target of 192-IgG-SAP is p75, LNGFr, which is only expressed on the rat basal forebrain cholinergic neurons. Those striatal neurons don’t express p75 in the adult. The NK1r is often expressed in the striatum, and you can use SSP-SAP (Cat. #IT-11) for them, but that’s the best we can do right now.

Related: 192-IgG-SAP (Cat. #IT-01), SSP-SAP (Cat. #IT-11)

Q: Your targeted toxin data sheet gives the following instruction for disposal: “Care in disposal is mandatory; autoclaving or exposure to 1 M sodium hydroxide will inactivate the material. All labware that comes into contact with this material should be likewise treated.” I am wondering if I can deactivate saporin by using 10% bleach or if everything has to be autoclaved?

A: Yes, you can use bleach to deactivate saporin prior to disposal or reuse of labware. If you are using nanogram quantities, these are too low to be toxic, so you can discard as you do your other non-hazardous laboratory materials without fear.

Related: Targeted Toxins

Q: Do conjugated toxins (dermorphin-saporin in particular) exhibit agonist effects? I’ve generated behavioral and tissue time course effects but have not established agonist effects for this conjugated toxin.

A: The peptide ligand toxins should exhibit agonist effects. They are constructed purposely to retain complete agonist activity, including for us the most important: internalization. So, for instance, SP-SAP (Cat. #IT-07) causes receptor internalization similar to SP, as reported in Mantyh et al.

As to dermorphin-SAP (Cat. #IT-12) specifically, it has agonist activity very much like dermorphin. This is reported in Porreca et al. in which it’s stated:

The bilateral microinjection of 3 pmol of dermorphin or of dermorphin-saporin directly into the RVM produced a robust antinociceptive effect in the 52°C hot-water tail-flick test. The peak antinociceptive effect of dermorphin, 78 ± 13.2% MPE, was not significantly different from that of the dermorphin-saporin conjugate, which was 59 ± 4.7% MPE (p > 0.5, Student’s t test).

Usually the amount needed to give a response is lower than the amount needed to kill a cell. Depending on what your system is; it may be a peculiarity of that system, but I would be a little concerned about not seeing an agonist effect. On the other hand, if you have demonstration of specific toxicity, it may not be all that crucial.

See: Targeted Toxins

Q: Your targeted toxin kits come with different controls. I’m not sure of the best way to use them. For example, with the anti-SERT-SAP kit (Cat. # KIT-23) 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: Yes, perhaps we do have a few too many options for controls; better too many than too few. For anti-SERT-SAP, the ideal control is mouse IgG-SAP (Cat. # IT-18). Anti-SERT-SAP is made from saporin conjugated to a mouse monoclonal IgG that has SERT as its antigen. So, mouse IgG-SAP – that is, saporin conjugated to mouse IgG that has no specific antigen for targeting – would be the best control, in my mind. For years, the unconjugated antibody and unconjugated saporin mixed together was the best control available (until we came out with the “irrelevant” control immunotoxins), and still might be considered a second good control, or useful in cases where down-regulation by the antibody is a concern.

Q: What about for the peptide toxins like orexin-SAP (Cat. # IT-20) or SP-SAP (Cat. #IT-07)— what controls are available for those?

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’s like shooting blanks; 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. The best use we have seen for Blank-SAP has been in Bugarith et al. 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!

See: Targeted Toxins, Control Conjugates

References

1. Bugarith K et al. Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Endocrinology 146(3):1179-1191, 2005.

Q: We injected anti-DBH-SAP (Cat. #IT-03) into the hypothalamus of Sprague-Dawley rats and sacrificed them 2 weeks later. We did not see any reduction in the DBH fiber staining.

When the drug arrived, we aliquoted it in 1-µl snap-cap tubes on ice, and stored them at -80°C. For injections, a 1-µl aliquot was diluted to a little over 10 µl so that we had a final concentration of 1 µg/10 µl. We administered two injections of 100 nl on each side (with 10 ng of anti-DBH-SAP) using a 0.5-µl Hamilton syringe attached to a stereotax. The needle was a 33-gauge with a blunt tip. I tried previously to use glass micropipette tips attached to a Hamilton syringe with the line filled with mineral oil, but found that the actual volume displacement was too unreliable.

A: We have not had any problems related to the stability of anti-DBH-SAP. In our work, failure to lesion is nearly always associated with a misplaced injection. From the information conveyed, I would suggest the following:

(1) It is possible that no drug was actually delivered to the brain. Two things could be done to ensure drug delivery. The first would be to add a tracer to the saporin solution that could be identified histologically. The second would be to visually monitor drug delivery using a calibrated tip. Air bubbles, pressure leaks and compression of the liquid can interfere with accurate delivery.

(2) It is possible that the anti-DBH-SAP was not delivered to the correct site, so that the expected uptake into the targeted terminals did not occur. Again, marking the site so it is clear where the injection was would help evaluate your accuracy. Establishing a reliable set of stereotaxic coordinates that work in your lab, in your rats and with your equipment and then using a dye to estimate the diffusion radius of your selected injection volume are always good ways to start. However, that being said, it should not be difficult to locate the injection site with such a large injector (33 g) – so #1 seems more likely to be the problem in the case you describe. Also, I would add that the larger the injector, the more nonspecific damage there will be. Glass capillary micropipettes are by far preferable to stainless steel cannulas in providing more reliable delivery of small volumes and in producing less nonspecific damage. Chronically implanted cannulas should be avoided, in my opinion, because gliosis at the cannula tip is apt to occur and this may alter the diffusion pattern of the injected substance, as well as interfering with lesion analysis.

(3) Try a different anesthetic. We have not tested a lot of anesthetics, but we have had problems getting a good lesion that we think are attributable to use of a ketamine/xylazine/acepromazine anesthetic cocktail. So we routinely avoid that one.

(4) I assume you are looking at fibers in the area of the injection. If not, it would be important to make sure the fibers being evaluated are associated with the same neurons innervating the terminal field at the injection site. Secondly, the 2-week wait mentioned between toxin injection and histology is critical for evaluating the lesion to assure that immunoreactive products are no longer present. Making sure that tissue processing controls are stringently adhered to so that controls and lesioned animals are run together in the same batch is also important.

(5) You might try injecting only one side and comparing terminal staining with the non-injected side in the same animal. This would not be a good idea, however, if the injection site is too close to the midline, so that both sides might be damaged from a unilateral injection.

Related: Anti-DBH-SAP (Cat. #IT-03)