FAQ

Q: You have stated that it was unlikely that saporin compounds or constituents would be excreted in urine or feces. However, you acknowledge that experimental data is lacking. Have there been any tests of animal urine or feces for saporin content? My animal care staff are concerned.

A: One of the reasons that no studies have been done on excretion of saporin is that there isn’t much on the theoretical side to cause concern. The primary issue is that the quantity used in mice (and even rabbits) is so small that when looked at in human terms (i.e., an animal 10 to 100-times larger), the dosage becomes insignificant. The LD50 for saporin in mice is 4-8 mg/kg;1 that would translate in humans to more than you’ll ever use! The immunotoxins, which contain only about 20% saporin by weight, really do not contain all that much saporin.

Looking at it another way, you need a concentration of about 100 nM to see even a vague hint of toxicity of saporin to cells. In human blood, that would correspond to 24 mg injected systemically into a person. It would be really expensive for anyone to get close to that number.

As far as urine and feces goes, the same calculations are appropriate, but there will be considerable degradation – the protein content in urine and feces is quite low and the probability is that you will be dealing with only saporin. Remember saporin is a plant protein that is related to proteins in foods that we eat (cucumbers, for example).

Q: Are there any studies which indicate what doses of saporin (by itself or compounded with an antibody) would be hazardous if ingested or injected (i.e. systemic dose level resulting in death or organ dysfunction).

A: When there is an antibody that does recognize a human epitope (the human p75-saporin immunotoxin that is used in rabbits, for example), at about 1 pM one sees the slightest bit of toxicity to cells. That translates, if injected by error into a human blood supply, to about 170 micrograms. That also is a gigantic dose. I am using very conservative numbers here, and the bottom line is that you cannot accidentally reach such dangerous levels under normal handling situations.

Having said all this, we still recommend that our customers take excellent care of themselves and we state clearly that precautions should be taken by people handling these materials, just as they should use precautions with all laboratory chemicals. Please refer to the data sheets provided with our products for safety instructions.

See: Saporin (Cat. #PR-01)

References

1. Stirpe F et al. Hepatotoxicity of immunotoxins made with saporin, a ribosome-inactivating protein from Saponaria officinalis. Virchows Arch B Cell Pathol Incl Mol Pathol 53(5):259-271, 1987.

Safety Instructions

Good laboratory technique must be employed for the safe handling of this product. This requires observation of the following practices:

1. Wear appropriate laboratory attire, including lab coat, gloves and safety glasses.

2. Do not pipet by mouth, inhale, ingest or allow product to come into contact with open wounds. Wash thoroughly any part of the body which comes into contact with the product.

3. Avoid accidental autoinjection by exercising extreme care when handling in conjunction with any injection device.

4. This product is intended for research use by qualified personnel only. It is not intended for use in humans or as a diagnostic agent. Advanced Targeting Systems is not liable for any damages resulting from the misuse or handling of this product.

For disposal: autoclave, or expose to 0.2 M NaOH, materials that come into contact with the toxin.

Q: We’re submitting a protocol to our IACUC to use IB4-SAP (Cat. #IT-10). We plan to inject the targeted toxin and then sacrifice the animal ten days later. What, if any, are the safety issues here?

A: The only danger to lab personnel from IB4-SAP would be accidental self-injection, and even then, at the doses typically used in rats, it would only produce very localized effects at the injection site.

Once injected into animals, the agent is rapidly rendered inaccessible to anyone else by binding, internalization and eventual catabolism. It is extremely unlikely that intact toxin would ever be excreted or recoverable from the rats. The components of the toxin, IB4 and saporin, by themselves are no toxic threat. We use no special precautions with such rats except appropriate care for whatever neurologic deficits they develop, i.e. foot drop, autotomy, etc.

One caveat: To the best of my knowledge the above statements are accurate, but I do not know of any experimental data that directly addresses the issues. I base my comments on our long experience with similar agents including ricin and volkensin which are much more toxic and unstable.

See: Targeted Toxins

Q: How long does it take to see the cell death occurring from the use of targeted toxins using saporin? Is there a time course of hours or days?

A: Details of the time course of early events have not been extensively studied. After ricin injections into the cervical vagus nerve, the proximal nerve becomes unresponsive to electrical stimulation between 36 and 48 hours. After septal injection of 192-Saporin (192-IgG-SAP, Cat. #IT-01), hippocampal theta rhythm begins to diminish on the third postoperative day and reaches a minimum by 7 days which is maintained indefinitely. Anatomical disintegration is complete within 10-14 days after injection of most toxins.

Q: Will this time course be the same regardless of the targeted toxin used or the method of administration?

A: Presumably, injection of toxin into the vicinity of target cell bodies and dendrites should produce effects somewhat sooner than toxin injections into axonal terminal fields where retrograde axonal transport must first deliver toxin to the perikarya. In the cervical vagus, based on transport times for ricin and inhibition of toxin transport by vincristine, we concluded that fast axonal transport is involved. Colchicine coinjected intraventricularly with 192-Saporin (192-IgG-SAP, Cat. #IT-01) prevents destruction of cholinergic basal forebrain neurons suggesting that fast axonal transport also is involved with i.c.v. toxin injections. Consequently, the delay introduced by injecting toxin into axon terminal fields is usually a few hours at most.

Q: What are some assays/methods to use to be able to graphically demonstrate cell death?

A: Toxin-induced cell death can be observed and documented with a variety of techniques. Often the easiest is simple Nissl staining because all of the RIP toxins (ricin, volkensin, saporin) produce profound chromatolysis that is readily apparent in Nissl stains (i.e. cresyl violet).

Electron microscopy can demonstrate details of neuron degeneration including loss of axon terminals at a distance from the cell body which can be useful in anatomic tracing studies.

Typically, target neurons express proteins that can be visualized with immunocytochemical techniques. Thus, immunofluorescence or peroxidase immunohistochemistry can be useful in detecting loss of staining for target molecules and co-expressed molecules in the neurons being targeted. The use of multiple markers is recommended to insure that cell loss occurred rather than down regulation of marker expression.

See: Targeted Toxins

Q: What are the options for delivery of targeted toxins?

A: The options for toxin delivery are varied and limited only by investigator ingenuity. Generally, injection has been the route of choice. Some toxins can be given intravenously, such as 192-Saporin (192-IgG-SAP, Cat. # IT-01) or anti-DBH-SAP (Cat. # IT-03), in which case all cells expressing p75 or dopamine beta-hydroxylase and exposed to the systemic circulation are potential targets. Intravenous injections will not deliver toxins to the CNS.

Subarachnoid injections have been used successfully for immunotoxins and peptide toxins such as SP-SAP (Cat. # IT-07).

Direct intraparenchymal injections have been used to restrict toxin application to just a few target cells. However, intraparenchymal injections require careful attention to injection technique and are impractical for large target structures.

Q: When injecting directly into tissue, are there any special techniques that should be used?

A: Direct injections into brain or spinal cord have been used successfully by some investigators. Specifics of toxin dose, concentration, injection volume and speed of injection have varied considerably. If a high concentration of toxin is deposited locally, lesion specificity is often lost. Presumably, if toxin concentration is too high, cellular uptake by non-specific bulk fluid-phase endocytosis (pinocytosis) can internalize enough saporin to be lethal.

There is currently interest in “convective” delivery techniques developed in the laboratory of Dr. Edward Oldfield at the NIH. The basic principle is to deliver a relatively large concentration slowly over an extended period, often using a rather dilute solution. The parameters for any given species and injection site need to be determined by pilot experiments.

Q: What sort of special care should be given to the animal after administration of the targeted toxin?

A: The toxins generally bind and internalize within minutes, although some immunotoxins circulate for longer periods if injected intravenously. However, no significant amount of active toxin is excreted. So, animals can be returned to group housing immediately after toxin injection. The only special requirements may derive from the specific target being studied. For example, rats given intraventricular 192-Saporin (192-IgG-SAP, Cat. # IT-01) develop decreased fluid and food intake for several days after injection. Since the adipsia is significant, providing the animals with fresh, juicy vegetables, such as cucumber or potatoes, can help.

Rats injected intraventricularly with anti-DBH-SAP (Cat. # IT-03) will lose considerable body weight and are slow to regain. They, too, may benefit from food supplements, including nuts and other high calorie appetizing treats. Otherwise, common sense care of any neurologic deficits is indicated depending on the target and toxin being used.

See: Targeted Toxins

Q: Can you use targeted toxins in vivo?

A: Yes, Molecular Neurosurgery is designed as a tool for in vivo use.

Q: How do you recommend administration of the targeted toxin?

A: There are several ways to administer the toxins depending on the cells being targeted:

1. Direct intraparenchymal pressure microinjection can be used to deliver the targeted toxin directly to target cells. This approach has been used successfully with several toxins, including SP-Saporin (SP-SAP, Cat. #IT-07), in the striatum to kill striatal interneurons that express the NK-1 receptor. Long slow infusions (0.1 µl/min) are probably the best way to do intraparenchymal injections. [1]

2. Targeted toxins can also be injected into terminal fields and retrogradely transported to the cell bodies. This approach has been used successfully to selectively destroy locus coeruleus noradrenergic neurons that project to the olfactory bulb by injecting anti-DBH-saporin (Anti-DBH-SAP, Cat. #IT-03) into the olfactory bulb.[2]

Intracortical injections of 192-Saporin (192-IgG-SAP, Cat. #IT-01) also have been used to destroy cholinergic basal forebrain neurons projecting to the injected patch of cortex.[3]

Lumbar subarachnoid injections of SP-Saporin (SP-SAP, Cat. #IT-07) can destroy lamina I neurons in the dorsal horn that express the NK-1 receptor.[4]

3. Lastly, SP-Saporin (SP-SAP, Cat. #IT-07) has also been applied directly to the surface of the spinal cord to kill lamina I neurons expressing NK-1 receptor. In all cases, pilot studies to determine optimal toxin dose and injection parameters are recommended.

See: Targeted Toxins

References

1. Wiley RG et al. Destruction of neurokinin-1 receptor expressing cells in vitro and in vivo using substance P-saporin. Neurosci Lett 230:97-100, 1997.
2. Blessing WW et al. Destruction of locus coeruleus neuronal perikarya after injection of anti-dopamine-beta-hydroxylase immunotoxin into the olfactory bulb of the rat. Neurosci Lett 243:85-88, 1998.
3. Wiley RG et al. Immunolesioning: Selective destruction of neurons using immunotoxin to rat NGF receptor. Brain Res 562:149-153, 1991.
4. Mantyh PW et al. Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278:275-279, 1997.

Q: What is a second immunotoxin?

A: ATS’s second immunotoxins are conjugations of a secondary antibody (as of December 2000, either goat anti-mouse IgG or goat anti-rabbit IgG) to the ribosome-inactivating protein, saporin.

Q: How does a second immunotoxin target?

A: The second immunotoxin uses the secondary antibody to “piggyback” onto your primary antibody in order to evaluate the ability of the primary antibody to internalize.

Q: What happens when the second immunotoxin gets inside the cell?

A: If the second immunotoxin is internalized, saporin will inactivate the ribosomes of the cell, thereby causing cell death.

Q: Are there different types of second immunotoxins available?

A: Yes, please see our catalog listing online for a complete list.

Q: What is the ratio of antibody to second immunotoxin for in vitro testing?

A: Both Mab-ZAP and Rab-ZAP have been shown effective in concentrations ranging from 0.5 to 2 moles of primary antibody per mole of second immunotoxin.

See: ZAP Conjugates (Second Immunotoxins)