FAQ

Q: You have stated previously 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 go, 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.

Q: We are re-examining some data collected using an immunotoxin not prepared by your company (VChAT-sap). Our results in vivo indicated that there were non-specific effects although the creators claimed it was specific. We ran a Western Blot and determined that about half the saporin was not bound to the antibody. This may have been the problem but I want to confirm the cytotoxicity of unbound saporin. Can you confirm that? Further, the antibody in question never bound selectively in ferret tissue. Does this suggest a problem as well? Can you give me information on the best way to design and test an immunotoxin.

A: Yes, saporin at higher concentrations can be cytotoxic. Without specific binding, you will only see non-specific cytotoxicity. The sequence of the immunogen for that antibody is from the rat protein, so I’m not sure if it would target the ferret protein (there usually is good sequence homology among transporters). We worked a lot with this antibody and with an immunotoxin (made by us), but never got any sort of results that would indicate that it was working. We also were greatly concerned that the epitope is an intracellular epitope, and so we have difficulty understanding, from a theoretical standpoint, how it even could work. Because of many concerns, we never commercialized it, and we believe all the effects in the literature were non-specific, but “credible” because of the unusual experimental system that was used.

The best way to design and test an immunotoxin is to talk to us. If your antibody has been tested and shown to be internalized by the cell you are targeting, there should be no problem with the activity. The conjugate will only work as well as your antibody does. We recommend that you try a second immunotoxin before having a custom conjugation performed. This allows you to use a secondary agent conjugated to saporin that “piggybacks” on your antibody and makes a second immunotoxin for use in vitro to test specificity and internalization of your antibody. You can check out the publication that talks about one of the secondary conjugates, Mab-ZAP (Cat. #IT-04). We also have many other secondary conjugates or you can biotinylate your material and use Streptavidin-ZAP (Cat. #IT-27). 

See: ZAP Conjugates, Custom 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: Why do your directions for SSP-SAP (Cat. #IT-11) state that it is to be used within hours after dissolution? To my knowledge, both proteins and peptides are stable in clean solution.

A: In fact, the two components of SSP-SAP (Stable Substance P and Saporin) are quite stable. However, we have found that many things happen in laboratories and some of them can impact stability. Probably the most severe is the loss of sterility. In that case, over time at room temperature or at 4°C, bacteria can grow on this rather excellent “medium.” This would cause inactivation. Because many laboratories, due to molecular biology work, have high levels of resident bacteria, we prefer to emphasize playing it safe.

Even if saporin is a stable protein, it is a protein and can suffer denaturation. This occurs more rapidly at room temperature than at 4°C, and hardly at all in the frozen state (really, it is stable for years when stored at -80°C). The maintenance of precise activity is of extreme importance to our customers who use these materials in vivo (their assays are very sensitive), and so we choose to advise the most conservative course.

Q: I understand that theoretically only one molecule of Saporin taken up by a cell is enough to induce cell death. I have been looking for literature on this topic but have not come across anything.

A: Definitely theoretical. The only article that we know of that states anything close to that is: Yamaizumi et al (1978) One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15(1): 245-250. As you can see, this article speaks to the enzymatic chain of diphtheria toxin, which has a slightly different mechanism of action for shutting down protein synthesis, but otherwise is similar to saporin. In fact, we test all sorts of toxins against cells in controlled conditions, and we have only one candidate that is in this range; all the rest are orders of magnitude away. It takes more than thousands per cell. Another question would be: how many actually get in?

In another FAQ, you addressed the question of one molecule of saporin killing a cell. Your response overlooked the data on ricin, abrin and modeccin (Eiklid, Olsnes and Pihl, Exp Cell Res, 126:321-326, 1980). In that paper, they showed that these RIP toxins applied to cells in culture produce all-or-none lethality. They used radioactive amino acid uptake and incorporation (as memory serves) and found only two types of cells, those with absolutely no uptake of label or those that were entirely normal – nothing in between. Also, if the data on ricin-induced apoptosis is correct (numerous authors), and I believe it is, then at low doses, the cells die from triggering apoptosis which seem possible with a single molecule of RIP free in the cytoplasm. To further complete your answer, someone (I haven’t found the article yet) showed that it took, on average, about 10,000 molecules of ricin/cell to kill cells in culture. This gives a hint at the efficiency of internalization and translocation in that cell type. I am not aware anyone else has looked at these issues with saporin conjugates.

A: Overlooking literature is actually a favorite sport of mine, but in this case I would respectfully point out conflicting information. There is a study of something that is quite between an all-or-none phenomenon: Barbieri et al. FEBS Lett, 2003 Mar 13;538(1-3):178-82. These authors document that ribosome-inactivating proteins have transforming activity on the classic FDA assay cell line: NIH3T3 cells. This would be a non-toxic activity that one presumes is due to internalization, and is somewhat on the none side of all or none, but hey, it’s an activity nonetheless. 

See: Targeted Toxins

Q: What dosage of 192-Saporin (192-IgG-SAP, Cat. #IT-01) should be used in the lateral ventricle to eliminate cholinergic neurons in the basal forebrain, including substantia innominata (SI)? I read that Calza et al [1] used 2 or 3 micrograms/4.5 µl and found this was highly effective.

A: It has been our experience that two- or three-micrograms into the lateral ventricle is necessary to obtain a maximum cholinergic basal forebrain (CBF) lesion. However, these doses typically kill some cerebellar Purkinje cells. Another issue is that some cholinergic neurons in the NBM region are never killed by 192-Saporin.

Q: Should we expect to be able to kill all or almost all ChAT SI neurons?

A: Mesulam’s lab has some data [2,3] to suggest that these neurons innervate the amygdala and adjacent cortex. Generally lesions of the septum and diagonal band are complete, but when you get more caudal, i.e. SI region, there will be some cholinergic neurons left. When you do ChAT or AChE stains, the amygdala and adjacent cortex are not denervated whereas the hippocampus, olfactory system and all the rest of the cortex are devoid of cholinergic terminals.

Q: Is there another toxin that will eliminate the remaining ChAT SI neurons?

A: There may be other targeted conjugates that could clean out the residual cells in the SI region if we knew what markers they co-express. For example, our SSP-saporin (Cat. #IT-11) conjugate is very good at removing cells that express the NK-1 receptor such as striatal cholinergic interneurons.

See: 192-IgG-SAP (Cat. #IT-01), SSP-SAP (Cat. #IT-11), Targeted Toxins

References

1. Calza L et al. Neural stem cells and cholinergic neurons: Regulation by immunolesion and treatment with mitogens, retinoic acid, and nerve growth factor. Proc Natl Acad Sci U S A 100(12):7325-7330, 2003.
2. Heckers S et al. Two types of cholinergic projections to the rat amygdala. Neuroscience 60:383-397, 1994.
3. Heckers S et al. Complete and selective cholinergic denervation of rat neocortex and hippocampus but not amygdala by an immunotoxin against the p75 NGF receptor. J Neurosci 14:1271-1289, 1994.

Q: I recently tried to order avidinylated-SAP (Cat. #IT-09) and was told that this product has been replaced with a new product, Streptavidin-ZAP (Cat. #IT-27). Why did you replace avidinylated-SAP?

A: We initially had good results with avidinylated-SAP. It combined well with biotinylated antibody to produce extremely potent cytotoxic materials, and had low toxicity itself. However, the weaknesses of avidin are well-documented. Probably the most severe is its high isoelectric point that has been suggested to cause nonspecific binding. As we produced more batches of avidinylated-SAP and completed comparative studies, we in fact, found this to be the case.

Q: I use avidinylated-SAP to demonstrate that my antibody internalizes. It worked quite well for me.

A: A couple of months ago we received two reports from customers that they were seeing that, even in batches that had performed well in quality control testing, there was a non-specific cytotoxicity with some cells and/or cell lines. Since a major use of this material is to demonstrate internalization of the biotinylated targeting agent, this was an unacceptable situation. We changed to streptavidin to overcome these specificity issues. As shown in the figure, streptavidin-SAP has an excellent capacity to transform a biotinylated reagent into a potent cytotoxic targeting vehicle, while streptavidin-SAP alone has no detectable cytotoxicity.

See: Streptavidin-ZAP (Cat. #IT-27)

Q: Are all lots of 192-Saporin (192-IgG-SAP, Cat. #IT-01) the same?

A: There are variances from lot to lot, and Advanced Targeting Systems includes a cytotoxicity graph on the data sheet with each product comparing the current lot with previous lots. Chemicon International also sells immunotoxins, and used to distribute ATS products. However, since early 2002 (according to Chemicon) they have been outsourcing their 192-Saporin from an un-named laboratory. A lot obtained from Chemicon was tested under ATS quality control conditions (see graph) and was found to be significantly less active than any of the ATS lots.

Q: How do I find out the optimal dosage?

A: For each new lot and each new application of immunotoxin, it is recommended that the end user perform preliminary tests to ascertain the proper dosage. The material used and the method of administration are important aspects of each experiment that should be carefully considered prior to beginning a full-blown project.

Q: What happens if I use too much immunotoxin?

A: Customers who had used the Chemicon material and then ordered 192-Saporin from ATS have reported that they needed to reduce the dosage level with ATS product. The Chemicon material was not as potent. Higher doses of 192-Saporin, (as described in Leanza et al.) cause deficits in hindlimb coordination and support, and ataxia. So it is important to use less if you’ve switched from Chemicon’s material to the ATS 192-Saporin.

See: 192-IgG-SAP (Cat. #IT-01)

References

1. Leanza G et al. Selective lesioning of the basal forebrain cholinergic system by intraventriculr 192-IgG-saporin: behavioural, biochemical and stereological studies in the rat. Eur J Neurosci 7:329-343, 1995.

Q: When performing intraparenchymal injections of immunotoxin, what is the proper volume to use? Is it better to induce two half-portions per hemisphere or is a higher concentration better? At what concentration do you expect necrosis or inflammation?

A: There is no one answer to the question of injection volume. Practically speaking, we have observed that large or extended structures such as the entire cholinergic basal forebrain (CBF) of rats are difficult to ablate with one single injection of 192-Saporin (192-IgG-SAP, Cat. #IT-01). The best results were obtained with 3-5 separate 0.5-1.0 µl injections. For even larger targets such as the CBF in primates, other strategies may be necessary. Oldfield and co-workers have reported success in delivering cytotoxic chemotherapy to large volumes of brain using long, slow infusions of solutions containing a low concentration of toxin (convective delivery). This procedure delivers toxin by bulk fluid flow rather than diffusion and avoids high local toxin concentrations around the infusion catheter or pipette. High local concentrations of toxin may compromise selectivity and produce non-specific cytotoxicity. This can occur when neurons and glia take up toxic amounts of saporin by bulk fluid phase endocytosis, rather than receptor-mediated endocytosis. With direct intraparenchymal injections, local necrosis can occur with surprisingly small doses of toxin. For example, 60 ng of SP-SAP (Cat. #IT-07) or dermorphin-SAP (Cat. #IT-12) into the rat striatum injected in 1 µl typically produces some necrosis in the center of the injection site. With immunotoxins such as 192-Saporin (192-IgG-SAP) or Anti-DBH-SAP (Cat. #IT-03), 200 ng in 0.5 µl may barely produce a trace of local damage.

Q: We are interested in the anti-Thy-1 nephritis model in rats. I want to know the titer of OX7-SAP (Cat. #IT-02) and how much we have to expend for each rat to establish the model?

A: The “titer” of OX7-SAP is rather difficult to define. It is not known precisely how many molecules of this immunotoxin are necessary to kill a thymic-derived (Thy-1-expressing) lymphocyte in vivo. Also, since OX7-SAP kills Thy-1-expressing lymphocytes, it may prove difficult to induce Thy-1 nephritis with the immunotoxin. In humans, proteinuria was reported in clinical trials using immunotoxins for treatment of cancer, but we do not know of any comparable data in rats. Probably the only way to determine the appropriate dose would be a dose ranging study.

Q: I have been doing research with 192-Saporin (192-IgG-SAP, Cat. #IT-01) for 2-3 years now. I have read in the literature that animals with cholinergic lesions often get sick following surgery and require potatoes, apples, lettuce and saline injections. They may even stop eating or drinking all together. I have followed these practices in the past, but stopped when it didn’t seem to make a difference. (I use a very small insignificant dose that is not prone to make animals ill). This is the first death I have had even remotely possibly related to the toxin. In short, the animal lost 64 grams over a period of 2 weeks, and expired 1-2 days thereafter. I weighed her at death and she was 139 grams (91 gram difference from her initial surgery weight). The rats in our colony are fed and given water ad libitum. However, we think that she dehydrated. She was given the same dose (1.1 microliters) as all of the other rats in the experiment. I do have other rats that were given injections from the same lot that do not appear to be sick or losing weight. I’m not sure what you can do with this information, but I would be grateful for whatever help you can offer.

In large series of intraventricular injections of 192-Saporin (192-IgG-SAP), I have never encountered quite the same sequence of events you describe. Death after intracranial toxin injection can reflect several possible misadventures including but not limited to:

1. Contamination of toxin solution with endotoxin resulting in death without awakening from anesthesia (usually due to bacterial contamination from prolonged exposure of toxin solution to room temperature),

2. Fatal intracranial hemorrhage which may result in delayed death depending on location and volume of bleeding,

3. Intracranial abscess (extremely unusual) from injection of contaminated solution, or

4. Unrelated bacterial, viral or parasitic systemic disease.

Q: Another one of my 192-Saporin-treated (or 192-IgG-SAP, Cat. #IT-01) rats is having an adverse reaction, including paralysis of the lower extremities. She has lost 40 grams in the past 3-4 days. Could this be Purkinje cell damage; does that happen after five weeks?

A: Purkinje cell damage after intraventricular injection of 192-Saporin (192-IgG-SAP) typically is manifest not by hind limb paralysis but rather tremor (shaking) and ataxia (clumsiness, poor balance). At less than lethal doses, 192-Saporin (192-IgG-SAP) does not produce paraparesis. Something else is going on. Rats develop hind limb paralysis from a variety of toxic or metabolic systemic insults in addition to specific nervous system disorders. The presence of rapid weight loss suggests the rat is systemically ill rather than an effect of a sub-lethal dose of 192-Saporin (192-IgG-SAP).

Selected references on convective delivery of toxin to brain:

Nguyen TT, Pannu YS, Sung C, Dedrick RL, Walbridge S, Brechbiel MW, Garmestani K, Beitzel M, Yordanov AT, Oldfield EH (2003) Convective distribution of macromolecules in the primate brain demonstrated using computerized tomography and magnetic resonance imaging. J Neurosurg 98(3):584-590.

Morrison PF, Chen MY, Chadwick RS, Lonser RR, Oldfield EH (1999) Focal delivery during direct infusion to brain: role of flow rate, catheter diameter, and tissue mechanics. Am J Physiol 277(4 Pt 2):R1218-R1229.

Lonser RR, Corthesy ME, Morrison PF, Gogate N, Oldfield EH (1999) Convection-enhanced selective excitotoxic ablation of the neurons of the globus pallidus internus for treatment of Parkinsonism in nonhuman primates. J Neurosurg 91(2):294-302.

Wood JD, Lonser RR, Gogate N, Morrison PF, Oldfield EH (1999) Convective delivery of macromolecules into the naive and traumatized spinal cords of rats. J Neurosurg 90(1 Suppl):115-120.

Lonser RR, Gogate N, Morrison PF, Wood JD, Oldfield EH (1998) Direct convective delivery of macromolecules to the spinal cord. J Neurosurg 89(4):616-622.

Laske DW, Morrison PF, Lieberman DM, Corthesy ME, Reynolds JC, Stewart-Henney PA, Koong SS, Cummins A, Paik CH, Oldfield EH (1997) Chronic interstitial infusion of protein to primate brain: determination of drug distribution and clearance with single-photon emission computerized tomography imaging. J Neurosurg 87(4):586-594.

Lieberman DM, Laske DW, Morrison PF, Bankiewicz KS, Oldfield EH (1995) Convection-enhanced distribution of large molecules in gray matter during interstitial drug infusion. J Neurosurg 82(6):1021-1029.

Bobo RH, Laske DW, Akbasak A, Morrison PF, Dedrick RL, Oldfield EH (1994) Convection-enhanced delivery of macromolecules in the brain. Proc Natl Acad Sci U S A 91(6):2076-2080.

Morrison PF, Laske DW, Bobo H, Oldfield EH, Dedrick RL (1994) High-flow microinfusion: tissue penetration and pharmacodynamics.Am J Physiol 266(1 Pt 2):R292-305.

See: Targeted Toxins

Q: I’m interested in using SAP to eliminate cells through retrograde transport, like OX7-SAP (Cat. #IT-02) and IB4-SAP (Cat. #IT-10) have been used. Can you explain how retrograde transport works and if it is possible for this to work with dermorphin-SAP (Cat. #IT-12)? What determines whether a targeted toxin will be able to be used in retrograde transport?

A: Current evidence indicates that effective suicide transport agents undergo endocytosis at nerve terminals followed by retrograde axonal transport of the endocytic vesicles containing the toxin. Experiments using vincristine have shown that the retrograde axonal transport of suicide transport toxins utilizes the fast transport system (microtubules). However, it is not known what determines whether or not a specific toxin-ligand undergoes axonal transport after internalization.

Empirically, it has been observed that immunotoxins (OX7-SAP, 192-Saporin or 192-IgG-SAP [Cat. #IT-01], anti-DBH-SAP [Cat. #IT-03]) and lectin-toxins (ricin, volkensin, IB4-SAP) all undergo retrograde axonal transport and are therefore effective suicide transport agents. This is not true, however, for neuropeptide-toxin conjugates, such as dermorphin-SAP. For example, in an unpublished study, we injected large doses (1-2 µg) of dermorphin-SAP into the lumbar intrathecal space of rats. After 2-3 days, rats were sacrificed and lumbar dorsal root ganglia examined for evidence of toxin effect (striking chromatolysis). None was found after examining numerous ganglia and >15,000 primary afferent neurons. Apparently, dermorphin-SAP is not retrogradely transported even if it is taken into the primary afferent terminals that express the mu opioid receptor (MOR).

Q: If a targeted toxin cannot be used in retrograde transport, will it only kill cell bodies in the injection site or will it also kill terminals?

A: Current evidence suggests that applying dermorphin-SAP (Cat. #IT-12) to the population of MOR-expressing neurons in the dorsal horn of the spinal cord results in destruction only of the neurons in lamina II and not the primary afferent terminals that also express MOR. This may be a general principle but it has not been tested in any other situation for dermorphin-SAP, nor have SP-SAP (Cat. #IT-07) and SSP-SAP (Cat. #IT-11) been evaluated for terminal uptake and suicide transport. Any saporin taken into a nerve terminal should not be toxic unless retrogradely transported to the cell body since there are no ribosomes (site of saporin action) or protein synthesis in the nerve terminal.

See: Targeted Toxins

Suggested Reading:

1. Oeltmann TN, Wiley RG (1986) Wheat germ agglutinin-ricin A-chain conjugate is neuronotoxic after vagal injection. Brain Res 377:221-228.
2. Wiley RG, Stirpe F, Thorpe P, Oeltmann TN (1989) Neuronotoxic effects of monoclonal anti-Thy 1 antibody (OX7) coupled to the ribosome inactivating protein, saporin, as studied by suicide transport experiments in the rat. Brain Res 505:44-54.
3. Contestabile A, Fasolo A, Virgili M, Migani P, Villani L, Stirpe F (1990) Anatomical and neurochemical evidence for suicide transport of a toxic lectin, volkensin, injected in the rat dorsal hippocampus. Brain Res 537(1-2):279-286.
4. Pangalos MN, Francis PT, Pearson RC, Middlemiss DN, Bowen DM (1991) Destruction of a sub-population of cortical neurones by suicide transport of volkensin, a lectin from Adenia volkensii. J Neurosci Methods 40(1):17-29.
5. Wiley RG (1992) Neural lesioning with ribosome-inactivating proteins: suicide transport and immunolesioning. Trends in Neurosci 15:285-290.
6. Roberts RC, Harrison MB, Francis SMN, Wiley RG (1993) Differential effects of suicide transport lesions of the striatonigral or striatopallidal pathways on subsets of striatal neurons. Exp Neurol 124:242-252.
7. Contestabile A, Stirpe F (1993) Ribosome-inactivating proteins from plants as agents for suicide transport and immunolesioning in the nervous system. Eur J Neurosci 5:1292-1301.
8. Wiley RG, Lappi DA(1994) Suicide Transport and Immunolesioning. R.G. Landes, Houston.
9. Roberts RC, Strain-Saloum C, Wiley RG (1995) Effects of suicide transport lesions of the striatopallidal or striatonigral pathways on striatal ultrastructure.Brain Res 710:227-237.
10. Wiley RG, Kline IVRH (2000) Neuronal lesioning with axonally transported toxins. J Neurosci Methods 103:73-82.

Q: How do I know my peptide will work as a targeted toxin?

A: There is rich literature that demonstrates peptides can usher proteins that inhibit protein synthesis (such as saporin) into cells and result in cell death. Peptide ligands that bind to the cell surface (i.e., to their receptors) are internalized-in fact, often quite rapidly. As with all saporin cytotoxins, internalization is necessary; antagonists that do not internalize would not be expected to be proper agents for a saporin cytotoxin. All agonists that have a decent affinity and internalization rate should work as a targeted toxin.

Q: How much peptide do I need to provide for a custom conjugation?

A: Actually, we will consult with you on the structure function properties of your peptide. We will need to synthesize an entirely new peptide for conjugation to saporin. We will, in collaboration with you and/or by examination of the literature, design the new peptide and have it synthesized (it is our plan to have peptide synthesis capabilities in 2003). We pass the price of the peptide, usually quite reasonable, directly to you without any increase.

Q: How much of the saporin conjugate will that give me?

A: We strive to give you 2-3 mg of peptide-saporin cytotoxin. These often are effective in the nanogram range.

Q: What is the ratio of saporin to antibody?

A: We synthesize the conjugate such that there is one mole of saporin per mole of peptide.

Q: What quality control is involved?

A: We monitor the reactions and purification by several means. The product is confirmed by gel electrophoresis. A data sheet will be provided when we ship the immunotoxin to inform you of final average molecular weight.

Q: What is the cost of a custom targeted toxin preparation? How long will it take to complete?

A: The standard cost of a peptide-saporin conjugation is US$3500.00 (as of March 2003), plus the price of the peptide. From the time we receive the peptide to the time we ship out the finished targeted toxin is 2-3 weeks.

See: Custom Conjugates

Q: How do I know my antibody will work in an immunotoxin?

A: We recommend that you use one of our second immunotoxins (secondary conjugates) to test for specificity and internalization of your antibody.

Q: How much antibody do I need to provide for a custom conjugation?

A: The required quantity is 8-15 mg of purified monoclonal antibody.

Q: How much of the saporin conjugate will that give me?

A: The yield is 15-30% in mg of immunotoxin from the number of mg of original antibody.

Q: What is the ratio of saporin to antibody?

A: The amount of saporin can vary between 1.5-3 moles saporin per mole of antibody.

Q: What quality control is involved?  Will you provide product specifications such as average saporin to antibody ratio?

A: We monitor the reactions and purification by several means.  The product is confirmed by gel electrophoresis. A data sheet will be provided when we ship the immunotoxin to inform you of final average molecular weight.

Q: What is the cost of a custom immunotoxin preparation? How long will it take to complete?

A: The standard cost of an antibody-saporin conjugation is US$4500.00 (as of Dec 2007).  From the time we receive the purified antibody to the time we ship out the finished conjugate is 2-3 weeks.

See: Custom Conjugates , ZAP Conjugates