Targeting Talk: Using Kit Controls

Q: Your targeted toxin kits come with different controls. I’m not sure of the best way to use them; 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: For mouse IgG-containing conjugates, the ideal control is Mouse IgG-SAP (Cat. # IT-18). Mouse IgG-SAP — that is, saporin conjugated to mouse IgG — that has no specific antigen for targeting is the best control. Unconjugated saporin is still considered a second good control, useful in cases where down-regulation by the antibody is a concern.

Q: What about for the peptide toxins like Octreotide-SAP?

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 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.

One of the best uses we have seen for Blank-SAP has been in:
Bugarith K, Dinh TT, Li AJ, Speth RC, Ritter S (2005) Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Endocrinology 146(3):1179-1191.

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!

Targeting Talk: Neurotransmitter Antibodies

Advanced Targeting Systems offers a full line of antibodies to neurotransmitters; everything from serotonin and L-DOPA, to noradrenaline and dopamine. These antibodies are an excellent choice for immunohistochemistry applications as they are ideally primed for use in tissues that have been perfused with a mixture of paraformaldehyde and gluteraldehyde.

Some frequently asked questions:

Q:  Could you confirm if Anti-Conjugated Caprylic Acid (Cat. #AB-T084) can detect free Caprylic acid or only when conjugated with BSA or another carrier protein?

A:  This antibody does not recognize BSA or need Caprylic acid to be conjugated to BSA in order for it to bind. However, it DOES need to be used in the presence of gluteraldehyde in order to create the proper epitope for the antibody to recognize the Caprylic acid.

Q:  I was wondering whether it could be possible to receive more information about the Gibberellic acid antibody (Cat. #AB-T130)? Is it possible to use this antibody to recognize free gibberellic acid by a direct ELISA system?

A:  The most common use for this product is immunohistochemistry. ELISA is used to test cross-reactivity and is not a recommended application for these antibodies. On its own, gibberellic acid is too small a molecule to provide a complete effective epitope, so for IHC the tissue is perfused with a gluteraldehyde component which provides the epitope complement needed for the antibody to recognize gibberellic acid.

Q:  For your conjugated neurotransmitter antibodies, what concentrations and sizes do you provide?

A:  All of these antibodies are sold in 50-mcl volumes. No concentrations are needed  for these products as they all have recommended dilutions for use listed on the data sheets.

Q:  What protocol should I use for these antibodies?

A:  We recommend using the protocols available on our website. Some have specific protocol recommendations which you can link to from the product page.

Questions? Ask a Product Manager

New Product Manager Alena Bishop

In this quarter’s Targeting Talk, ATS is proud to talk about more than new products, a new Product Manager! Her name is Alena Bishop and although originally from the San Francisco Bay Area, she recently graduated from UCSD in June of this year. With a newly minted degree in Molecular Biology and ATS’s need for a Research Associate with just such a pedigree, it has proved to be an excellent match. Since starting in July, Alena has taken quickly to the innovative work being done in the lab and appreciates participating in the development of products that play such an important role in the everyday research of so many scientists globally. Eager to dive into the customer-facing aspects of our products, she looks forward to engaging with each of you regarding any technical questions you may have.

alenaheadshot

She’s here to tell you, in her own words, what she is working on and what products to look forward to:

“One of the projects I have been working on involves a novel use of specific strains of Clostridium botulinum that inhibits cells by blocking the release of neurotransmitters. This effect is long-lasting but not permanent, and this attribute gives the toxin great potential as a new targeted payload. This toxic payload could be used to temporarily prevent cell function without actually killing the cells, and coupled with ATS’s targeting technology this treatment could be administered specifically to cells of interest. Avoiding killing the cells completely would provide a life-like model to study the effects of temporary loss of cell function in organisms. After a certain period of time, the effects of the toxin would disappear, restoring normal neuronal cell function. My immediate goal is to put the finishing touches on an assay kit for quantifying the activity of Clostridium botulinum useful for a variety of strains. Look for this activity assay kit to be released in the coming quarter!”

Targeting Talk: Streptavidin-ZAP Applications

Q: I’ve been looking at your secondary conjugates and want to see if my targeting agent is specific to certain cells. Which secondary conjugate should I use?

A: It depends on two factors: 1) the type of assay you want to use, and 2) the kind of targeting agent you want to use.

For in vitro assays, in particular, internalization assays, you can use any of the ZAP Internalization Kits (Z-Kits that include all the materials necessary to test your targeting agent). For the Secondary Antibody Z-Kits, you use your primary antibody and select the appropriate secondary antibody species (e.g. for a human antibody, use Hum-ZAP (Cat. #KIT-22-Z), Fab-ZAP human (Cat. #KIT-51-Z), FabFC-Human (Cat. #KIT-65-Z), Hug-M-ZAP (Cat. #KIT-43-Z), or Fab-ZAP Hug-M (Cat. #KIT-78-Z), depending on the isotype of your primary antibody (See page 8 Promos). Or you can biotinylate your antibody and use Streptavidin-ZAP (Cat. #KIT-27-Z).

For in vivo applications, it depends on the kind of targeting agent you want to use. Regardless of whether you use an antibody, peptide or ligand, you will need to biotinylate the material first. ATS offers a biotinylation service that is efficient and economical (See page 8 Promos).

If you are using a biotinylated peptide, you will use a kit that includes the appropriate control — Blank-Streptavidin-SAP. Order Cat. #KIT-27-B (25 mcg, 100 mcg, 250 mcg, or 1 mg).

If you are using a biotinylated antibody, you will use the Streptavidin-ZAP kit that includes the appropriate antibody species control: BIgG-SAP Human, BIgG-SAP Mouse, BIgG-SAP Rabbit, BIgG-SAP Rat. Select the kit that matches the species of your biotinylated primary antibody. Each kit is available in 25, 100, 250 or 1000 mcg sizes.

Have questions? Contact us.

Targeting Talk: Saporin Safety

Over the years, ATS has frequently been asked about Saporin’s safety for use in the lab as well as when used clinically. Residual awareness of alternate Ribosome-Inactivating Proteins (RIPs) and ‘toxins’ such as Ricin have caused some researchers new to the use of RIPs to question the belief that Saporin is safe.  Unlike Type 2 RIPs (such as Ricin), Type I RIPs, like Saporin have no binding chain and consequently no means of entering the physiological space necessary for the protein to act as a toxin.  The following is a review of safety in handling and potential toxicity within the human body for systemic events not related to normal research applications of Saporin conjugates, including Substance P-Saporin (SP-SAP), which is a therapeutic under development for the treatment of chronic pain.

The acute LD50 for saporin in mice (25 g) is 6.8 mg/kg;[1] that would translate in humans (75 kg) to 510 mg! A concentration of about 100 nM is the threshold to see even a vague hint of saporin toxicity. In human blood, that would correspond to 24 mg injected systemically into a person. The fermentation process to produce recombinant saporin has a titer of 2 mg/L meaning that the production broth itself contains no more than 67 nM concentration of saporin. Furthermore, the final protein concentrations from production batches of recombinant Saporin used in our drug are 4 mg/ml, meaning 6 mL of final material would need to accidentally end up in a human before the ‘hint of toxicity’ threshold would potentially be met.

The toxicology studies of SP-SAP contained within ATS’s IND prior to the current human Phase I clinical trial evaluated effects related to the intended method of administration, intrathecal local injection. SP-SAP is not expected to ever be a self-administered therapy, so the effects of gross off-target events, such as accidental auto-injection, swallowing, spillage, or immersion were not considered.

The table below[2] highlights antibody-saporin conjugates approved by the FDA for Phase I/II clinical trials in humans. The therapeutics listed below were administered intravenously and imply what the FDA accepted as non-toxic levels of saporin-based conjugates in these studies.

Looking more closely at the study by French et al.,[3] several milligrams of antibody conjugate were repeatedly injected into human patients under a FDA regulated clinical trial and peak serum levels tested, demonstrating rapid clearing of saporin from the system.

As a company that specializes in Saporin, our two-plus decades of experience working with the protein in research, preclinical, and clinical environments has taught us that with minimal standard laboratory precautions users are not at any real risk of toxic effects. Even our CSO, after 30+ years of working with Saporin exhibits undetectable levels of Saporin antibodies in his blood!

References

1. Thorpe, P.E.; Brown, A.N.; Bremner, J.A., Jr.; Foxwell, B.M.; Stirpe, F. An immunotoxin composed of monoclonal anti-Thy 1.1 antibody and a ribosome-inactivating protein from Saponaria officinalis: Potent antitumor effects in vitro and in vivo. J. Natl. Cancer Inst. 1985, 75, 151–159.

2. Polito L, Bortolotti M, Pedrazzi M, Bolognesi A. Immunotoxins and other conjugates containing saporin-s6 for cancer therapy. Toxins (Basel). 2011 Jun;3(6):697-720.

3. French, R.R.; Bell, A.J.; Hamblin, T.J.; Tutt, A.L.; Glennie, M.J. Response of B-cell lymphoma to a combination of bispecific antibodies and SAP. Leuk. Res. 1996, 20, 607–617.

Targeting Talk: Safety of the Toxin

Q: Our QA group wants to know about the safety of the toxin in your conjugates? What precautions should we take in handling saporin products?

A: Saporin is a Type 1 ribosome-inactivating protein (RIP), due to its N-glycosidase activity, from the seeds of Saponaria officinalis. It was first described by Fiorenzo Stirpe and his colleagues in 1983 in an article that illustrated the unusual stability of the protein.[1] Among the RIPs are some of the most toxic molecules known, including ricin and abrin (the latter is the poison preferred by the characters in movie The Blue Lagoon). These toxins contain a second protein strand that inserts the RIP into a cell, making it able to enzymatically inactivate the ribosomes, shutting down protein synthesis and resulting in cell death, and eventually causing death of the victim.

Saporin does not possess a cell-binding chain[2] and has no method of internalization without a targeting agent to escort it into a cell. It is this fact that also adds to the safety of its use in the lab. Autoclaving or exposure to 0.2 M NaOH is sufficient to decontaminate material that has been in contact with Saporin and its conjugates. The LD50 for Saporin in mice is 4-8 mg/kg;[3] this dosage amount would be insignificant in humans. Hundreds of articles in the scientific literature (search “Saporin” in Pub Med) have demonstrated tremendous specificity in targeting neuronal cells with many different Saporin conjugates and by many different scientists.

REFERENCES

  1. Stirpe F, Gasperi-Campani A, Barbieri L, Falasca A, Abbondanza A, Stevens WA. (1983). Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort) of Agrostemma githago L. (corn cockle) and of Asparagus officinalis (asparagus) and from the latex of Hura crepitans L. (sandbox tree). Biochem J 216(3): 617–625. PMID 6667259
  2. Barthelemy I, Martineau D, Ong M, Matsunami R, Ling N, Benatti L, Cavallaro U, Soria M, Lappi DA. The expression of saporin, a ribosome-inactivating protein from the plant Saponaria officinalis, in Escherichia coli. J Biol Chem. 1993;268(9):6541-8. PMID: 8454624.
  3. Stirpe F, Derenzini M, Barbieri L, Farabegoli F, Brown AN, Knowles PP, Thorpe PE. Hepatotoxicity of immunotoxins made with saporin, a ribosome-inactivating protein from Saponaria officinalis. Virchows Arch B Cell Pathol Incl Mol Pathol. 1987;53(5):259-71. PMID: 2889289.

Targeting Talk: Retrograde Transport

Q: I’m trying to find out if enough Anti-DBH-SAP will be retrogradely transported and taken up by non targeted sympathetic neurons by bulk fluid-phase endocytosis. Does saporin become degraded after it kills the neuron or does it enter the extracellular matrix?

A: It is very unlikely that a targeted toxin such as Anti-DBH-SAP is freed from the targeted neuron in a meaningful condition. There has never been a reported identification of a targeted toxin, functionally or not, after it has eliminated its targeted neuron. 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.

Targeting Talk: Suicide Transport and Immunolesioning Book

Q: Hello, I have used your Anti-DBH-SAP (Cat. #IT-03) conjugate, and I’m having a hard time finding this citation: R.G. Wiley, D.A. Lappi. Suicide Transport and Immunolesioning. Molecular Biology Intelligence Unit, R.G. Landes Co, Austin, TX (1994). Do you know where I could find a copy?
A: This book is available in many university libraries and can also be purchased here: http://www.amazon.com/Suicide-Transport-Immunolesioning-Molecular-Intelligence/dp/1570590958

Targeting Talk: in vitro cytotoxicity assays

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.

Targeting Talk: Using Kit Controls

Q: Your targeted toxin kits come with different controls. I’m not sure of the best way to use them; 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: For mouse IgG-containing conjugates, the ideal control is Mouse IgG-SAP (Cat. # IT-18). Mouse IgG-SAP — that is, saporin conjugated to mouse IgG — that has no specific antigen for targeting is the best control. Unconjugated saporin is still considered a second good control, useful in cases where down-regulation by the antibody is a concern.

Q: Which control is best to use with Octreotide-SAP?

A: The best control to use with Octreotide-SAP is Blank-SAP (Cat. #IT-21). Blank-SAP serves as a control for all our peptide-targeted SAP conjugates. Listed below are the appropriate controls to use with our primary saporin conjugates.

Blank-CTA (IT-61) peptide-targeted CTA conjugates SP-CTA and Neurotensin-CTA
Blank-SAP (IT-21) peptide-targeted SAP conjugates SSP-SAP, MOR-SAP, CRF-SAP, NPY-SAP, CCK-SAP, Galanin- SAP, Bombesin-SAP, Oxytocin- SAP, Neurotensin-SAP, NK3- SAP, Dyno-SAP and NMB-SAP
Fab IgG-SAP (IT-67) goat IgG Fab-ZAP secondary conjugates Fab-ZAP mouse, Fab-ZAP human, Fab-ZAP rat, Fab-ZAP rabbit and FabFc-ZAP human
Goat IgG-SAP (IT-19) goat IgG-containing targeted toxins Mab-ZAP, Rab-ZAP, Hum-ZAP, Rat-ZAP, Anti-M-ZAP, Hug-M- ZAP and gPIG-ZAP
Human IgG-SAP (IT-49) human IgG-containing targeted toxins custom conjugates
Mouse IgG-SAP (IT-18) mouse IgG-containing targeted toxins 192-IgG-SAP, OX7-SAP, Anti- DBH-SAP, ME20.4-SAP, Anti- SERT-SAP, Anti-CD25-SAP human, Mac-1-SAP rat, Anti- CD22-SAP, Anti-6 His-ZAP, Anti- GFP-ZAP, Anti-Basigin2-SAP, Anti-V5-ZAP, and Anti-FLAG (M5)-ZAP
Mouse IgM-SAP (IT-41) mouse IgM-containing targeted toxins Anti-M-ZAP
Rabbit IgG-SAP (IT-35) rabbit IgG-containing targeted toxins mu p75-SAP, GAT1-SAP, Goat- ZAP, Anti-ChAT-SAP, Melanopsin-SAP and Chick-ZAP
Rat IgG-SAP (IT-17) rat IgG-containing targeted toxins Mac-1-SAP, Anti-DAT-SAP, Anti- CD25-SAP mouse and Anti- CD103-SAP