custom-conjugates

Q: When we contacted you to find out more about having a custom saporin conjugation performed with our primary antibody, you recommended that we use the ATS secondary conjugate system to determine that our antibody was specific to the population we want to eliminate. We looked more at the website, and it seems that we are supposed to start with Anti-M-ZAP, Cat. #IT-30 (our primary Ab is a mouse IgM), and use the Mouse IgM-SAP, Cat. #IT-41 for control. Is this correct?

A: If your primary antibody is a mouse IgM, then you are correct that Anti-M-ZAP (Cat# IT-30) is the appropriate secondary conjugate to use. As for control conjugates, the best control would be a secondary conjugate using an IgM isotype control mixed with Anti-M-ZAP. An alternative would be to use Goat IgG-SAP (Cat# IT-19) made with normal goat IgG that mimics Anti-M-ZAP without the specific affinity for mouse IgM.

Once you determine you need a direct conjugate made between your mouse IgM primary antibody and saporin, then you would want to use the Mouse IgM-SAP (Cat# IT-41) as a control toxin just as you use your direct conjugate.

Related: ZAP Conjugates, Control Conjugates, Custom Conjugates

Zikri NN, Schumer E, Wang JJ, Gaughan A, Hadley GA, Moffatt-Bruce SD (2010) Induction of CD4(+)CD25(+) T regulatory cells with CD103 depletion. J Surg Res 163(1):162-168. doi: 10.1016/j.jss.2010.04.021

Summary: CD8+ T cells expressing CD103 have been shown to play a key role in the rejection of renal allografts. Use of M290-SAP (a custom saporin conjugation) allows allograft tolerance even in a completely mismatched islet cell transplant model. Use of 1 mg M290-SAP/kg body weight in mice allowed the authors to characterize the kinetics of M290-SAP and its induction of CD4 CD25 regulatory T cells.

Related Products: Custom Conjugates

Zhang L, Moffatt-Bruce SD, Gaughan AA, Wang JJ, Rajab A, Hadley GA (2009) An anti-CD103 immunotoxin promotes long-term survival of pancreatic islet allografts. Am J Transplant 9:2012-2023. doi: 10.1111/j.1600-6143.2009.02735.x PMID: 19645708

Objective: To determine whether blockade of the CD103 pathway represents a viable therapeutic strategy for intervention in organ allograft rejection and graft-vs-host disease.

Summary: These data document that depletion of CD103 expressing cells represents a viable strategy for therapeutic intervention in allograft rejection.

Usage: Recipient C57BL/6 mice received 2.0 mg/kg M290-SAP or Rat IgG-SAP on day 3 post-transplantation.

Related Products: Anti-CD103-SAP (Cat. #IT-50), Rat IgG-SAP (Cat. #IT-17)

Foehr ED, Lorente G, Kuo J, Ram R, Nikolich K, Urfer R (2006) Targeting of the receptor protein tyrosine phosphatase beta with a monoclonal antibody delays tumor growth in a glioblastoma model. Cancer Res 66(4):2271-2278. doi: 10.1158/0008-5472.CAN-05-1221

Summary: The receptor protein tyrosine phosphatase ß (RPTPß) is overexpressed in astrocytomas, and is a potential target for tumor therapy. After testing antibodies against an extracellular domain of RPTPß in vitro with Mab-ZAP (Cat. #IT-04), two custom conjugates, 7E4B11-SAP and 7A9B5-SAP, were created by Advanced Targeting Systems. The authors tested the custom conjugates, using anti-DAT-SAP (Cat. #IT-25) as a positive control, and mouse IgG-SAP (Cat. #IT-18) as a negative control. The 7E4B11-SAP conjugate displayed significant antitumor activity in mice engrafted with U87 glioma cells.

Related Products: Mab-ZAP (Cat. #IT-04), Anti-DAT-SAP (Cat. #IT-25), Mouse IgG-SAP (Cat. #IT-18), Custom Conjugates

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.

Gunhan-Agar E, Choudary P, Landerholm TE, Chalupa LM (2000) Depletion of cholinergic amacrine cells does not perturb the segregation of on and off cone bipolar cell projections. Neuroscience 2000 Abstracts 119.3. Society for Neuroscience, New Orleans, LA.

Summary: The pathways signaling onset and offset of light are segregated in the retina with On-cone and Off-cone bipolar cells terminating on the stratified dendrites of On and Off retinal ganglion cells (RGCs). During development the axons of On and Off cone bipolar cells form two strata in the IPL in a remarkably precise manner, without any obvious refinements. Moreover, such a precise ingrowth pattern occurs even after RGCs have been depleted (J. Neurosci., 2000, 201:306-314). Here we show by immunostaining that two bands of cholinergic processes are present in the rat retina as early as P1, some 7 days before the formation of segregated bipolar inputs. Double labeling of retinal sections with the antibody to recoverin (that recognizes On and Off cone bipolar cells) and the antibody for VACHT (which labels cholinergic processes) revealed that the segregated terminals of cone bipolar cells are juxtaposed with the two bands of cholinergic fibers. These observations suggested that the cholinergic fibers could serve as a scaffold for the later ingrowing bipolar cell axons. To test this hypothesis, we devised a novel method for depleting retinal cholinergic amacrine cells with a VACHT-saporin immunotoxin. A single treatment of the developing retina with this immunotoxin was found to eliminate virtually all cholinergic cells and processes. Recoverin labeling of bipolar cells showed that the axons of these neurons still form two stratified terminal bands within the IPL. Thus, neither RGCs nor cholinergic amacrine cell processes are required for the formation of segregated ON and Off cone bipolar cell projections.

Related Products: Custom Conjugates

Wu M, Shanabrough M, Leranth C, Alreja M (2000) Cholinergic excitation of septohippocampal GABA but not cholinergic neurons: implications for learning and memory. J Neurosci 20(10):3900-3908. doi: 10.1523/JNEUROSCI.20-10-03900.2000 PMID: 10804229

Summary: It has long been assumed that the drug-induced enhancement of learning and memory in both young and aged rats was accomplished through a cholinergic pathway in the hippocampus. Wu et al. used a fluorescent labeling molecule, 192-IgG conjugated to Cy3 (Custom Service from ATS) to visualize these neurons. They found that the effects of cognition-enhancing drugs are not facilitated through action on cholinergic neurons. Instead, activation of GABA neurons is implicated in this model.

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #AB-N43FL3)