custom-conjugates

Ogawa S, Nathan FM, Parhar IS (2014) Habenular kisspeptin modulates fear in the zebrafish. Proc Natl Acad Sci U S A 111(10):3841-3846. doi: 10.1073/pnas.1314184111

Summary: The peptide kisspeptin can be found in several areas of the brain, but its role in regions other than the hypothalamus has not been studied. Zebrafish express kiss1 mRNA which is a conserved ortholog of the mammalian KISSI/KissI making zebrafish a viable model for investigating the role of kisspeptin in various brain systems. Animals received 1 μg of the custom conjugate kiss-SAP (see NK3-SAP, Cat. #IT-63) via an intracranial injection. Blank-SAP (Cat. #IT-21) was used as a control. Reducing Kiss1 immunoreactivity in the habenula and the raphe reduced an invoked fear response, indicating a role for kisspeptin in fear inhibition.

Related Products: Blank-SAP (Cat. #IT-21), NKB-SAP (Cat. #IT-63), Custom Conjugates

Quadros EV, Nakayama Y, Sequeira JM (2013) Saporin conjugated monoclonal antibody to the transcobalamin receptor TCblR/320 is effective in targeting and destroying cancer cells. J Cancer Ther 4(6):1074-1081. doi: 10.4236/jct.2013.46122

Summary: Although the transcobalamin receptor (TCb1R) is expressed in most cell types, the expression levels are increased in actively proliferating cells, and decreased in quiescent cells. This expression profile makes the TCb1R an attractive target for cancer therapy. The authors used a custom conjugate of antibodies generated against the TCb1R and saporin to eliminate cancer cell lines in culture. When applying the conjugate to cells in a dosing range of 0.156-5 nM, 2.5 nM was found to have the optimal effect. Given that the level of toxicity was determined by the level of TCb1R expression, targeting the TCb1R may have potential as part of a cancer treatment strategy.

Related Products: Custom Conjugates

Mishra SK, Hoon MA (2013) The cells and circuitry for itch responses in mice. Science 340(6135):968-971. doi: 10.1126/science.1233765

Summary: Although previous work implicated neurons expressing the GRP (gastrin-releasing peptide) receptor were in the pruritic, or itch pathway, transgenic mice lacking natriuretic polypeptide b (Nppb) were almost completely insensitive to itch. Using the custom conjugate Nppb-SAP (Cat. #IT-69), the authors eliminated itch in response to a wide range of pruritic substances in normal mice through the administration of 5 μg of conjugate into the intrathecal space. Even after this lesion, the scratching response to intrathecal GRP was not changed, indicating that the role of GRP is at a later stage than previously hypothesized.

Related Products: Nppb-SAP (Cat. #IT-69)

Kato J, O’Donnell RT, Abuhay M, Tuscano JM (2012) Efficacy and toxicity of a CD22-targeted antibody-saporin conjugate in a xenograft model of non-Hodgkin’s lymphoma. Oncoimmunology 1(9):1469-1475. doi: 10.4161/onci.21815

Summary: CD22 is a B-cell-specific antigen found on many B-cell malignancies. It is not expressed by stem cell precursors, and is rapidly internalized when bound by an antibody. In this work, the authors use a custom conjugate of anti-CD22 (mAb HB22.7) and saporin in a cytotoxicity assay on non-Hodgkin’s lymphoma cell lines, as well as in a mouse tumor model. The dosing for the tumor model was 1 mg conjugate per kg of animal. Mouse IgG-SAP (Cat. #IT-18) was used as a control. The results indicate that CD22 is a potential therapeutic target for cancer therapy.

Related Products: Mouse IgG-SAP (Cat. #IT-18), Custom Conjugates

Daniels-Wells TR, Helguera G, Rodriguez JA, Leoh LS, Erb MA, Diamante G, Casero D, Pellegrini M, Martinez-Maza O, Penichet ML (2013) Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1. Toxicol In Vitro 27(1):220-231. doi: 10.1016/j.tiv.2012.10.006

Summary: The antibody-avidin fusion protein ch128.1Av has been shown to target the human transferrin receptor 1 (TfR1) and kill malignant B cells by blocking the use of iron. Combination of this construct with a mono-biotinylated saporin custom conjugate produces an iron-independent toxicity to TfR1-expressing cells, even those that are resistant to ch128.1Av alone. The saporin-containing conjugate induces a transcriptional response consistent with oxidative stress and DNA damage. The data also show that the saporin conjugate is not toxic to human hematopoeietic stem cells.

Usage: An antibody-avidin fusion protein (ch128.1Av) was mixed with MonoBiotin-ZAP to make an immunotoxin that targets the human transferrin receptor 1 (TfR1).

Related Products: MonoBiotin-ZAP (Cat. #BT-ZAP), Custom Conjugates

Mishra SK, Holzman S, Hoon MA (2012) Neuromedin B serves a role in nociceptive signaling. Neuroscience 2012 Abstracts 471.22. Society for Neuroscience, New Orleans, LA.

Summary: We are interested in identifying new somatosensory signaling molecules and used an array based differential screen. In order to subtract genes not involved in signaling processes, we compared expression profiles in trigeminal ganglia (TG) with those of the geniculate ganglia (GG); a ganglia similar in structure but with different function. One gene we uncovered was neuromedin B (NMB), as expected from the differential expression, neuropeptide NMB is expressed in TG and dorsal root ganglia (DRG), but not in GG. Double labeling experiments, revealed NMB is expressed in a subset of sensory neurons that co[[unable to display character: ‐]]label with CGRP and TRPV1, suggestive of a role for NMB in nociception. Indeed, administration of NMB[[unable to display character: ‐]]antagonist greatly attenuates edema and nerve sensitization following stimulation of peripheral nerves with mustard oil, demonstrating that NMB contributes to neurogenic inflammation. Moreover, direct injection of NMB causes local swelling and nociceptive sensitization. Interestingly, we also found the receptor for NMB is expressed in interneurons in the superficial layers of the dorsal horn. We used NMB[[unable to display character: ‐]]saporin to specifically eliminate NMB-receptor expressing spinal cord cells and determined that they are required for responses to noxious heat, but not for reactions to mechanical and pruritic stimuli. Thus, NMB may be a neurotransmitter that is selectively involved in the perception of thermal stimuli, and has a role in neurogenic inflammation.

Related Products: Custom Conjugates

Kato J, Satake N, O’Donnell RT, Abuhay M, Lewis C, Tuscano JM (2013) Efficacy of a CD22-targeted antibody-saporin conjugate in a xenograft model of precursor-B cell acute lymphoblastic leukemia. Leuk Res 37(1):83-88. doi: 10.1016/j.leukres.2012.09.010

Summary: Most cases of acute lymphoblastic leukemia (ALL) are of B-cell lineage. Although children with ALL have a high survival rate, there is a subset of children with a much lower survival rate, and long-term side effects from treatment are problematic. CD22 has been suggested as a therapeutic target because it is not present on hematopoietic stem cells, therefore allowing regeneration of normal B cells following depletion of malignant B cells. The authors used a custom conjugate of the antibody HB22.7 and saporin to demonstrate specific toxicity against pre-B ALL cell lines. Mouse IgG-SAP (Cat. #IT-18) was used as a control.

Related Products: Mouse IgG-SAP (Cat. #IT-18), Custom Conjugates

Q: We are interested in having a custom conjugation of saporin and our antibody. Do you remove unconjugated antibody from the final material you send us?

A: We do remove the unconjugated antibody and saporin from the final product that we send to you. And perhaps to answer a question you may not be asking, but may be curious about, the unconjugated material is not particularly usable, after it has been removed from the final product as it has been slightly modified in preparation for the conjugation.

See also: Custom Conjugates

Smith MA, Williams H, Krajewski SJ, Mcmullen NT, Rance NE (2011) Arcuate NK3 receptor-expressing KNDy neurons are essential for estrogen modulation of LH secretion and body weight in the female rat. Neuroscience 2011 Abstracts 712.07. Society for Neuroscience, Washington, DC.

Summary: Arcuate kisspeptin, neurokinin B, and dynorphin (KNDy) neurons have been proposed to mediate estrogen negative feedback in multiple species. To determine if these neurons are essential for this feedback, we ablated KNDy neurons in the arcuate nucleus of female rats using [MePhe7]Neurokinin B, a selective NK3 receptor (NK3R) agonist, conjugated to Saporin ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). The specificity of this conjugate for NK3R-expressing KNDy neurons is described in a separate abstract (see Krajewski et al., Soc. Neurosci. Abstr. 2011). Twenty-four female rats were ovariectomized (OVX) and received bilateral arcuate microinjections of either [MePhe7]NKB-SAP or a scrambled peptide conjugated to Saporin (Blank-SAP controls). 20-23 days later, animals were implanted with s.c. silastic capsules containing 17β-estradiol (E2), and animals were sacrificed 11 days later. Blood samples for RIA of serum LH were taken at time of OVX and injections (baseline), 20-23 days post-OVX, and 11 days after E2-treatment. Because OVX and E2-treatment have well-described effects on body weight, animals were weighed at the same three time points. In control animals, OVX induced a 13-fold rise in serum LH, which returned to baseline 11 days after E2 replacement. In contrast, OVX had no effect on serum LH in [MePhe7]NKB-SAP animals. There was a small decrease in serum LH 11 days after E2 replacement in [MePhe7]NKB-SAP animals, but the magnitude of this change was much less than seen in control animals. Control animals also exhibited a 20% increase in body weight 20-23 days after OVX, followed by a significant reduction after E2 replacement. Surprisingly, neither OVX nor E2 replacement affected body weight in [MePhe7]NKB-SAP-treated animals. Rather, these animals showed a steady increase in body weight throughout the experiment, at rates comparable to intact female rats or OVX rats treated with E2 (Williams et al., Endocrinology, 2010). Immunohistochemical studies showed near-complete destruction of KNDy neurons in the arcuate nucleus of [MePhe7]NKB-SAP animals. There was preservation of proopiomelanocortin and neuropeptide Y immunoreactivity in the arcuate nucleus and GnRH-immunoreactive fibers in the median eminence. These data provide compelling evidence that arcuate KNDy neurons play an essential role in estrogen negative feedback on LH secretion as well as the estrogen modulation of body weight.

Related Products: Custom Conjugates, Blank-SAP (Cat. #IT-21)

ATS Poster of the Year Winner

Krajewski SJ, Smith MA, Williams H, Ciofi P, Lai JY, Mcmullen NT, Rance NE (2011) Ablation of NK3 receptor-expressing KNDy neurons in the rat arcuate nucleus using [MePhe7]Neurokinin B-Saporin. Neuroscience 2011 Abstracts 712.09. Society for Neuroscience, Washington, DC.

Summary: A subpopulation of neurons expressing kisspeptin, neurokinin B and dynorphin (KNDy neurons) has been shown to reside within the arcuate nucleus of many mammalian species. Although these peptides are critical for reproductive function, the precise role of the arcuate KNDy neurons is not fully understood. Here we describe a method to ablate KNDy neurons based on their co-expression of the Neurokinin 3 receptor (NK3R, Burke et al., J. Comp. Neurol, 2006). Saporin, a molecular neurotoxin, was conjugated to [MePhe7]Neurokinin B, a selective NK3R agonist ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). Binding studies revealed that the conjugation of saporin did not alter the affinity of [MePhe7]NKB to NK3R in rat cerebral cortex membranes. To investigate the specificity of this conjugate for ablation of NK3R neurons, stereotaxic surgery was used to bilaterally inject [MePhe7]NKB-SAP into the arcuate nucleus of female rats. Control rats were injected with saporin conjugated to a scrambled peptide (Blank-SAP, Advanced Targeting Systems). Rats were sacrificed 31-34 days later and the brains were processed for immunohistochemical studies. Nissl stained sections from [MePhe7]NKB-SAP-treated rats showed no signs of inflammation at the injection sites and no qualitative changes in cell density compared to Blank-SAP control rats. Immunohistochemistry revealed near-complete loss of NK3R-immunoreactive (ir) neurons throughout the arcuate nucleus of [MePhe7]NKB-SAP rats. When the injection site was dorsal to the arcuate nucleus, there was also variable loss of NK3R-ir cells in the lateral hypothalamus and zona incerta. In the arcuate nucleus, [MePhe7]NKB-SAP injections resulted in a 98% and 94% reduction in the number of kisspeptin and neurokinin B-ir neurons, respectively, compared to Blank-SAP controls. The number of dynorphin-ir neurons in the arcuate nucleus of [MePhe7]NKB-SAP-treated rats was reduced by 67%, a value consistent with the co-expression of NK3R on dynorphin neurons in our previous study (Burke et al., J. Comp. Neurol, 2006). In contrast, arcuate proopiomelanocortin and neuropeptide Y immunoreactivity were preserved in [MePhe7]NKB-SAP rats. Moreover, there was no difference in GnRH-ir fiber density in the median eminence between the two groups. These results document the utility of [MePhe7]NKB-SAP for selective ablation of NK3R-expressing KNDy neurons in rat hypothalamus. These rats were used to examine the role of KNDy neurons in the estrogen regulation of LH secretion and body weight in the female rat (see Smith et al., Soc. Neurosci. Abstr. 2011).

Related Products: Custom Conjugates, Blank-SAP (Cat. #IT-21)

ATS Poster of the Year Winner