Fab-pHast products

Fab-pHast products bind to your primary antibody via a secondary antibody or Streptavidin cross-linked to a pH-dependent fluorescent reporter.  This fluorescent reporter increases intensity as the pH of its surroundings becomes more acidic, as evident when exposed to the environment inside a cell.

SLC46A3 as a Potential Predictive Biomarker for Antibody-Drug Conjugates Bearing Noncleavable Linked Maytansinoid and Pyrrolobenzodiazepine Warheads.
Kinneer K,  et al. Clin Cancer Res, 24 (24):6570-6582, 2018.

Objective:  To develop biomarkers to uncover the underlying mechanism of resistance by certain cell lines for ADCs.

Summary: Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376.

Dose:  For Lysosomal trafficking, ADCs were labeled with Fab-pHast human (Cat. #PH-01). Cells were incubated with 3 mg/mL of labeled ADCs at 37°C for desired time points and fluorescence quantified by flow cytometry.

pHast Ab Internalization Assay
Parental HEK-293 cells, and HEK-293 cells transfected with the p75 receptor, were plated in a 96-well plate overnight. Titrated 192-IgG antibody (Cat. #AB-N43) was incubated at RT with 50 nM of Fab-pHast Mouse (Cat. PH-02) for 20 min prior to addition to cells. Plates were incubated overnight to allow maximum internalization, but a few hours is sufficient for detection.

Please let us know if we can answer any questions about this promotion or any of our products.

Orexin-SAP Model of Sleep Disorders

Discovery and Development of N-[4-(1-Cyclobutylpiperidin-4-Yloxy)Phenyl]-2-(Morpholin-4-Yl)Acetamide Dihydrochloride (Suvn-G3031): A Novel, Potent, Selective, and Orally Active Histamine H3 Receptor Inverse Agonist with Robust Wake-Promoting Activity. (2019). J Med Chem, 62 (3):1203-1217. Nirogi R, Shinde A, Mohammed AR, Badange RK, Reballi V, Bandyala TR, Saraf SK, Bojja K, Manchineella S, Achanta PK, Kandukuri KK, Subramanian R, Benade V, Palacharla RC, Jayarajan P, Pandey S, & Jasti V.

IT-20:  Orexin-SAP

Objective:  To examine the effects of clinical candidate, 17v on sleep/wake activity in Orexin-SAP lesioned male Wistar rats.
Summary:  17v demonstrated high receptor occupancy and marked wake-promoting effects with decreased rapid-eye-movement sleep in Orexin-SAP lesioned rats supporting its potential therapeutic utility in treating human sleep disorders.
Dose:  Intracerebral injections of Orexin-SAP (100 ng/0.5 μl) in lateral hypothalamus of rats produce selective disruption of neurons containing orexin-B receptors and produces symptoms that are characteristic of narcolepsy.

Leptin responsiveness in mediating weight loss

Leptin receptor-expressing neurons in the ventromedial nucleus of the hypothalamus contribute to weight loss caused by fourth ventricle leptin infusions. (2019). Am J Physiol Endocrinol Metab  PMID: 31361549
Seamon M, Ahn W, Li A-J, Ritter S, & Harris RBS.

IT-47:  Leptin-SAP

Objective: To test the importance of VMH leptin responsiveness in mediating weight loss caused by fourth ventricle leptin infusion.
Summary:  Leptin did not inhibit food intake and respiratory exchange ratio in rats treated with Leptin-SAP.  VMH leptin receptors do not play a significant role in maintaining energy balance in basal conditions, but limit weight gain during positive energy balance.
Dose: Bilateral VMH 75-nl injections of 260 ng/ml of Leptin-SAP or Blank-SAP.

Neuroprotective Effects of Exercise

Neuroprotective Effects of Exercise on the Morphology of Somatic Motoneurons Following the Death of Neighboring Motoneurons. (2019). Chew C, & Sengelaub DR. Neurorehabil Neural Repair, epub ahead of print 1545968319860485. 


Objective:  To explore whether exercise shows the same neuroprotective effect on induced dendritic atrophy as that seen with androgen treatment.
Summary:  Exercise following neural injury exerts a protective effect on motoneuron dendrites comparable to that seen with exogenous androgen treatment.
Dose:  Motoneurons innervating the left vastus medialis muscle were selectively killed by intramuscular injection of CTB-SAP (2 μL, 0.1%).  Saporin injection reduced the weight of the vastus medialis muscle; exercise had no effect on muscle weight.

Latest Itch research using Targeted Toxins

Spinal Neuropeptide Y1 Receptor-Expressing Neurons Form an Essential Excitatory Pathway for Mechanical Itch. (2019). Cell Reports, 28 (3):625-639.e626. Acton D, Ren X, Di Costanzo S, Dalet A, Bourane S, Bertocchi I, Eva C, & Goulding M.

Objective:  To determine the central pathway for mechanical itch.
Summary:  NPY-Y1 signaling regulates the transmission of innocuous tactile information by establishing biologically relevant thresholds for touch discrimination and mechanical itch reflexes.  neither the evoked nor spontaneous scratching seen following activation of Y1Cre neurons was affected by ablation of the GRPR+ neurons.  NK1R+ neuron ablation failed to modulate mechanically evoked itch.
Dose:  P28 mice were given a single intrathecal (i.t.) injection of either Bombesin-SAP (400 ng in 5 mL 0.9% sterile saline) to ablate GRPR+ cells or SSP-SAP to ablate NK1r+ neurons (100 ng in 5 mL 0.9% sterile saline). Littermate controls received Blank-SAP (equal mass in 5 mL 0.9% sterile saline).

Also see:

Cross-Talk between Distinct Receptors Shapes Itch Behavior in the Spinal Cord (September 14, 2018). Available at http://dx.doi.org/10.2139/ssrn.3249822. Meng, Qing-Tao et al.

Summary: Consistently, Nppb-SAP ablated spinal Npr1 and Npr3 neurons and impaired histamine-, but not CQ-evoked, itch. Thus, the findings identify the role of BNP-NPRC signaling in modulation of histamine-evoked itch via NPRC-NMBR cross-talk independent of GRP-GRPR signaling. Our studies reveal distinct modes of action for bombesin-related peptides and NP in itch transmission.

Latest Alzheimer’s Disease research using Targeting Tools

3D Reconstruction of the Neurovascular Unit Reveals Differential Loss of Cholinergic Innervation in the Cortex and Hippocampus of the Adult Mouse Brain. Nizari S, Carare RO, Romero IA, & Hawkes CA. (2019). Front Aging Neurosci, 11 (172).  IT-16: mu p75-SAP

Objective:  To further characterize the effect of the loss of cholinergic innervation on the NVU (neurovascular unit) in Alzheimer’s Disease.
Summary:  Significantly less ChAT staining was detected in the medial septum of saporin-treated mice at 45 days post-surgery. This was accompanied by a significant decrease in cholinergic nerve fiber density in the hippocampus and the cortex. As expected, p75 NTR-negative neurons in the striatum were not affected by mu p75-SAP treatment.
Dose:  In this study, the mu-p75-SAP was used to induce death of basal forebrain cholinergic neurons and their fiber projections.  mu p75-SAP 0.5 µL (0.596 µg/µL) or 0.9% saline (n = 19) was injected into each ventricle.

Degeneration of ipRGCs in Mouse Models of Huntington’s Disease Disrupts Non-Image-Forming Behaviors Before Motor Impairment. Lin M-S, Liao P-Y, Chen H-M, Chang C-P, Chen S-K, & Chern Y.  (2019). J Neurosci, 39 (8):1505. AB-N38: Anti-Melanopsin

Summary:  results show that M1 ipRGCs were susceptible to the toxicity caused by mutant Huntingtin. The resultant impairment of M1 ipRGCs contributed to the early degeneration of the ipRGC–SCN pathway and disrupted circadian regulation during HD progression.
Dose:  Immunostaining (1:3000)

Targeting p75 LNGFr neurons

Latest research using 192-IgG-SAP to eliminate p75-positive neurons.

Nitric Oxide Donor Molsidomine Promotes Retrieval of Object Recognition Memory in a Model of Cognitive Deficit Induced by 192 IgG-Saporin. (2019). Behav Brain Res, 366 108-117. Hernández-Melesio MA, Alcaraz-Zubeldia M, Jiménez-Capdeville ME, Martínez-Lazcano JC, Santoyo-Pérez ME, Quevedo-Corona L, Gerónimo-Olvera C, Sánchez-Mendoza A, Ríos C, & Pérez-Severiano F.  
Summary:  Results showed that 192-IgG-SAP reduced the immunoreactivity of cholinergic septal neurons (41%), compared with PBS-receiving control rats (p < 0.05).

Co-Treatment with Rivastigmine and Idalopirdine Reduces the Propensity for Falls in a Rat Model of Falls in Parkinson’s Disease. Lid – 10.1007/S00213-018-5150-Y [Doi]. (2019). Psychopharmacology (Berl), [Epub ahead of print] (1432-2072 (Electronic). Koshy Cherian A, Kucinski A, Wu R, de Jong IEM, & Sarter MA-Ohoo.
Objective: The authors used a longer and more taxing rotating beam apparatus to determine the potential therapeutic efficacy of idalopirdine when combined with  rivastigmine.
Summary: The results extend the prediction that the combined treatment with idalopirdine and an AChE inhibitor improves complex movement control and reduces propensity for falls in patients with movement disorders.
Dose: Basal forebrain cholinergic neurons situated in the nucleus basalis and substantia innominata were targeted with 192-IgG-SAP in aCSF infused bilaterally (200 ng/μL; 0.8 μL/hemisphere).

Basal Forebrain Chemogenetic Inhibition Disrupts the Superior Complex Movement Control of Goal-Tracking Rats. (2019). Behav Neurosci, 133 (1):121-134. 2019/01/29. Kucinski A, Kim Y, & Sarter M.
Dose: Basal forebrain cholinergic neurons situated in the nucleus basalis and substantia innominata were targeted with 192-IgG-SAP in aCSF infused bilaterally (200 ng/μL; 0.8 μL/hemisphere).

Partial Depletion of Septohippocampal Cholinergic Cells Reduces Seizure Susceptibility, but Does Not Mitigate Hippocampal Neurodegeneration in the Kainate Model of Epilepsy. (2019). Brain Res, 1717 235-246. Soares JI, Da Costa C, Ferreira MH, Andrade PA, Maia GH, & Lukoyanov NV.
Objective:  To examine how the inhibition of epilepsy-related cholinergic plasticity may be reflected inseizure susceptibility and/or in the development of chronic epilepsy and its neurological consequences.
Summary:  These data suggest that seizure-induced plasticity of cholinergic cells may indeed enhance seizure susceptibility and contribute to epileptogenic processes. They do not support the hypothesis that epilepsy-related hypertrophy of cholinergic neurons may potentiate hippocampal cell loss and respective behavioral impairments.
Dose:  Bilateral lesions of cholinergic cells were made by infusing 0.5 μl of SAP (0.08 μg/μl saline solution) into the hippocampus.

Disruption of Medial Septum and Diagonal Bands of Broca Cholinergic Projections to the Ventral Hippocampus Disrupt Auditory Fear Memory. (2018). Neurobiol Learning Memory, 152 71-79. Staib JM, Della Valle R, & Knox DK.
Objective:  To determine which efferent projections are critical for contextual fear memory discrimination and extinction memory.
Summary:  The results of this study suggest that MS/vDBB cholinergic neurons are critical for fear and extinction memory.
Dose:  192-IgG saporin was infused into all brain regions at a concentration of 0.2 μg/μL dissolved in 0.2 M  PBS. The total volume of each injection was 0.5 μL.  Sham surgeries were accomplished using the same volume (0.5 μL) of PBS.

Targeting Stem Cells

Stem cell research has great potential to understand, treat and cure diseases.  By watching stem cells mature into cells in bones, heart muscle, nerves, and other organs and tissue, researchers can determine how diseases and conditions develop.  Here are some recent publications that use ATS products to advance research involving stem cells.

Figure Legend.  Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nat Biotechnol. 2016 Jun 6. doi: 10.1038/nbt.3584. [Epub ahead of print] PMID: 27272386.  Hematoxylin and eosin staining of femur marrow sections of non-treated control, 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice 2 days post-conditioning.  See Targeting Trends cover article.

Targeting the niche: depleting haemopoietic stem cells with targeted therapy. (2019). Bone Marrow Transplantation. DOI 10.1038/s41409-019-0445-0. Abadir E, Bryant C, Larsen S, & Clark GJ.
Review:  Anti-mouse CD45 ADC (clone 104-Saporin) Depletes mature lymphoid cells and HSPC, conditioning allows for high level sustained multilineage engraftment of congenic mice.
Anti-mouse ADC (CD117-saporin) Combined with T cell depleting agents allowed for significant and durable engraftment in an immunocompetent mouse allo-HSCT model.

Immunological Properties of Neural Crest Cells Derived from Human Induced Pluripotent Stem Cells. (2018). Stem Cells Devel, 28 (1):28-43.  Fujii S, Yoshida S, Inagaki E, Hatou S, Tsubota K, Takahashi M, Shimmura S, & Sugita S. 
AB-N07: Anti-NGFr  ME20.4; Dose:  IHC; 1:200 dilution

Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation. (2019). Nat Commun, 10 (1):617-617.  Czechowicz A, Palchaudhuri R, Scheck A, Hu Y, Hoggatt J, Saez B, Pang WW, Mansour MK, Tate TA, Chan YY, Walck E, Wernig G, Shizuru JA, Winau F, Scadden DT, & Rossi DJ.   
IT-27: Streptavidin-ZAP
Objective:  To investigate a safe and effective method for hematopoietic stem cell transplantation.
Summary:  CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects.
Dose:  The CD117-ADC was prepared by combining biotinylated anti-CD117 (clone 2B8) with Streptavidin–ZAP.  A dose of 1.5 mg/kg of CD117-ADC (~12 µg Streptavidin-ZAP) optimally resulted in depletion of >99% of immunophenotypic and functional HSCs.

Viability of Mouse Retinal Explant Cultures Assessed by Preservation of Functionality and Morphology. (2019). Invest Ophthalmol Visual Sci, 60 (6):1914-1927. Alarautalahti V, Ragauskas S, Hakkarainen JJ, Uusitalo-Järvinen H, Uusitalo H, Hyttinen J, Kalesnykas G, & Nymark S.
AB-N38:  Anti-Melanopsin
Summary: Organotypic retinal explant cultures have been used to study retinal development, retinal diseases and injuries, drug screening, and retinal stem cell therapies.
Dose:  The amount of ganglion cells and melanopsin-expressing intrinsically photosensitive RGCs (ipRGCs) were detected by staining of RNA-binding protein.  Melanopsin detection by Immunostaining.  1:2500

Hematopoietic chimerism and donor-specific skin allograft tolerance after non-genotoxic CD117 antibody-drug-conjugate conditioning in MHC-mismatched allotransplantation. (2019). Nature Commun, 10 (1):616.  Li Z, Czechowicz A, Scheck A, Rossi DJ, & Murphy PM.
IT-27:  Streptavidin-ZAP
Objective:  To develop a conditioning protocol for fully MHC-mismatched bone marrow allotransplantation in mice involving transient immunosuppression and selective depletion of recipient hematopoietic stem cells.

Summary:  CD117-ADC conditioning promotes skin allograft tolerance.
Dose:  Biotinylated monoclonal antibodies directed against mouse CD117 were coupled to Streptavidin-ZAP.  Each mouse was injected with 1.5 mg/kg of ADC in a total volume of 300 mcl PBS.

Novel Application of Human Periodontal Ligament Stem Cells and Water-Soluble Chitin for Collagen Tissue Regeneration: In Vitro and In Vivo Investigations. (2011). Tissue Engineer Part A, 18 (5-6):643-653.  Jung IH, Park JC, Kim JC, Jeon DW, Choi SH, Cho KS, Im GI, Kim BS, & Kim CS. 
AB-T044: Trans-hydroxyproline Rabbit Polyclonal, Conjugated; Western blot 1:1000

An ID2-dependent mechanism for VHL inactivation in cancer. (2016). Nature, 529 (7585):172-177. 2016/01/07. Pmc5384647  Lee SB, Frattini V, Bansal M, Castano AM, Sherman D, Hutchinson K, Bruce JN, Califano A, Liu G, Cardozo T, Iavarone A, & Lasorella A.
AB-T044:  Trans-hydroxyproline Rabbit Polyclonal; Western blot.

Targeting Glioblastoma

Glioblastoma, or glioblastoma multiforme (GBM), is a rare disease, with a global incidence of <10 per 100,000 people; the prognosis is very poor.  Here are a couple of recent studies testing targeted saporin therapies.

Integrin α10, a Novel Therapeutic Target in Glioblastoma, Regulates Cell Migration, Proliferation, and Survival.
Munksgaard Thorén M, Chmielarska Masoumi K, Krona C, Huang X, Kundu S, Schmidt L, Forsberg-Nilsson K, Floyd Keep M, Englund E, Nelander S, Holmqvist B, & Lundgren-Åkerlund E.
Cancers, 11 (4):587, 2019
Objective:  To investigate the potential of integrin α10β1 as a therapeutic target in  glioblastomas (GBMs).
Summary:  integrin α10β1 has a crucial role in the migration, proliferation, and survival of GBM cells and that an integrin α10β1 antibody–drug conjugate induced cell death of GBM cells both in vitro and in vivo.
Dose:  Infusions of anti-10-SAP or Anti-ctrl-SAP were made icv (1 µg/2 L per infusion).

Expression of Different Neurokinin-1 Receptor (NK1R) Isoforms in Glioblastoma Multiforme: Potential Implications for Targeted Therapy.
Cordier D, Sailer M, Gerber A, Kluba C, Bauman A, Hutter G, Mindt TL, Mariani L.
Cancer Biother Radiopharm Epub2014.
The neurokinin-1 receptor (NK1r) has been found to be consistently over-expressed in gliomas, making it a potential target for therapeutic strategies. However, treatments with therapies utilizing substance P (SP), the ligand for the NK1r, have at best yielded uneven results. In this work the authors investigated factors that may predict the response to therapies directed at NK1r gliomas. SSP-SAP (Cat. #IT-11) was used at a concentration of 1 nM in cytotoxicity assays on several different glioma cell lines. Using this and other data it was shown that only the cell line with the most full-length NK1r RNA transcripts displayed high levels of binding, internalization, and cell killing necessary for NK1r to be a therapeutic target using SP.

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

Q1 2019 Scientific Review

IT-01: 192-IgG-SAP

    • Co-treatment with rivastigmine and idalopirdine reduces the propensity for falls in a rat model of falls in Parkinson’s disease. LID – 10.1007/s00213-018-5150-y [doi]. Koshy Cherian A et al. (2019). Psychopharmacol (Berl), [Epub ahead of print]  Dose: Basal forebrain cholinergic neurons situated in the nucleus basalis and substantia innominata were targeted with 192-IgG-SAP in aCSF infused bilaterally (200 ng/μL; 0.8 μL/hemisphere.

    • Basal forebrain chemogenetic inhibition disrupts the superior complex movement control of goal-tracking rats. Kucinski A et al. (2019). Behav Neurosci, 133 (1):121-134. 2019/01/29. 

IT-03: Anti-DBH-SAP

   • Ablation of brainstem C1 neurons improves cardiac function in volume overload heart failure. Andrade DC et al. (2019). Clin Sci, [Epub ahead of print] CS20180589. Dose: (7.5ng/100nl of sterile saline solution) was injected bilaterally into the RVLM.

IT-06: Mac-1-SAP

   • Inflammatory macrophages in the sciatic nerves facilitate neuropathic pain associated with type 2 diabetes mellitus. Saika F et al. (2019). J Pharmacol Exper Ther 02 Jan 2019.  Dose: Injections of Mac-1-SAP or unconjugated Saporin (10 μl) were administered 3 times every 2 days. Perineural administration of Mac-1-SAP improved high-fat diet (HFD)-induced mechanical allodynia.

IT-10: IB4-SAP

   • Non-Peptidergic Nociceptive Neurons Are Essential for Mechanical Inflammatory Hypersensitivity in Mice. Pinto LG et al. (2019). Mol Neurobiol, Epub 23 Jan  Dose:  IB4-SAP (0.16–3.2 μg/5 μl, i.t.), unconjugated Saporin (as control, 1.8 μg/5 μl, i.t.), or saline (vehicle, 5 μl/i.t.) were injected into the subarachnoid space on the midline between the L5 and L6 vertebrae.

IT-20:  Orexin-SAP

   • 0054 SUVN-G3031, a Histamine H3 Receptor Inverse Agonist Produces Wake Promoting Effect in Orexin-2-saporin Lesioned Rats. Benade V et al. (2019). Sleep, 42 (Supplement_1):A22-A23. 

IT-27: Streptavidin-ZAP

   • Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation. Czechowicz A et al. (2019). Nat Commun, 10 (1):617-617. Dose:  The CD117-ADC was prepared by combining biotinylated anti-CD117 (clone 2B8) with Streptavidin–ZAP.  A dose of 1.5 mg/kg of CD117-ADC (~12 µg Streptavidin-ZAP) optimally resulted in depletion of >99% of immunophenotypic and functional HSCs.

   • Identification of lineage-specific markers for therapeutic targeting of mast cells. Plum T. (2019). (Ph.D.), Ruperto-Carola University of Heidelberg, Germany, Aachen, Germany.    Dose: Mice were injected i.v. with either 100 µg of 1:1 molar mixture of biotinylated CD63 antibody and Streptavidin-ZAP (60 µg mAb + 40 µg SAP) or with 40 µg SAP alone. All injections were performed in 200 µl PBS.

IT-63: NK3-SAP

   •Glutamatergic Neurokinin 3 Receptor Neurons in the Median Preoptic Nucleus Modulate Heat-Defense Pathways in Female Mice. Krajewski-Hall SJ et al. (2019). Endocrinology, 160 (4):803-816. 

IT-69: Nppb-SAP

   • Spinal somatostatin-positive interneurons transmit chemical itch. Fatima M et al. (2019). PAIN, 160 (5)  Dose:  Npra receptor–expressing spinal cord interneurons were ablated through intrathecal injection of Nppb-SAP (5 μg/10 μL) or control Blank-SAP in lumbar segment 3 to 4. Behavioral analyses were performed 1 week after the toxin injection.

AB-N38: Anti-Melanopsin

   • Degeneration of ipRGCs in Mouse Models of Huntington&#039;s Disease Disrupts Non-Image-Forming Behaviors Before Motor Impairment. Lin M-S et al. (2019). J Neurosci, 39(8):1505.  Dose:  Immunostaining (1:3000)

Custom Saporin Conjugate

   • Integrin α10, a Novel Therapeutic Target in Glioblastoma, Regulates Cell Migration, Proliferation, and Survival. Munksgaard Thorén M et al. (2019). Cancers, 11 (4):587.