Multiplexed Protein Array for Immunologists

Assay 1,631 Antibodies in Under 8 Hours!
The RayBio® Immunome Protein Arrays are multiplex protein immunoassays for numerous research applications. Correctly folded, functional proteins are immobilized onto a solid glass slide surface. The slides can then be probed with serum or small molecules and used in protein-antibody studies, small-molecule inhibition of kinase studies, protein-protein studies, methylation studies or DNA binding studies. Our main immunome array product offering contains 1,631 proteins. Smaller custom arrays are also available.

Each 75mm x 25mm glass slide is spotted with 4 identical protein arrays (also called “subarrays”), each with 1631 correctly folded and functional spotted proteins! Each protein is attached to the streptavidin-coated slide exclusively via a biotinylated affinity tag, specifically the biotin carboxyl carrier protein (BCCP) domain of the E. coli acetyl CoA carboxylase. This affinity tag was chosen because the BCCP domain must be folded into its native 3-dimensional structure in order to become biotinylated. If the expressed protein mis-folds the BCCP tag will also mis-fold, preventing biotinylation. If the protein folds correctly then folding of the BCCP tag should be unhindered and biotinylation will result. This property allows only correctly folded proteins to be bound to the slide.

Research Applications

• Screening protein-antibody interactions 
• Performing small-molecule-kinase inhibition experiments 
• Screening protein-protein interactions 
• Performing DNA-binding experiments Performing methylation assays

How it Works

Questions? Please contact me directly-Pete Shuster, CEO and Owner. 612-801-1007 or pshuster@neuromics.com.

Immune System/Stem Cell Health and Aging

Looking, feeling and  performing your best depends on balanced immunity and stem cell vitality

As we age we encounter:

• Slower healing
• Longer recovery time from vigorous exercise
• More general aches and pains
• Longer recovery from illness
• Wrinkled, dry and thinning skin

Why is that? What are the causes? Answers can be found by a better understanding of immune/inflammatory response and stem cell systems. As we age our immune system weakens or becomes dysregulated (autoimmunity) and our stem cell "bank balances" deplete. My friend and world class stem cell therapies expert, Dr. Neil Riordan, gives an excellent description of the process: Your Body’s Stem Cell Bank Account.

To oversimplify, the immune system is responsible for cleansing the body of pathogens, toxins, allergens and damaged tissues/cells. This creates a healthy environment for stem cells to repair and regenerate new, healthy cells and tissue. We call this process the immunoLink^TM.

immunoLink Therapies is a new company of mine slated to launch in the spring of 2015. Our goal is to slow the aging process by offering solutions that balance your immunity and increase your stem cell vitality. Our vanguard product (currently available) is Stem-Kine. It is a blood stem cell booster which increases the level of immune factors and red blood cells in your cardiovascular system. The result is faster healing/recovery and increased endurance via better oxygen delivery.

If you desire to learn more, do not hesitate to call (612-801-1007) or e-mail: pshuster@neuromics.com. Thank you. Pete Shuster

Cytokines and Neuropathic Pain

I have been frequently posting results of testing blood serum of our clients suffering from Autoimmune (including Neuroimmuno) Diseases and Autistic Children. Our testing includes both standard medical testing (serum levels of toxins, metals, pathogen loads and markers like Cortisol, CRP, CDs, ALPS, NSE, S100b). In addition, we also doing testing using our Quantibody® Antibody.

Many of the clients report chronic mild to severe neuropathic pain.

By definition, this pain encompasses a series of heterogeneous conditions with some similar clinical manifestations. Peripheral examples include traumatic nerve injury, diabetic peripheral neuropathy and chemotherapy-induced peripheral neuropathy or Multiple Sclerosis. (see: Front Pharmacol. 2013; 4: 142.Published online Nov 22, 2013. doi: 10.3389/fphar.2013.00142). It is hard to unravel the cause and effects of this pain,

However, in analyzing the results from our clients' test we notice a relationship between dysregulation of some key cytokines and levels of pain (more data is required to confirm this). Here're our results (n=5)

Figure: Clients reporting pain vs healthy controls.

We will continue posting results here. We are releasing a Custom Quantibody Neuroimmuno-Pain Array which will include more bio-markers suspected to play a role in pain signaling.

Immune/Inflammatory Response and Autism

Autism Spectrum Disorder (ASD) Children and Immune/Inflammatory Response Markers

Persistent chronic inflammation/immune response and oxidative stress are hallmarks of ASD. Like autoimmune diseases, an unbalanced immune system leads to unwanted assaults on healthy tissue. In ASD Children, inflammatory/immune response proteins could cross the Blood-Brain Barrier resulting in ongoing neuro-inflammation. This throws out of balance the natural pruning and repair cycle in the brain and could drive the related behaviors and symptoms of ASD Children.

We have been working with ASD children from central Europe residing in areas of heavy industry. All have unhealthy levels of metals in there blood stream and most have persistent viral and/or bacterial infection. This testing is part of our treatment program. Our treatment strategy involves first removing heavy metals and related toxins and the adding stem cell activators (stem cells are key players in modulating the immune system and repairing damage).

Previously I reported results from our testing for neuro growth/repair and oxidative stress markers using our custom ASD array. Here, I report on results from our measuring well known immune/inflammatory markers in 6 of these ASD children. The markers studied were: (IL-2, IL-6 and TNF-alpha). All of the markers were elevated when compared to healthy children. Here're the results:

Graphs: ASD vs Healthy Controls (*Healthy Control Data from Kim et al. Journal of Translational Medicine. 2011: 9:113)

Here's more on these markers:

•  IL-2 is involved in immune regulation. It is one of the key factors in transplant regulation. It is also elevated in many children suffering from ASD. This elevation could be caused by toxins, chronic infection or genetic abnormalities. It should be also noted that IL-2 can cross the blood brain barrier making it a culprit in the symptoms of ASD. 
• IL-6 is a potent pro-inflammatory agent that plays a crucial role in the pathogenesis of systemic inflammatory diseases like ASD.
• TNF-a has been implicated as main effector of the functional consequences of neuroinflammation on neurodegeneration.

We are seeing improvements in symptoms and behaviors in the ASD Children undergoing treatment. We are early in the our Stem Cell Activating treatment phase. Our plans call for a cycles of testing and treating. If the tests yields movement of these markers to healthy levels, this will confirm how well our these treatments are working. Our plans our to openly share data.

If you would like to learn more about our testing and treatment regimes, I can be reached @ pshuster@neuromics.com or 612-801-1007.

Autism Spectrum Disorder (ASD) Custom Array Results

Blood Serum Levels and Key Markers

We have been testing immune/inflammatory response, oxidative stress and growth factor markers in ASD children from central Europe. Most showed high levels of  related cytokines and chemokines.

From our initial testing and published results, we have developed a custom Quantibody Array to test ASD children. The markers in this array are: BDNF, HSP-70, Leptin, RAGE and TGF-beta1.

Figure: Serum Levels of Key Markers in Tested ASD Children

Here's more on each marker:

• BDNF is a protein involved in making healthy new neurons. This protein is dis-regulated in autism. It is shown to be decreased in some studies and increased in others. These variations could be a function of age. During brain development, BDNF regulates the birth and differentiation of brain cells, or neurons. Some of BDNF’s target cells, such as cortical interneurons, which transmit information between different layers of the brain cortex, have been implicated in autism. BDNF is also a regulator of brain growth, and children with the disorder tend to have abnormally large brains during early development. Vigorous exercise, for example, increases BDNF levels in blood and studies have linked this increase to growth of new healthy neurons in the hippocampus region of the brain. We found all ASD children tested showed low levels of BDNF. All had moderate to high levels of heavy metals and viral/bacterial pathogens. Most also had evidence of leaky blood brain barriers. Could this low level be due to consumption of BDNF demanded by the chronic need for neuro-repair or is genetic in origin or perhaps both?
• HSP70 is strongly upregulated by heat stress and toxic chemicals, particularly heavy metals such as: aluminum, arsenic, cadmium, copper, mercury, etc. This upregulation in ASD could be linked to difficulty is in clearing toxins. Children with highest tested HSP70 levels also had the highest concentration of heavy metals. The 3 children with healthy levels have been undergoing ongoing treatment to clear metals.
• Leptin modulates appetite and energy tough elevated levels of leptin present in cases of autism might be an important sign of immune processes, particularly those related to inflammation. It is also suggested leptin may be a link between autism and epilepsy that provides an avenue for novel or better management of autistic children with epilepsy. All were in the range of published healthy controls (mean=2065 pg/ml).
• RAGE is hypothesised to have a causative effect in a range of inflammatory diseases such as diabetic complications, Alzheimer's Disease and even some tumors. In ASD, it is thought to be a master switch for chronic inflammation. Including in the brain. RAGE is a receptor for S100B so it is also elevated in ASD and is reflective of neurological damage. All showed elevated serum levels of rage.
• TGF-beta1 is a protein that controls proliferation, cellular differentiation, and other functions in most cells. This control includes tissue repair in the nervous system. Decreased transforming growth factor beta1 in autism is a potential link between immune dysregulation and impairment in clinical behavioral outcomes. 6 of 8 of the children had low TGF-beta1 levels. Though the correlation between symptoms and behaviors is not conclusive.

We plan to significantly increase the samples of ASD children tested using this custom Quantibody Array. . 

Our ultimate goal is to use this array as a tool to determine the efficacy of therapies we are developing. Our goals is to develop natural products based therapies that are proven to activate stem cells. These cells could catalyze immune response modulation and tissue repair. The process is to first clear metals and pathogens, then treat with stem cell activators and test and fine tune treatments and test again. 

I will be posting results on an ongoing basis. I also welcome any and all comments. I am available for direct contact at pshuster@neuromics.com or 612-801-1007.

Quantibody Arrays for Tracking Patient Health

Neuromics is working with Dr. Joe Smarda, a renowned Immunologist, to track levels of cytokines in the blood serum of his clients. We have selected RayBiotech's Quantibody® Arrays for these assays. The Clinics in Joe's network treat his clients for autoimmune related disorders.

Our regime is:

1. Test clients pre-treatment
2. Treat
3. Test
4. Refine treatment
5. Test 

The specified treatment regime is continued until clients have blood serum cytokine levels that are in the range of our healthy controls. Here's data from our Quantibody® T-helper cell Cytokine Arrays (pre-treatment).

Figures IL-6, IL-1 beta, MCP-1 and PAI1 Array results in 4 clients with Autoimmune related Diseases.

We plan on posting these serial  testing results. They are designed to monitor status and indicate therapeutic effectiveness.

We will also be sharing some of the specific therapies being used. These will include treatments aimed at mobilizing endogenous stem cells. These cells have natural immune suppression/anti-inflammatory properties. Stay tuned.

Our Antibody Arrays and ELISA Manufacturing Partner Receives ISO Certification

RayBiotech Awarded ISO 13485: 2003 Certification 

Our partner, RayBiotech, provides us Antibody Arrays and ELISA Kits. This should give potential customers confirmation that this kit are rock solid.

 NORCROSS, GA -- (Marketwired) -- 09/11/13 -- RayBiotech, Inc. announced today that the company has been awarded ISO 13485: 2003 certification with respect to its compliance in the manufacture of in vitro diagnostics kits to be provided to the research community. The ISO 13485 award applies to multivariant antibody arrays, enzyme-linked immunosorbent assays (ELISAs) as well as membrane and glass format arrays and reagents. ISO 13485 is the International Organization for Standardization's certification that the fundamentals of quality management are in place and actively implemented as formal systems within the organization. The ISO 13485 compliance certification is in addition to the company's formal compliance under Good Laboratory Practices (GLP) and Good Manufacturing Practices (GMP), and it further confirms RayBiotech's commitment to quality control and quality assurance for all of its products and services.

Figure: Multiplex antibody arrays-how they work

We will be posting customers feedback on Bazaarify as they becomes available.

Quantibody® Cytokine Arrays

Cytokines, Adipokines, Growth Factors and Neurotrophins

We are pleased to announce addition of Antibody Arrays to our catalog. These are excellent tools for study apoptosis, inflammation, angiogenesis and  immune response and related autoimmune, inflammatory and degenerative diseases plus many forms of cancers. Check out this video.

Specific Research Applications Include:
•High-throughput profiling of cytokine expression
•Validation of semi-quantitative antibody array results
•Identifying potential molecular targets for drug development
•Identifying the molecular mechanisms of drug action
•Identifying crucial factors involved in disease processes
•Discovering biomarkers for disease management
•Discovering expression patterns for molecular classification of diseases

How Quantibody Arrays Work

We will be aggresively adding new arrays to our product offerings in June. Stay tuned for more.





ASIC3 and Osteoarthritis

ASIC3 modulates pain and disease progression

Neuromics' foundation is built on providing reagents for pain researchers. I have posted the twists and turns via key publications and related data. Here's yet another success story with one of our Pain and Inflammation Research Antibodies.

Acid sensing ion channels (ASICs) are sodium-selective ion channels activated by low extracellular pH, and belong to the degenerin/epithelial Na+ channel superfamily. ASIC3  is the most sensitive to such a pH change [2,3], abundantly expressed in dorsal root ganglia (DRG) [4], and strongly correlated with pain. Here researchers show the role of ASIC3 in osteoarthritis: Masashi Izumi, Masahiko Ikeuchi, Qinghui Ji, Toshikazu Tani. Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis. Journal of Biomedical Science 2012, 19:77 doi:10.1186/1423-0127-19-77.
Highlights: OA was induced via intra-articular mono-iodoacetate (MIA) injection, and pain related behaviors were evaluated including weight bearing measured with an incapacitance tester and paw withdrawal threshold in a von Frey hair test. OA rats showed not only weight-bearing pain but also mechanical hyperalgesia outside the knee joint (secondary hyperalgesia). ASIC3 expression in knee joint afferents was significantly upregulated approximately twofold at Day 14. Continuous intra-articular injections of APETx2 inhibited weight distribution asymmetry and secondary hyperalgesia by attenuating ASIC3 upregulation in knee joint afferents. Histology of ipsilateral knee joint showed APETx2 worked chondroprotectively if administered in the early, but not late phase.

Images: Fast Blue labeling and immunohistochemistry staining for ASIC3 : (a-b) Naïve- model, (c-d) OA-model, (e-f) APETx2 administration to OA-model in early phase. Photos in each row are the same DRG. In (b),(d),(f), large arrows indicate Fast Blue labeled, ASIC3 immunoreactive (ASIC3-ir) DRG cells, while ASIC3-ir cells that were not labeled by Fast Blue are indicated by small arrowheads. More than 100 FB-labeled neurons were analyzed from 4 rats in each group. The percentage of ASIC3-ir knee joint afferents was 18 ± 3% (mean ± SD) in naïve models, 46 ± 4% in OA-models (p = 0.003), and 20 ± 5% in the early-phase APETx2 group (p = 0.006), respectively. Scale bar: 50 μm

Protocol: The [DRG] sections were blocked in 3% normal goat serum for 1 h, then incubated in primary antibody of ASIC3 (Neuromics; Edina, MN, GP 14015, 1:500) overnight in a humid chamber. The next day, the sections were incubated in the secondary antibody (Vector; Burlingame, CA, FI-7000, 1:500, FITC tagged) for 2 h. All antisera used were diluted in PBS containing 1% normal goat serum and 0.05% Triton X-100. Before, between, and after each incubation step, the sections were washed 3 times for 5 min in PBS. Finally, all sections were mounted with Vectashield (Vector, Burlingame, CA).
1. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M: A proton-gated cation channel involved in acid-sensing. Nature 1997, 386:173–177. 2. Lingueglia E: Acid-sensing ion channels in sensory perception. J Biol Chem 2007, 282:17325–17329. note: see http://neuromics.net/weblog/post/tag/dr-eric-lingueglia/ for research using our siRNA transfectio reagent for ASIC3 gene expression analysis. 
3. Wemmie JA, Price MP, Welsh MJ: Acid-sensing ion channels: advances, questions and therapeutic opportunities. Trends Neurosci 2006, 29:578–586.
4. Voilley N, de Weille J, Mamet J, Lazdunski M: Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci 2001, 21:8026–8033.

MOR and NMDAR Interplay-Implications in Pain Control

Our Opioid Receptor Antibodies continue to be referenced in publications by Pain Researchers. Many of these studies provide a greater understanding of how opioids alleviate pain and what modulates this ability.

For example, the capacity of opioids to alleviate inflammatory pain is negatively regulated by the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). This study drills down into the specifics of this regulation and references use of Neuromics' MOR1C Antibody: María Rodríguez-Muñoz, Pilar Sánchez-Blázquez, Ana Vicente-Sánchez, Esther Berrocoso and Javier Garzón. María Rodríguez-Muñoz, Pilar Sánchez-Blázquez, Ana Vicente-Sánchez, Esther Berrocoso and Javier Garzón. The Mu-Opioid Receptor and the NMDA Receptor Associate in PAG Neurons: Implications in Pain Control. Neuropsychopharmacology , (3 August 2011) | doi:10.1038/npp.2011.155.

Abstract: The capacity of opioids to alleviate inflammatory pain is negatively regulated by the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). Increased activity of this receptor complicates the clinical use of opioids to treat persistent neuropathic pain. Immunohistochemical and ultrastructural studies have demonstrated the coexistence of both receptors within single neurons of the CNS, including those in the mesencephalic periaqueductal gray (PAG), a region that is implicated in the opioid control of nociception. We now report that mu-opioid receptors (MOR) and NMDAR NR1 subunits associate in the postsynaptic structures of PAG neurons. Morphine disrupts this complex by protein kinase-C (PKC)-mediated phosphorylation of the NR1 C1 segment and potentiates the NMDAR–CaMKII, pathway that is implicated in morphine tolerance. Inhibition of PKC, but not PKA or GRK2, restored the MOR–NR1 association and rescued the analgesic effect of morphine as well. The administration of N-methyl-D-aspartic acid separated the MOR–NR1 complex, increased MOR Ser phosphorylation, reduced the association of the MOR with G-proteins, and diminished the antinociceptive capacity of morphine. Inhibition of PKA, but not PKC, CaMKII, or GRK2, blocked these effects and preserved morphine antinociception. Thus, the opposing activities of the MOR and NMDAR in pain control affect their relation within neurons of structures such as the PAG. This finding could be exploited in developing bifunctional drugs that would act exclusively on those NMDARs associated with MORs.

I will continue to post these studies. They give hope for pain sufferers as many propose potential new druggable targets.

MCP-1 promotes lung inflammation

Providing reaserchers Immune/Inflammatory Response Markers is a growing business for us. Publications referencing their use is a key validation that they can do the job.

Here's a recent pub that references use of our goat anti-mouse RAGE polyclonal antibody. More importantly, it studies the role of Monocyte chemoattractant protein 1 (MCP-1) in modulating inflammatory response in the lungs: Marieke A.D. van Zoelen, Marleen I. Verstege, Christian Draing, Regina de Beer, Cornelis van’t Veera, Sandrine Florquin, Paul Bresser, Jaring S. van der Zee, Anje A. te Velde, Sonja von Aulock and Tom van der Poll. Endogenous MCP-1 promotes lung inflammation induced by LPS and LTA. doi:10.1016/j.molimm.2011.04.001.

Potential Therapeutic Targets for Bone Cancer Pain-P2X Receptors

Cancer pain is difficult to treat as it appears to be driven simultaneously by inflammatory, neuropathic and tumorigenic mechanisms. I have reported on multiple occasions publication referencing use of our Pain and Inflammation Research Antibodies in studying bone cancer pain.

I would like to alert you to the latest reference:

Timothy K. Y. Kaan, Ping K. Yip, Sital Patel, Meirion Davies, Fabien Marchand, Debra A. Cockayne, Philip A. Nunn, Anthony H. Dickenson, Anthony P. D. W. Ford, Yu Zhong, Marzia Malcangio, and Stephen B. McMahon Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats. Brain, September 2010; 133: 2549 - 2564.

......Slides were then incubated with rabbit anti-P2X3 (1:2000, Neuromics) and sheep anti-calcitonin gene-related peptide (1:1000, Biomol...anti-beta-III-tubulin (1:4000, Promega) and guinea pig anti-P2X3 (1:100, Neuromics). The next day, after three washes with phosphate-buffered......

Summary: Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone. Using a well-established rat model of bone cancer pain, AF-353, a recently described potent and selective P2X3 and P2X2/3 receptor antagonist, was administered orally to rats and found to produce highly significant prevention and reversal of bone cancer pain behaviour. This attenuation occurred without apparent modification of the disease, since bone destruction induced by rat MRMT-1 carcinoma cells was not significantly altered by AF-353. Using in vivo electrophysiology, evidence for a central site of action was provided by dose-dependent reductions in electrical, mechanical and thermal stimuli-evoked dorsal horn neuronal hyperexcitability following direct AF-353 administration onto the spinal cord of bone cancer animals. A peripheral site of action was also suggested by studies on the extracellular release of adenosine triphosphate from MRMT-1 carcinoma cells. Moreover, elevated phosphorylated-extracellular signal-regulated kinase expression in dorsal root ganglion neurons, induced by co-cultured MRMT-1 carcinoma cells, was significantly reduced in the presence of AF-353. These data suggest that blockade of P2X3 and P2X2/3 receptors on both the peripheral and central terminals of nocioceptors contributes to analgesic efficacy in a model of bone cancer pain. Thus, systemic P2X3 and P2X2/3 receptor antagonists with central nervous system penetration may offer a promising therapeutic tool in treating bone cancer pain.

Related Reagents:

All Purinergic Receptors
Neurotransmission Research Antibodies

RAGE and Pneumonia

We are working with customers and collaborators to strengthen our product offerings for Immune Response Researchers.

An interesting finding on Receptor for Advanced Glycation End Products (RAGE) and response to S. pneumoniae pneumonia infection just crossed our radar. Dr. Marieke A. D. van Zoelen and team published evidence that RAGE plays a detrimental role in the host response to S. pneumoniae pneumonia by facilitating the bacterial growth and dissemination and concurrently enhancing the pulmonary inflammatory and procoagulant response. Data include use of our RAGE-Cat#: GT15030.

Here's the publication and related data:

Marieke A. D. van Zoelen, Marcel Schouten, Alex F. de Vos, Sandrine Florquin, Joost C. M. Meijers, Peter P. Nawroth, Angelika Bierhaus, and Tom van der Poll. The Receptor for Advanced Glycation End Products Impairs Host Defense in Pneumococcal Pneumonia. J. Immunol., Apr 2009; 182: 4349 - 4356.

...Endogenous peroxidase activity was quenched using 1.5% H2O2 in PBS. Primary Abs used were goat anti-mouse RAGE polyclonal Abs (Neuromics), and secondary Abs were biotinylated rabbit anti-goat Abs (DakoCytomation). ABC solution (DakoCytomation) was used as the...

Images: Expression of RAGE in lungs during S. pneumoniae pneumonia. Representative view of a lung from a normal, uninfected Wt mouse (A) displaying ubiquitous expression of RAGE on the surface of endothelium. B, Absence of RAGE positivity in the lung of a RAGE–/– mouse. C and D, Lungs from a Wt mouse 48 h after the inoculation of S. pneumoniae. Arrow indicates bronchial epithelium in healthy lungs (A); asterisk indicates neutrophils in an area with confluent pneumonia (D), both being negative for RAGE staining. RAGE staining: original magnification x10.

Realted Reagents:
RAGE Mouse Mononclonal
Immune Response Antibodies

Immune Response Proteins