Sensitive, Specific and Cost Effective Protein Expression Analysis

Reaching for Cell Based Assay Solutions Excellence

We have been receiving positive feedback from clients using our Quantibody® and RayBio® C-Series Membrane-Based Antibody Arrays. Our manufacturing partner, Raybiotech, is ISO 9000 certified insuring all arrays are made to exacting specification. The cost for measuring a single cytokine runs as low as 16 USD/Cytokine!

We use these arrays to test the blood serum of clients with autoimmune diseases to determine levels of cytokine and growth factor expression vs healthy controls. We also use them to determine proteins secreted by our  USB Derived hMSCs cultured in our hMSC Media. This gives us insight as to the protein expression patterns in our hMSC, Chondrogenesis and Osteogenesis Assays.

Figure: The graph shows cytokine secretion of hMSCs after a continuous 24 day culture. These results were obtained using our select markers from our cytokine and bone metabolism arrays. Data courtesy of Dr. Jim Musick and Tiana Tonrey, Vitro Biopharma.

We are pleased to announce the addition of these new human and mouse arrays:

Quantibody® Human Growth Factor+Neurotrophin+Cytokine Array Q1-10. Precisely measures: Amphiregulin, BDNF, bFGF, BMP-4, BMP-5, BMP-7, beta-NGF, EGF, EGFR, EG-VEGF (PK1), FGF-4,FGF-7 (KGF), GDF-15, GDNF, Growth Hormone, HB-EGF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-1, Insulin, M-CSF R, NGFR (TNFRSF16), NT-3, NT-4, Osteoprotegerin (TNFRSF11B), PDGF-AA, PLGF, SCF, SCF R (CD117/c-kit), TGF alpha, TGF beta 1, TGF beta 3, VEGF-A, VEGFR2, VEGFR3 and VEGF-D.
Mouse C-Series Cytokine Array. C1-2-Semi-quantitavely measures: GCSF,GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-6, IL-9, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-17A, IFN-gamma, MCP-1 (CCL2), MCP-5, RANTES (CCL5), SCF, TNF RI (TNFRSF1A), TNF alpha, Thrombopoietin, (TPO) and VEGF-A.

Images: Representative images obtained with RayBio® C-Series Antibody Arrays. These membranes were probed with conditioned media from two different cell lines. Membranes were exposed with Kodak X-Omat® film at room temperature for 1 minute. Note the strong signals of the Positive Control spots in the upper left and lower right corners.

Questions? Do not hesitate to contact me: pshuster@neuromics.com or direct phone-612-801-1007. Thank you. Pete Shuster, CEO and Owner, Neuromics

Treating Autism-Immune System Balancing and Stem Cell Activating Therapies

Linking the Immune System with Stem Cell Health to Treat ASD Children

We have been testing immune/inflammatory response, oxidative stress and growth factor/neurotrophin biomarkers in the blood sera of Children with Autism Spectrum Disorder (ASD). We did this testing using our Quantibody Arrays.

The purpose of these tests were to establish a baseline for the state of these children's immune system and stem cell health. In this baseline testing, we found that all had high immune/inflammatory response, oxidative stress and low levels of the growth factors/neurotrophins needed for repairing and regenerated damaged cells/tissue (stem cell health).

We then treated these children with customized combinations of immune balancing and stem cell activating agents with the goal of showing that any improvement in ASD related symproms would be confirmed by positive modulations of biomarkers that are dysregulated in this disorder. We re-test at 6 months after initiation of the treatment plans.

The goal of our treatment strategies is to balance the immune system and activate the natural cell/tissue repair and regeneration (stem cell) processes. We call this the immunoLink^TM.

We are seeing improvement in the symptoms of the first 2 children recently retested and a marked improvement in the levels of their biomarkers. Here's data from one of the children. Note that key immune/inflammatory response markers have now moderated to the levels of healthy controls.

Graphs: Comparison of initial testing (red) and 6 month re-test (green) vs healthy controls (blue) for one of our ASD Children

We are developing a website that will have a wealth of information to help parents determine treatment strategies for ASD Children. We are also making our treatment plans and strategies available to interested parents. The website ecosystem will be named immunoLink^TM Therapies.

In the meantime, should you want to investigate if we could help your child, do not hesitate to contact me e-mail: pshuster@neuromics.com or cell: 612-801-1007. Pete Shuster, CEO and Owner.

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.

Autism, Heavy Metals, BDNF and HSP-70

Autism Spectrum Disorder (ASD) Testing using Custom Quantibody® Array

We recently completed testing blood serum from ASD children. These children were all from Central Europe and lived in cities with heavy industries. All had elevated levels of heavy metals like Aluminum and Copper. We believe that these levels may be contributing to the pathogenesis of the ASD symptoms.

Though the sample was small, the results were striking. The elevated levels of Heat Shock Protein 70 (HSP 70) is consistent with high heavy metal toxicity. Studies have reported both elevated and decreased levels of Brian Derived Neurotrophic Factor (BDNF) vs healthy controls, but is always reduced in subjects with high heavy metal blood serum levels. Could these elevated heavy metals be a root factor ASD Children?

Based on these findings, we have proposed a 2 pronged treatment strategy:

1. Bring heavy metal serum levels into acceptable balance.
2. Treat ASD children with natural stem cell enhancing substances

Stem Cells are immune/inflammation supressive and capable of repairing tissue damage. Our theory is that this could result in improvement in ASD related symptoms. Testing serum levels of BDNF, HSP-70 and other select bio-markers would confirm therapeutic efficacy.

I will be posting results updates here so stay tuned.

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.

Proud Distributor of RayBiotech's Antibody Arrays and ELISAs

Pioneer and Market Leader

I am pleased to announce we are a distribution partner for RayBiotech. The solutions we offer include Antibody Arrays and ELISA Kits. With these additions, you now have many options for the either the qualitative, semi-quantitative or quantitative measurement of protein expression. From single antibody pair ELISAs to multiplexed arrays. The arrays enable you to simultaneously analyze up to 80 cytokine, inflammatory response, growth factor or angiogenesis related proteins.

In this posting, I focus on our new C-Series Antibody Array Kits. The kits utilize the sandwich immunoassay principle, wherein a panel of capture antibodies is printed on a nitrocellulose membrane solid support (usualy 2.5 cm x 3 cm). The array membranes are processed similarly to a Western blot (chemiluminescent readout). Signals are then visualized on x-ray film or a digital image, allowing densitometry data collection and calculation of fold-changes for each detected protein. The entire procedure can be completed in 1 day, and is simple enough to permit even the novice researcher to successfully collect data with very few pitfalls and little or no optimization.

They are proven and frequently published. You can now analyze the protein expression in you assays for a fraction of the cost of tradition methods. Here's an example: Simona Giorgini, Daniela Trisciuoglio, Chiara Gabellini, Marianna Desideri, Laura Castellini, Cristina Colarossi, Uwe Zangemeister-Wittke, Gabriella Zupi and Donatella Del Bufalo. Modulation of bcl-xL in Tumor Cells Regulates Angiogenesis through CXCL8 Expression. doi: 10.1158/1541-7786.MCR-07-0088. Mol Cancer Res August 2007 5; 7

Figure: bcl-xL overexpression in ADF glioblastoma and M14 melanoma cells increases CXCL8 expression. Image of membranes from a protein array. Membranes are probed with CM from the (A) glioblastoma control (AN8) and bcl-xL–overexpressing (AXL42 and AXL74) clones and (B) melanoma control (Mneo) and bcl-xL–overexpressing (MXL12 and MXL90) clones. In the negative control (NEG), the CM is replaced with an appropriate mock buffer according to array protocol. The intensity of protein signals for each membrane was compared with the relative positive signals by densitometric analysis. C. Schematic representation of proangiogenic factors that can be detected by the use of the membrane. The CXCL8, TGF-1β, TIMP1, TIMP2, and VEGF protein signals (two spots) are indicated by red, blue, yellow, violet, and green rectangles, respectively, in each image....The Human Angiogenesis Antibody Array I (RayBiotech. Inc.) was used according to the manufacturer's protocol to evaluate the secretion of 20 angiogenic factors into the CM of the different lines. A schematic representation of the proangiogenic factors that may be detected by the use of the array has been reported in the above figure. Membranes spotted in duplicate with antibodies against angiogenic factors were blocked with blocking buffer and then were incubated overnight with CM. Next, membranes were washed with wash buffer, incubated with biotin-conjugated antibodies against proangiogenic factors, washed with wash buffer, and incubated with horseradish peroxidase–conjugated streptavidin. The signals on the membranes were detected by chemiluminescence. Membranes, blocking and wash buffer, and antibodies against proangiogenic factors were all provided with the kit. The intensity of protein signal (two spots for each protein) was compared with the relative positive signals by densitometric analysis.

Here's an excellent video that provides more detail on Antibody Array Kit capabililities:

I will continue to post information on new addition with related pubs and data.