Figure 1. Autologous and allogeneic T cell immunotherapy.

Cellular HCPs in genetically engineered drugs may cause different human reactions when administered, because specific HCPs have potential “adjuvant effects”, generate immune responses against impurities in HCPs, or cause allergic reactions or even anaphylactic shock. In addition to safety issues, the presence of HCPs can also affect product quality. For example, HCPs can trigger aggregation or fragmentation of therapeutic proteins. In addition, it was found that certain HCPs promote the degradation of polysorbates, a type of non-ionic surfactant widely used in the preparation of buffers to stabilize proteins. The degradation of polysorbates will affect the stability of protein drugs. The protease activity in HCPs can also affect the protein composition in the culture supernatant, thereby affecting the subsequent protein purification process or the stability of long-term storage of protein drugs. In addition, HCPs may undergo post-translational modifications, making quantification and characterization more difficult. In view of the fact that cellular HCPs in genetically engineered drugs may cause safety and effectiveness issues, the detection of their removal efficiency and cell limit is an important parameter for drug release and clinical research, and is one of the critical product quality attributes (CQA) of drug quality control. Therefore, although only a few clinical adverse events have so far been attributed to HCPs impurities, in order to ensure product safety, HCPs must be accurately characterized and quantitatively analyzed through highly sensitive analytical methods.

Factors Influencing HCPs

The quantity and composition of HCPs are affected by the entire production process. Among them, the expression pathway is very important. Depending on the host cells and culture conditions, the types of HCPs vary greatly, ranging from hundreds to thousands. Many host cells such as E. coli, mammalian cells, NSO, SP2/0 and the human embryonic kidney cell line HEK293 have been used for biopharmaceutical production. During mammalian cell culture, recombinant proteins are often secreted from cells into cell culture fluid (CCF), which contains HCPs. In addition, due to the death of some cells, soluble intracellular proteins are released into CCF, and some artificial operations (such as centrifugation, filtration, etc.) can also cause cell lysis. Therefore, harvested CCF usually contains secreted HCPs and intracellular HCPs. When this protein mixture is incubated in a fermenter, other changes in HCPs occur due to enzymatic activity (such as proteases or sialidases).

The main factors affecting HCPs are shown in the table below:

HCPs analysis provides information about the composition of substances entering the downstream process and the removal rate of HCPs at each purification step. In some cases, HCPs can even be combined with or co-purified with certain protein drugs. Process characterization and validation studies are needed to illustrate which process steps can remove HCPs and also to demonstrate the stability of these steps in removing HCPs. Therefore, detection of HCPs is an important part of purification process development and helps ensure production consistency. Finally, stable and highly sensitive detection methods are needed to detect HCPs of drug proteins.

Taking the CHO expression system as an example to introduce the analysis method of HCPs:

Quality Control Content and Requirements

Currently, there is no unified international standard for cell limits for HCPs, and biopharmaceutical companies use risk control to control the cell volume of HCPs. After the production and purification of any genetically engineered drug, trace amounts of HCPs are usually found in the final drug protein. The US FDA stipulates that when using a highly sensitive method to detect HCPs in pharmaceuticals, the content should be lower than the relative value of the lower detection limit of 100ppm (100ng·mg^-1 total protein), which is usually considered an important reference point for process development. HCPs levels should be monitored in: 1) preclinical batches used for toxicological evaluation; 2) all batches during clinical development; and 3) process validation samples during final production.

Research Methods

During process development and production, appropriate methods must be selected for the detection of impurities such as HCPs, and their quantitative methods have always received attention. Currently, qualitative or semi-quantitative methods may be sufficient for process sample and batch testing. Typically, methods used for HCPs analysis include immunoassay methods (such as Western Blot and ELISA) or other methods (such as electrophoresis and mass spectrometry), and multiple techniques can be studied in parallel or in combination. In addition, new detection methods have emerged but are not yet widely used due to their immaturity.

ELISA Method

Among methods for HCPs analysis, anti-HCPs-ELISA is considered the gold standard. This method (1-100ppm) is suitable for product development and process control. However, unlike conventional ELISA, the target of anti-HCPs ELISA is not single, and the total HCPs “antigen” is a complex protein group. Therefore, when producing HCPs antibodies/standard reagents, it is necessary to ensure: 1) a specific production cell line and production process; 2) its protein concentration must be accurately quantified. The general method for preparing HCPs antibodies is to use host cells containing empty expression vectors to prepare antigen-immunized animals, obtain antiserum, and isolate and purify polyclonal antibodies for ELISA detection. Since the immunogenicity of HCPs as antigens depends on factors such as the structure, properties, abundance, and relative molecular mass of the protein itself, there will be different detection results during the analysis process, which to a certain extent will lead to inaccurate quantitative results.

In addition, commercial kits cannot cover all process-specific HCPs and cannot identify changes in HCPs caused by process changes, etc. If other binding and detection antibodies are used, it is recommended to test the detection antibody coverage. HCPs coverage assessment helps evaluate the antibody’s ability to identify HCPs in the standard as well as those present in the manufacturing process and protein drug product. The design and validation of immunoassays for HCPs is a challenge due to: 1) the method has a narrow dynamic range (<100), long development cycle, high cost, and high risk of failure; 2) a wide variety of possible kinds of HCPs in genetically engineered drugs; 3 ) Generally, polyclonal antibody reagents are used for detection; 4) Different proteins have similar epitopes, leading to false positives; 5) Hook effect, high-abundance proteins lead to detection saturation; 6) The antigen composition of HCPs should be comprehensive enough to withstand changes in the normal production process of the product; 7) There is a lack of completely matching quantitative standards. Since the HCPs test is used to detect samples containing HCPs, any cross-reactivity between anti-HCPs antibodies and drug proteins will affect the test and lead to biased results. Therefore, contamination by HCPs antigens must be avoided. In the future, it can be expected that ELISA will remain the gold standard for detecting HCPs, but in order to make this detection method more accurate, it is crucial to select appropriate antibodies.

Gel Electrophoresis/Western-Blot Method

Gel electrophoresis is commonly used in biopharmaceutical development laboratories to perform semi-quantitative analysis of different proteins present in a sample. For HCPs analysis, one-dimensional SDS-PAGE gels do not have high resolution but are used in combination with Western-Blot. Two-dimensional electrophoresis (2-DE) is commonly used for upstream or downstream process development and characterization, with its enhanced ability to resolve different HCPs on a single gel. The principle of 2-DE is to separate based on isoelectric point (first dimension) first, and then separate based on size (second dimension). Spot analysis in gel does not require immunoblotting, avoids the problem of membrane transfer, and can achieve the separation of trace HCPs impurities and products. Provides approximate relative molecular mass and isoelectric point information of protein spots, but excess protein may obscure HCPs spots. Mass spectrometry is often used to further identify in-gel proteins. Often, gel electrophoresis is used as a complementary method for the detection of non-immunogenic HCPs.

Western-Blot is used for consistent screening of large numbers of samples and detection of unknown proteins that react with anti-HCPs antibodies. It is not only suitable for detecting HCPs, but also provides approximate information on the relative molecular weight of HCPs and can be used as part of a GMP control system. However, Western-Blot also has big problems: 1) It requires high-quality HCPs polyclonal antibodies; 2) A large amount of overloaded target proteins in the gel will cause non-specific staining of the bands, thereby masking the possible bands of HCPs; 3) SDS-induced protein denaturation may lead to the loss of conformational epitopes; 4) Western-Blot cannot accurately quantify HCPs, and the sensitivity of the experiment depends on the quality of the polyclonal antibody.

Proximity Ligation Analysis (PLA)

In the PLA experiment, oligonucleotide chains (i.e., PLA probes) are labeled on 2 antibodies targeting different epitopes. These probes are connected by ligase when they are close to each other, and then amplified by qPCR to quantify the target proteins (i.e. HCPs).

Biosensor Method

Researchers report the use of biosensors for the detection of typical impurities in biopharmaceutical production. Biosensors can measure target molecules online making them promising analytical tools in process analytical technology (PAT). However, unlike traditional immunoassays, biosensors require regeneration, so the use of weak-affinity antibodies is recommended, but at the expense of sensitivity.

Mass Spectrometry

In recent years, mass spectrometry (MS) has been increasingly used in the detection of HCPs to help identify possible risk factors and evaluate purification processes. Although ELISA is most commonly used to detect the total amount of HCPs, it cannot identify HCPs outside the detection range of the ELISA kit, and compared with traditional ELISA methods, the liquid chromatography mass spectrometry (LC-MS) method has a short development time, It can specifically confirm and quantify all HCPs, detect all impurity proteins in an unbiased manner, and has the advantages of high method dynamic range (up to 6 orders of magnitude) and fast method adjustment.

Related Products

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Classification of TGase in Mammals

There are many kinds of TGase, which are distributed in various tissues and perform different functions. The discovered TGase can be divided into 8 categories from the gene level (Factor XIII a, TGase 1, TGase 2, TGase 3, TGase 4, TGase 5, TGase 6, TGase 7), 6 of which have been separated, purified and characterized at the protein level.

Relationship Between TGase and Diseases

Figure 1.TG2 multifunctional roles.

TGase and Inflammatory Diseases

TGase is closely related to a series of inflammatory diseases, including wound healing, tissue repair, fibrosis and autoimmune status.

TGase and Wound Healing

Several different types of TGase are involved in wound healing, through their synergistic effect, complete wound healing in trauma or pathological process. The most important of these is Factor XIIIa. Factor XIIIa can reduce blood loss after trauma by increasing the stability of fibers during blood coagulation, activating platelets, and participating in the regeneration of granulation tissue. TGase type 1 and type 3 are involved in the repair of epidermal trauma. TGase2 is involved in angiogenesis and stabilizing the extracellular matrix in the process of trauma repair by acting as a cell adhesion protein or an integrin-binding receptor.

TGase and Tissue Fibrosis

TGase is closely related to several chronic inflammatory states in liver diseases (cirrhosis, liver fibrosis, alcoholic liver and hepatitis C) and fibrosis of kidney and lung. In different stages of liver injury and fibrosis, the mRNA level of TGase2 increases. In vitro experiments have proved that under the condition of exogenously induced liver injury, nuclear factor kappaB will bind to the promoter of TGase2, thereby increasing the expression of TGase2. TGase2 is mainly involved in the formation of liver fibrosis by catalyzing the cross-linking of extracellular matrix.

TGase and Autoimmune Diseases

TGase is associated with a series of autoimmune diseases, which are typically characterized by the production of antibodies against TGase. TGase and Chronic Neurodegeneration

TGase plays a very important role in chronic neurodegeneration. They are characterized by the formation of aggregates of highly cross-linked insoluble proteins. Mainly include Alzheimer’s type senile dementia, polyglutamine (polyQ) tail diseases, such as Huntington’s disease, Parkinson’s disease, progressive supranuclear palsy.

TGase and Neoplastic Diseases

Research in this field is quite active at present, and many reports are related to this problem. The general view is that the activity of TGase2 in tumor cells is lower than that in corresponding normal tissues observed in vitro, which contains normal forms of TGase, also contains modified forms of TGase, and sometimes even inactive forms, and subcellular localization may also occur Change. The decline of TGase activity in tumors is often a biological indicator of poor prognosis, and may be related to tumor metastasis, making it easier for cells to sneak into the bloodstream through the basement membrane.

Application of TGase in Diagnosis and Therapy

The diagnosis of celiac disease can use ELISA method to detect the antibody against TGase. Experiments have shown that the activity of TGase in AD cerebrospinal fluid is higher than that in normal brain tissue, so that TGase can be used as a diagnostic indicator for some neurodegenerative diseases. TGase was first used in the treatment of FXIIIa as a replacement therapy for the treatment of rare blood coagulation gene defect diseases. Purified enzymes are used as exogenous bioglue to help repair surgical wounds, fractures and cartilage damage. In addition, some people use retinoids as a specific inducer to induce the expression of TGase2 for the treatment of malignant tumors and acne.

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Structural Properties and Hazards of SEB

SEB is mostly found in spoiled milk and meat products in food. Some studies have reported that the content of SEB in the supernatant of Staphylococcus aureus (strain S-6) culture is as high as 0.2 mg/mL, and the LD50 (half lethal dose) of mice is 0.2 mg/mL. The molecular weight of SEB is 28.4 KDa, which contains 239 amino acid fragments. The structure of SEB is mainly composed of 2 regions, of which the 129 amino acid residues in region 1 contain 2 β-sheets and 3 α-helices (α1, α2 and α3). The 2 β-sheets form a cylindrical structure, the inner walls are lined with hydrophilic residues, and form a “cross” with each other. Region 2 is divided into 2 distinct parts, one contains 2 α-helices (α4 and α5) and 2 very short β-strands, and the other has α-helices located between the curved sheet and the β-cylinder. One side of α4 and α5 is exposed to the solvent, and the two domains are connected by a loop structure. Compared with other enterotoxins, SEB has stronger heat resistance, its structure and function do not change significantly after heating at 100 ℃ for 30 min, and it can resist the hydrolysis of intestinal proteases. Therefore, food contaminated with enterotoxins can still cause potential toxic effects even after cooking and human digestion.

Enterotoxin B acts as a superantigen substance that simultaneously forms bridges by cross-linking with major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) to stimulate T cell, and does not involve antigen recognition. It can generate receptor complexes with MHC II sites on APCs and T cells. It can effectively bypass the treatment of conventional APCs, induce T cells to produce cytokines, and cause the activation and proliferation of T cell subsets. Relevant studies further confirmed that SEB increased the expression of components of the arachidonic acid pathway, leading to inflammation, edema and shock. Current studies have found that ingesting a small amount of SEB in humans will produce other symptoms such as immune disorders, organ damage, and clinical manifestations such as fever, respiratory diseases (cough, dyspnea, and chest pain) and digestive tract diseases. Because SEB has the characteristics of high temperature resistance, acid and alkali resistance, and easy preparation of aerosols, it has been listed as a B-class potential biological warfare agent by the Centers for Disease Control and Prevention (CDC) of the United States, and it is also listed in the United Nations “Prohibition of Biological Weapons”.

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Although the association between poor nutrition and illness has long been recognized, there is a lack of reliable, objective, short-term screening methods to evaluate nutritional risk. Determination of the prealbumin level is a cost-effective and objective method of assessing severity of illness in patients who are critically ill or have a chronic disease.

A prealbumin test may be used to:

* Find out if you are getting enough nutrients, especially protein, in your diet

* Help diagnose certain infections and chronic diseases

Low prealbumin scores mean that you are likely to need a nutritional assessment. Low prealbumin scores may also be a sign of liver disease, inflammation, or tissue death (tissue necrosis). High prealbumin scores may be a sign of long-term (chronic) kidney disease, steroid use, or alcoholism.

TABLE 1 Prealbumin Risk Stratification

Source:  Prealbumin in Nutritional Care Consensus Group. Nutrition 1995;11:170.

Test results may vary depending on your age, gender, health history, the method used for the test, and other things. Your test results may not mean you have a problem. Ask your healthcare provider what your test results mean for you.

Creative Diagnostics can support your testing by providing native prealbumin antigens and several monoclonal antibodies. Our new high-affinity mouse monoclonal antibodies have been validated both in ELISA and Immunoturbidimetric. Clones can be paired with each other and are suitable for the development of diagnostics assays.

Featured Prealbumin/TTR Antigens and Antibodies

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Most existing treatments for individual proteins rely on interactions with specific activity regulation of the target protein, such as enzyme inhibition or ligand blockade. However, there are some treatment-related proteins whose activities are unknown or unavailable, so the strategies above cannot be used to treat some proteins. In addition, strategies involving protein degradation platforms such as proteolytic targeting chimeras and other platforms (such as dTAGs, Trim-Away, chaperone-mediated autophagy targeting, and SNIPERs) have been developed for proteins that are generally difficult to target. However, these methods require the use of intracellular protein degradation mechanisms, so they are limited to proteins containing intracellular domains, and ligands can bind to these domains and recruit necessary cellular components.

Studies have found that extracellular proteins and membrane-associated proteins account for 40% of all protein-coding genes and are key factors in cancer, aging-related diseases and autoimmune diseases. Therefore, the general strategy of selective degradation of these proteins may improve human health . In this study, the researchers used a conjugate that binds to the lysosomal shuttle receptor on the cell surface and the extracellular domain of the target protein to determine a targeted degradation strategy for extracellular and membrane-associated proteins. These researchers have developed a new class of molecules that can shuttle unwanted proteins from the cell surface or the surrounding environment to the lysosome, which is the cell compartment dedicated to protein degradation. These molecules are called lysosomal targeted chimeras (LYTACs).

Mechanism of action of lysosome targeted chimera (LYTAC)
Existing studies have shown that cation-independent mannose-6-phosphate receptor (CI-M6PR) is involved in the biochemical pathway that mediates the internalization of cell line cargo. LYTAC uses this mechanism to transport labeled targeting molecules to the lysosome for degradation. The LYTAC molecule consists of an oligosaccharide peptide group (which binds to the cell surface receptor CI-M6PR) and an antibody that binds to a specific transmembrane protein or extracellular protein. The antibody can also be replaced by a small protein binding molecule. When combined with CI-M6PR and target protein at the same time, the resulting complex is swallowed by the cell membrane to form a transport vesicle. This brings this complex to the lysosome, after which the target protein is degraded and the receptor CI-M6PR is recycled. By degrading treatment-related proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71, and programmed death ligand, it proves that the platform has a wide range of effects.

The key to this tool’s function lies in its dual-function design. One side of the LYTAC molecule can be customized to bind to any protein of interest. On the other side of it is a short amino acid sequence, or peptide, inlaid with a sugar called mannose-6-phosphate. This sugar acts as a label for the cell. When a cell contains proteins that are sent to the lysosome for degradation, it adds this sugar to ensure they reach their destination. There are receptors on the cell surface that interact with this sugar. When they grab the LYTAC molecule and pull it into the cell, the labeled protein will also be dragged in with it. In the process of attaching this tag to the protein, LYTAC hijacks a natural cell shuttle mechanism designed to escort newly synthesized lysosomal proteins to their new home. However, lysosomal proteins are tough enough to survive in the presence of degrading enzymes encountered in lysosomes, and most proteins cannot do this, so those that are labeled proteins by the LYTAC method are usually destroyed. This selective degradation can help scientists study and treat diseases such as cancer and Alzheimer’s disease related to surface proteins by targeting and degrading proteins that play an important role. More interestingly, the tethered end of LYTAC can be anything that binds to proteins, such as antibodies or existing drugs, so in the future, many other proteins and diseases can be target attacked.

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Studies have found that the regulatory center of these epigenetic changes is located in the mitochondria. Mitochondria not only produce a large amount of 5′-adenosine triphosphate (ATP) through tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) to maintain cell homeostasis, they also participate in the biosynthesis of some biological molecules, such as lipids and heme , Iron-sulfur clusters and intermediate metabolites, these intermediate metabolites can be used as signals to participate in the regulation of the rest of the cell, so that mitochondria become the center of a variety of biological processes. Through the continuous signal communication between the energy control center mitochondria and the nucleus, cells and organisms can monitor and integrate the body’s nutrient utilization and energy demand information, thereby ensuring the body’s metabolic stability.

Although the complete loss or nearly complete loss of mitochondrial function is unfavorable for cells, studies have found that partial inhibition of mitochondrial activity can promote the longevity of worms, flies, and mice. Studies on Caenorhabditis elegans indicate that in the early stages of life, the reduction of electron transport chain (ETC) activity in the mitochondria leads to extensive chromatin reorganization, which is essential for activating the mitochondrial unfolded protein response (UPRmt). This pathway can promote the restoration of mitochondrial protein homeostasis and stress. Specifically, the overall chromatin compaction induced by mitochondrial stress is manifested by the use of histone H3K9 methyltransferase SETDB1/MET-2 and its cofactors ATF7IP/LIN-65 to methylate non-essential genes to avoid the transcription of non-essential genes under stress conditions. The activation of stress-related UPRmt genes is demethylated and activated by histone H3K27 demethylases JMJD-1.2 and JMJD-3.1, thereby causing a sustained response and prolonging lifespan.

UPR mt is a transcriptional response that can induce the expression of mitochondrial chaperones and proteases, and then repair or degrade the misfolded proteins in mitochondria, and ultimately restore protein homeostasis in mitochondria. In addition, studies also found that UPR mt can also promote the reconnection of cell metabolism to reduce mitochondrial stress and promote cell survival. In Caenorhabditis elegans, when mitochondrial protein homeostasis is disrupted, UPR mt is induced, resulting in a decrease in the efficiency of mitochondrial entry, so that the transcription factor (TF) ATFS-1 that would otherwise enter the mitochondria for degradation enters the nucleus. Another TF and chromatin remodeling factor DVE-1 is also involved in UPR mt signal transduction, but unlike ATFS-1, its nuclear translocation does not depend on the efficiency of mitochondrial entry.

With the deepening of research, people have increasingly realized that many metabolic intermediates such as folic acid, acetyl-CoA (acetyl-CoA) and α-ketoglutarate (α-KG) not only affect the energy and material metabolism of cells , It also has a profound and dynamic impact on the overall chromatin modification. These intermediate metabolites can serve as substrates for enzymes that modify signal proteins, metabolic enzymes, and chromatin proteins. As the main source of epigenetic regulation of metabolites, such as, acetyl-CoA which is the source of histone and protein acetylation, and s-adenosylmethionine is the epigenetic methyl donor. In addition, α-KG produced in the TCA cycle of mitochondria is an essential cofactor for histone demethylase and DNA methylase. In contrast, succinic acid and fumaric acid inhibit these α-KG-dependent enzymes. In short, mitochondrial disturbances can change the nuclear epigenome by affecting various mitochondrial-derived metabolites, and ultimately affect cell proliferation and aging.

Recently, scientists have studied TF DVE-1, which is essential for UPR mt and contains homology domains, and identified nucleosome remodeling and histone deacetylases ( NuRD) complex that mediate the subcellular localization of DVE-1 in response to mitochondrial stress. The results show that after the mitochondrial function is disturbed, the level of mitochondrial-derived acetyl-CoA decreases, and the level of histone acetylation decreases, so that the NuRD complex can achieve overall chromatin reorganization. Restoring acetyl-CoA levels by providing the substrates and nutrients needed to produce acetyl-CoA can prevent the reduction of histone acetylation and inhibit chromatin reorganization under mitochondrial stress. In general, acetyl-CoA is a signal of mitochondrial dysfunction, which regulates body aging through NuRD-mediated chromatin remodeling.

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Zonulin is a protein, synthesized in intestinal and liver cells, that reversibly regulates intestinal permeability. Zonulin modulates the permeability of tight junctions between cells of the wall of the digestive tract. It was discovered in 2000 by Alessio Fasano and his team at the university of maryland school of medicine. The classic symptom of cholera is profuse, watery, debilitating diarrhea. One of the bacterial toxins associated with cholera, called zonula occludens toxin, rapidly and reversibly opens the tight junctions between intestinal cells, temporarily causing leaky gut. Dr. Fasano and his colleagues found that cells in the human intestine produce a protein that is almost identical to the zonula occludens toxin, and they named it zonulin. Dr. Fasano’s group then isolated zonulin from human intestines and found it to increase intestinal permeability in primates.

Zonulin and Leaky Gut

Leaky gut, or “intestinal permeability,” is a condition in which the lining of the small intestine becomes damaged, causing undigested food particles, toxic waste products and bacteria to “leak” through the intestines and flood the blood stream. The foreign substances entering the blood can cause an autoimmune response in the body including inflammatory and allergic reactions such as migraines, irritable bowel, eczema, chronic fatigue, food allergies, rheumatoid arthritis and more.

Zonula occludens toxin (ZOT – R) is one of the toxins released by Vibrio cholerae which cause the severe diarrhoea experienced by those with cholera, and acts by loosening the tight junctions of the gut. As these weaken, water can rapidly flow back into the gut putting sufferers at severe risk of dehydration. When studying this toxin, researchers identified that there was a human analogue which our own gut cells could release in order to regulate tight junction structure and function, which they named zonulin (R). Importantly, zonulin remains the only modulator of intracellular tight junctions expressed so far that can affect gut function and health and associated immune response and so it is widely investigated. High levels of zonulin have been associated with increased intestinal permeability, as zonulin induces the breakdown of the tight junctions between intestinal epithelial cells. Several autoimmune, inflammatory, and neoplastic diseases have been associated with elevated levels of zonulin or evidence of increased intestinal permeability. These include celiac disease, type 1 diabetes, and juvenile nonalcoholic fatty liver disease. In addition, evidence is accumulating to support an association with multiple sclerosis, rheumatoid arthritis, asthma, and inflammatory bowel disease.

When we say that zonulin increases intestinal permeability, it is helpful to think of the lining of the intestines as like a cheesecloth. Only the tiniest particles should pass through. Zonulin makes the holes of the cheesecloth bigger and allows large particles to pass into the bloodstream and through the body. We call this increased intestinal permeability, or leaky gut. The process of leaky gut is thought to contribute to inflammation throughout the body and even to the development of autoimmune disease.  Consistent with the theory of leaky gut and autoimmune disease, excess zonulin production is found in a variety of autoimmune diseases. It is also found during flare-ups of celiac disease. Whether a person has an autoimmune disease or not, the 2 most important triggers for zonulin release are bacteria and gluten. Even in people who do not have celiac disease, the gluten and gliadin proteins that are found in wheat can trigger the release of zonulin and increase intestinal permeability.

Zonulin Elisa Kit

Creative diagnostics provides high quality zonulin elisa kits for the quantitative determination of human haptoglobin concentrations in serum-free cell culture supernates, serum, plasma, saliva, and urine. It is only for research use.

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The target protein is TRPM4 (the melatonin-related transient receptor potential), a type of important cation channel family located on the cell membrane. In recent years, it has been found that TRPM4 plays a key role in maintaining certain physiological functions and is closely associated with human disease. TRPM4 is found in all tissues of the body, including the brain, heart, kidney, colon and intestinal tissue, etc, where it plays a major role in regulating blood flow to the body through vasoconstriction, as well as setting the rhythm of the heart and moderating body immune response.

Figure 1. Human TRPM4 bound with the agonist Ca+ and modulator DVT

Wei Lu, Ph.D., an assistant professor at VARI and lead author on a study describing TRPM4’s structure, said it was important to understand the role TRPM4 played in the regulation of the circulatory system, but for years the research had been limited by a lack of insight about its molecular architecture. “Our findings not only provide a detailed, atomic-level map of this critical protein, but also reveal completely unexpected facets of its makeup.”

TRPM4 is primarily involved in the regulation of blood supply to the brain, which only accounts for about 2% of the body’s total body mass but requires 15% -20% of the blood supply. To block blood flow in the brain for several diseases, such as stroke, traumatic brain injury, cerebral edema and high blood pressure, can have serious consequences on the health of patients. Of course, these diseases are also important public health problems currently facing mankind.

Lu explained that many safe guards exist in the circulatory system of the brain to protect the body against a sudden disruption in blood supply, and one of the guards is TRPM4, “We hope that a better understanding of what this protein looks like will give scientists a molecular blueprint on which to base the design of more effective medications with fewer side effects.”

The researchers also expressed that the structure of TRPM4 is distinctly different from other molecular structures in the TRP superfamily, which is a special class of proteins that mediate the body’s response to sensations and sensory stimuli, such as pain, pressure, vision, temperature and taste. Known as the ion channels, proteins like the TRP nestle within the cell membrane acting as safeguards to regulate the intensity of the chemical signals that pass in and out of the cell.

The TRP superfamily contains eight molecules, and TRPM4 appears to be wholly unique. For the first time, this study mapped the structure of the TRPM4 protein at the atomic level. It is a crown-like structure, with four peaks that make up a large N-terminal domain, which is also a major hallmark of TRPM4 protein. This region, found at the start of the molecule, is a major site of interaction with cellular environment and other molecular. At the other end of the TRPM4 protein, called the C-terminal domain, the researchers found an umbrella-shaped structure supported by a “”pole” and four helical “ribs”, a feature that has never been observed.

Researchers clearly observed the atomic level structure of TRPM4 protein using an advanced cryogenic electron microscopy suite that enables scientists to observe the details of the smallest components of life. Dr. Lu believes they will do more in-depth studies to elucidate the key role of TRPM4 in the development of a variety of diseases to provide potential targets for the development of novel disease therapies.

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In hospital, we always been taught to C-reactive protein is “inflammation” synonymous in the blood test, especially in the case of cold. When the C-reactive protein increased, indicating that patients with inflammation. But is C-reactive protein only indicate to the issue? C-reactive protein is one of the most important and sensitive markers of human acute phase response. There are more clinical significance. Pregnancy, autoimmune arthritis, lupus, infections, and cancer can cause an especially high CRP.

1 CRP and infection

CRP is an important factor in determining the etiology of infection. The level of CRP can be significantly higher in bacterial infections. It can distinguish between bacteria and virus infections. A value higher than 100mg/L strongly suggests bacterial infections, whereas that below 10 mg/L indicates viral infection.

2 CRP during normal pregnancy

CRP does not cross the placental barrier and therefore, will be useful in diagnosing infections in newborns. It has been shown that CRP is present in amniotic fluid and fetal urine, and the elevated levels are associated with adverse pregnancy outcome.

3 CRP and cardiovascular risk

A C-reactive protein test can help doctors determine your risk for heart disease or stroke. CRP is unlikely to contribute directly to cardiovascular disease as a pathogenic factor. Similar conclusions were drawn from recent Mendelian randomization studies. Using widely available high-sensitivity assays, CRP levels of 1, 1 to 3, and 3 mg/L have been classified as low, moderate, and high-risk groups for future cardiovascular events. Individuals with LDL cholesterol below 130 mg/dL and CRP levels of 3 mg/dL represent a high-risk group.

4 CRP and cancer

CRP levels have been used to predict the risk of cancer, detect cancer recurrence, and in prognosis. CRP is a biomarker of inflammation and indicator of the immune response to tumors. Its role as a predictor of survival has been shown in multiple myeloma, melanoma, lymphoma, ovarian, renal, pancreatic, and gastrointestinal tumors.

5 CRP and diabetes

Elevated levels of CRP and IL-6 predict the development of type 2 diabetes. This association supports a possible role for inflammation in diabetogenesis. CRP is a powerful independent predictor of diabetes, after adjustment for obesity, clinical risk factors, and fasting insulin levels. Minor increase in CRP level has also been reported to be associated with a number of medical conditions that do not appear to be associated with inflammation. Elevated CRP is also observed with several genetic polymorphisms of the CRP and other genes, ethnicity, dietary patterns and obesity.

Creative Diagnostics provides high quality CRP antibodies and CRP elisa kits for CRP related research, these products have been tested and validated by our in-house team of scientists for sensitivity and reproducibility.

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There are three main kinds of cytoskeletal filaments: microfilaments, microtubules, and intermediate filaments in Eukaryotic cells. Intermediate filaments help to provide structure to cells, and are involved in cell movement. Vimentin, a 57kDa intermediate filament protein, which is a class III intermediate filament protein and often found in mesenchymal cells in eukaryotes, or cells that contain a distinct nucleus, and predominately expressed in developing embryo and in cells. Vimentin intermediate filaments are in general present in mesenchymal cells. Vimentin is found not only in eukaryotic cells, but also in bacteria, where it helps form the cytoskeleton. Vimentin is encoded by VIM gene, 57kDa Protein, and have 466 amino acids, VIM gene is highly conserved in vertebrates, VIM gene also involved in the immune response, and controls the transport of low-density lipoprotein (LDL)-derived cholesterol from a lysosome to the site of esterification.

Function of Vimentin

Vimentin plays a significant role in holding cellular structures of the organelles in the cytosol, this protein has a flexible nature, allowing it to respond to mechanical stress. It interacts with other structural proteins, like microtubules, to make the cell rigid and sturdy. Studies performed on cells without vimentin found that they were functional, but very easily damaged when exposed to pressure. It was found that cells without vimentin are extremely delicate when disturbed with a micropuncture. Vimentin is attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally. As an organizer of a number of critical proteins, vimentin function involved in attachment, migration, and cell signaling.

Except for it maintains cellular integrity, stabilizes cytoskeleton interactions and provides resistance to avoid cells damage, vimentin also has an important clinical significance as a tumor marker. All intermediate filament proteins are expressed in a highly developmentally-regulated fashion; vimentin is the major cytoskeletal component of mesenchymal cells. vimentin is a widely expressed and highly conserved that is constitutively expressed in mesenchymal cells. Because of this, vimentin is often used as a marker of mesenchymally-derived cells or cells undergoing an epithelial-to-mesenchymal transition (EMT) during both normal development and metastatic progression. Vimentin IF proteins have been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-to-mesenchymal transition, and the metastatic spread of cancer.

VIM antibody usually as a marker used for mesenchymal in histopathological diagnosis, and often with keratin to distinguish epithelial and mesenchymal tumors, such as identification of malignant melanoma, lymphoma and thymoma; VIM antibody and leukocyte common antigen to distinguish lymphoma and other mesenchymal tumors; VIM antibody and myogenic cell markers often identifiy myogenic and fibrous tumors, etc.

Vimentin Products

Creative Diagnostics offers a wide range of Vimentin related products such as vimentin proteins, antibodies, elisa kits, hybridomas, engineered antibodies and cDNA products for use in common research applications: ELISA, Flow Cytometry, Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Immunoprecipitation, Intracellular Staining by Flow Cytometry, Peptide ELISA, Sandwich ELISA, Simple Western, Western Blot.

Vimentin antibodies

Vimentin ELISA kits

Vimentin hybridomas

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