TechNotes

Adiponectin TechNotes

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Adiponectin TechNotes

Adiponectin is an abundant protein hormone which belongs to a family of so-called adipokines. Adiponectin is expressed mostly by adipocytes and is important regulator of lipid and glucose metabolism. It is established that adiponectin is a insulin-sensitizing hormone with anti-diabetic, anti-inflammatory and anti-atherogenic properties. Potential diagnostic usage of adiponectin was a subject of increasing interest in recent years. It was shown that decreased serum adiponectin concentration indicates insulin resistance and type 2 diabetes. Besides, hypoadiponectinemia was shown to be associated with coronary artery disease. Several authors point out that high level of circulating adiponectin reduce risk of coronary heart disease among type 2 diabetes patients and is associated with reduced risk of myocardial infarction in apparently healthy men. So, there is growing interest among medical professionals to use adiponectin for insulin resistance diagnosis and predicting of cardiovascular complications in subjects with type 2 diabetes.

Several oligomeric forms of native adiponectin circulating in the blood are described in literature: trimers (low-molecular weight form, LMW), hexamers (medium molecular weight form, MMW) and higher order multimers (high molecular weight form, HMW). Concentration of total adiponectin in the blood is about 3-30 μg/ml, whereas concentration of the closest structural homolog of adiponectin, C1q, is about 80-200 μg/ml. It is therefore of utmost importance that anti-adiponectin antibodies would have no cross-reactivity with human C1q. Some authors describe significant gender differences in adiponectin level in healthy adults and these differences are believed to contribute to discrepancies in adiponectin concentration reported by various authors. It was shown, that biologic activity of adiponectin is mediated by high-molecular weight form and, not surprisingly, it has been suggested recently that concentration of HMW form of adiponectin or ratio HMW/total adiponectin (sum of three types of oligomers) in serum correlates stronger than total adiponectin with insulin resistance and other measures of type 2 diabetes.

HyTest offers new generation of anti-human adiponectin monoclonal antibodies suitable both for research purposes (Western blotting, direct ELISA) and for the development of adiponectin-specific sandwich immunoassays.

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References:
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Antibodies for New Type of BNP immunoassay TechNotes

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Antibodies for New Type of BNP Immunoassay TechNotes.

Brain natriuretic peptide (BNP) is an acknowledged marker of heart failure (HF) that is widely used in clinical practice for HF diagnosis and patient management.
BNP is known as an unstable molecule (1, 2). Several recent studies have revealed that BNP is presented by multiple forms in HF patients’ plasma, truncated from both N- and C-termini and only a small portion of BNP circulates as a full-size BNP32 molecule (3). The majority of commercially available BNP assays are designed as sandwich-type immunoassays utilizing two MAbs specific to distantly located epitopes. At least one of these two antibodies is specific to the ring structure, while the other one is usually specific to the C terminus of the BNP molecule. Recent data regarding BNP instability in circulation suggests that immunoassays utilizing at least one MAb specific to the terminal epitope could underestimate the real BNP content in the blood sample.

HyTest specialists have recently developed antibodies for a brand new type of BNP immunoassay - the “Single Epitope Sandwich” immunoassay (SES assay) - which differs from all commercially available “conventional”- type sandwich BNP assays (4).
In the SES assay the capture antibody (MAb 24C5, epitope 11-17) which is specific to the relatively stable ring part of BNP molecule recognises antigen. The detection antibody is specific only to the complex of the capture antibody with the BNP (or proBNP) and does not recognize these two molecules (capture antibody and BNP) separately. Therefore only a single epitope of BNP molecule is needed for this novel type of sandwich BNP immunoassay. This feature provides additional advantages to the SES assay over conventional BNP assays in terms of a higher apparent stability of BNP antigen in the sample or bloodstream.

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Additional product information:
Human ProBNP and proBNP-derived peptides (BNP and NT-proBNP) TechNotes

References:
Search BNP references from PubMed
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Cystatin C TechNotes

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Cystatin C TechNotes.

Cystatin C is a low molecular weight (13.4 kDa) cytoplasmic protein functioning as an inhibitor of various cystein proteases in the bloodstream. It inhibits both endogenous proteases, such as lysosomal cathepsines, and proteases of parasites and microbes. Cystatin C binds to the target molecule in µM to sub pM range in competitive reversible manner (1). Due to its important function cystatin C is synthesized at the stable level by most nucleated cells. Cystatin C consists of 120 amino acid residues encoded by a 7.3 kb gene located in chromosome 20 (2). Mutation in the cystatin C protein sequence is directly linked to the development of hereditary cystatin C amyloid angiopathy (HCCAA) in which the patients suffer from repeated cerebral hemorrhages (3).

In clinical practice cystatin C is known as a well-described serum marker of renal failure that is not dependent of age, sex or lean muscle mass (4, 5). At the same time cystatin C is becoming increasingly known marker of elevated risk of death from cardiovascular causes, myocardial infarction and stroke (5). Stable production rate and free filtration by the renal glomeruli due to the low molecular weight, and positive charge (pI 9.3) are strong advantages of cystatin C as a serum marker of renal function. Creatinine-based equations to estimate the glomerular filtration rate (GFR) are sensitive to some nonrenal factors, such as age, sex, race and lean muscle mass. There is a growing number of reports demonstrating that cystatin C is more preferable measure of GFR than creatinine as long as it doesn't depend on these factors (5). Cystatin C is also more sensitive marker of mild renal dysfunction than creatinine (6). The concentration of plasma (serum) cystatin C in healthy individuals ranges around 0.8–1.2 mg/l, depending on analytical methods (7). Change in the serum concentration of cystatin C has been proposed as an index of kidney function: increased serum levels are almost exclusively associated with a reduction in GFR. Serum concentration of cystatin C is increased near 2-fold during various renal disorders (7). Elevated serum cystatin C level is also a strong predictor of the risk of death and cardiovascular events in elderly persons (5).

The urinary concentration of cystatin C is low and the cystatin C concentration in urine from healthy subjects is about 100 µg/l, since the protein is metabolized by the proximal tubule after filtration in the renal glomerulus. However, the concentration of cystatin C in urine from patients with renal tubular disorders is raised approximately 200-fold (8).

HyTest offers a set of high-affinity monoclonal antibodies specific to different epitopes of human cystatin C molecule. We also supply our customers with information about best MAb combinations to be used in sandwich immunoassays for quantitative measurements of cystatin C in body fluids.

References:
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D-dimer and High Molecular Weight Fibrin Degradation Products TechNotes

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D-dimer and High Molecular Weight Fibrin Degradation Products TechNotes.

Fibrinogen is a main protein of the blood coagulation system. It consists of two identical subunits, each of which contains three polypeptide chains: a, b and g. The process of blood coagulation results in the conversion of ﬁ brinogen into ﬁbrin by thrombin and subsequent ﬁbrin polymerization. The fibrin clot is then digested by plasmin and ﬁbrin degradation products with different molecular weights are released into the bloodstream. D-dimer is a final product of ﬁ brin degradation (MW 180 kDa, Fig. 1). It consists of the remnants of all three chains that are cross linked by disulﬁde bonds. The dimeric structure is held by two isopeptide bonds between the C-terminal parts of g-chains forming a cross-linked region. The D-dimer level in healthy individuals is less than 0.5 mg/ml. Elevated levels of D-dimer were found in the blood of patients with pulmonary thromboembolism, deep vein thromboses, atherosclerosis and other cardiovascular diseases. The elevated level of D-dimer in blood indicates a risk of myocardial infarction and is believed to be a reliable marker of pathological coagulation that underlies pathogenesis of most cardiovascular diseases (1, 2). It is widely used to exclude deep vein thrombosis (3). Furthermore, elevated levels of D-dimer without thrombotic symptoms makes it possible to suspect malignancy. For the accurate determination of D-dimer, the assays must not detect fibrinogen and its degration products (D-monomer). HyTest offers D-dimer produced from clotted fibrinogen by means of plasmin digestion and anti-D-dimer MAbs.

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References:
Search D-dimer references from PubMed
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Related products:
Cat# 4D30; D-dimer, human, antibody
Cat# 8D70; D-dimer

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Detection of Transferrin - Transferrin Receptor Complex

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Transferrin and Transferrin receptor TechNotes.

Cells obtain iron from plasma where it circulates in a complex with a carrier protein transferrin (Tf). To be transported into cells, iron loaded Tf is bound to transferrin receptor (TfR), and their complex passes into cells by means of internalization, where iron releases by pH-dependent mechanism. Transferrin receptor is a transmembrane protein that participates in iron transport from plasma into cells. It consists of two identical subunits of 95 kDa linked by two disulfide bonds. Each TfR subunit contains an N-terminal cytoplasmic domain (1-67 amino acid residues), a transmembrane domain (68-88 amino acid residues) and a C-terminal extracellular domain (89-760 amino acid residues).

The main pool of TfR molecules is located on erythroblasts which demand a lot of iron for hemoglobin synthesis. After the erythroid cells have matured, the extracellular part of the TfR molecule is truncated from the cell surface by cleavage of an R100 – L101 bond. TfR released into the blood stream consists of 101-760 amino acid residues of cell TfR and is called soluble (or serum) transferrin receptor (sTfR). The expression of transferrin receptor depends on the concentration of iron in the cellular cytoplasm. The concentration of soluble transferrin receptor (sTfR) has been reported to be proportional to the total amount of cell-associated transferrin receptor. In blood, soluble TfR is completely bound to Tf and circulates as sTfR-Tf complex.

The determination of the sTfR level in blood has become widely used in clinical practice. The normal concentration of sTfR in blood ranges within 2 – 5 μg/ml. An increase in the sTfR level was found in iron deficiency anemia, autoimmune hemolytic anemia, hereditary spherocytosis, b-thalassemia, sickle cell anemia and some others. Soluble TfR is indispensable marker of iron deficiency anemia and is mainly used for the differentiation between iron deficiency anemia (accompanied by an increase in the sTfR level) and anemia of chronic
disease (proceeded at the normal sTfR level). The measurement of Tf is also widely used in diagnosis of anemia together with the determination of sTfR, ferritin and iron concentration in serum. Soluble transferrin receptor and transferrin are measured in plasma and serum by immunoassays based on the specific anti-Tf or anti-TfR antibodies.

HyTest offers anti-TfR and anti-Tf MAbs, allowing detection of TfR, Tf and sTfR-Tf complex in human blood.

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Enzyme and Reagents for molecular bioloby TechNotes

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Enzymes and Reagents for molecular biology TechNotes.

More than 2 decades have passed after revolutionary invention of Polymerase Chain Reaction (PCR). During this period PCR has become a powerful tool in science and laboratory practice.

For more than 10 years Hytest has been involved in the development and production of high quality products for molecular biology. Hytest's main task is to be in touch with customers and support them in conversion of their ideas into reality.

In this booklet you can find the list of high quality products you need for better work in molecular biology, genetics, diagnostics and other PCR-related fields of life science. See table 1 for comparison of basic characteristics of the thermostable polymerases that we are offering.

GAPDH TechNotes

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GAPDH TechNotes.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well-known as one of the key enzymes involved in glycolysis. It catalyzes the reversible oxidative phosphorylation of glyceraldehydes-3-phosphate. Besides functioning as a glycolytic enzyme in cytoplasm, recent evidence suggest that mammalian GAPDH is also involved in a great number of intracellular processes such as membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and repair. During the last decades many findings have been made concerning the role of GAPDH in different pathologies including prostate cancer progression, programmed neuronal cell death and age-related neuronal diseases such as Alzheimer’s and Huntington’s diseases.

GAPDH molecule is a homotetramer composed of 36 kDa subunits. Thus the molecular weight for the whole molecule is 144 kDa. Since it is constitutively and stably expressed in almost all tissues at high level, GAPDH became a well-established “housekeeping” protein and is widely used as a loading control for protein normalization in such procedures as Western blotting. It is also useful for cells visualizing in microscopy assays. Some physiological factors, such as hypoxia and diabetes, can increase GAPDH expression in certain cell types.

Anti-GAPDH monoclonal antibodies developed by HyTest, especially well characterized MAb 6C5, are suitable for GAPDH immunodetection in Western blotting, sandwich immunoassays and immunocytochemical applications.

Additional product information:
GAPDH Western blot Set TechNotes

References:
Search GAPDH references from PubMed
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GAPDH Western blot Set TechNotes

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GAPDH WB Set TechNotes. Get Quote

HyTest has developed a new, fast and sensitive method for GAPDH immunodetection in Western blot. You no longer need secondary antibodies; just use our direct MAb 6C5 -HRP-conjugate and incubation time will take only 5 minutes. Read more about this method from our GAPDH Western blot set TechNotes.

Additional product information:
GAPDH TechNotes

References:
Search GAPDH references from PubMed
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Heart type fatty acid binding protein (H-FABP) TechNotes

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Heart type fatty acid binding protein (H-FABP) TechNotes.

Fatty acid-binding proteins (FABPs) are a group of relatively small cytoplasmic proteins (12 to 15 kDa), which are abundant in tissues with active fatty acid metabolism, including the heart. All FABPs exhibit rather complicated tertiary structures and their 10 anti-parallel ß-strands are organized into 2 nearly orthogonal ß-sheets, which form a slightly elliptical ß-barrel, with two 8-10 residue helixes linking the first 2 ß-strands together.

Heart-type fatty acid-binding protein (H-FABP or FABP3) consists of 133 amino acid residues with a molecular mass of 14858 Da and theoretical pI 6.29. For almost two decades FABP has been used in clinical practice as an early, highly sensitive marker of myocardial injury. It is widely applied in emergency triage of patients with acute coronary syndromes. Although H-FABP is a small protein abundant in the cytosol, it is readily released into the circulation following myocardial damage. After acute myocardial infarction (AMI), H-FABP is released from damaged myocytes much more rapidly than, for example, troponins, which are the most specific markers of cardiac injury. H-FABP elevation in the patient’s blood could be detected within 1 to 3 hours after symptom onset, reaching its peak within 6 hours, and then returning to a normal level within 12 to 24 hours. On the other hand, H-FABP is evidently more cardiac specific than myoglobin, the other early marker of cardiac injury. H-FABP distribution in the heart (about 0.6 mg/g) is almost 10-fold lower than in skeletal muscle tissue, whereas myoglobin’s distribution in heart (2.7 mg/g) is equal to or even lower than that in skeletal muscle tissue (2.2 - 6.7 mg/g).

This difference in tissue distribution helps to differentiate between myocardial and skeletal muscle injury. Previous studies suggested that H-FABP can be used as a reliable marker for hypertrophic and dilated cardiomyopathy, heart failure, early estimation of infarct size, a reperfusion marker and for the early detection of postoperative myocardial tissue loss in patients undergoing coronary bypass surgery, stroke, and obstructive sleep apnea syndrome. Moreover, H-FABP levels were associated with the risk of death during the long-term follow-up in patients with chronic thromboembolic pulmonary hypertension. In addition, H-FABP appears to be a marker that will enable the successful detection of cardiac injury in the early asymptomatic period in patients with metabolic syndrome.

The imperfection of H-FABP as a diagnostic marker of AMI includes the relatively lower (than for troponins) cardiospecificity, smaller diagnostic window (1-24 hours following the onset of the chest pain) and its elimination from plasma mainly by renal clearance, possibly causing a falsely high value in the case of kidney malfunction.

However, these drawbacks can be overcome through the simultaneous utilization of late and more specific markers, such as cTnI.

A new generation of unique anti-FABP monoclonal antibodies, which was recently produced by HyTest, makes possible the development of highly sensitive and rapid sandwich immunoassays. Our in-house assays have a linear detection range from 0.15 to 500 mg/L (with detection limit of 0.05 mg/L). All recommended MAb combinations were evaluated in small-scale clinical trials with blood samples from AMI patients.

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References:
See all Fatty acid binding protein (FABP) References

Human papillomavirus (HPV) TechNotes

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HPV TechNotes.

Human papillomavirus (HPV) belongs to Papillomaviruses, a diverse group of DNA-based viruses that infect the skin and mucous membranes of humans and a variety of animals. Over 100 different human papillomavirus (HPV) types have been identified on the basis of difference in the virus genome nucleotide sequences (e.g. type 1, 2 etc.) Today genital HPV infection is one of the most widespread sexually transmitted diseases. Approximately 20 million people around the world are currently infected with HPV. At least 50 percent of sexually active men and women acquire genital HPV infection at some point in their lives. By age 50, at least 80 percent of women will have acquired genital HPV infection. In accordance with WHO information, genital HPV infection was a reason of over 99% of cervical cancer cases, i.e. about 1.4 million women were affected worldwide and 239 000 of them died each year.

All HPVs are transmitted by skin-to-skin contact. A group of about 30-40 HPVs is typically transmitted through sexual contact and infect the anogenital region. Some sexually transmitted HPVs, types 6 and 11, may cause genital warts. However, other HPV types which may infect the genitals do not cause any noticeable signs of infection.

Persistent infection with a subset of about 13 so-called “high-risk” sexually transmitted HPVs, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 — different from the ones that cause warts — may lead to the development of cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia (AIN). These are precancerous lesions and can progress to invasive cancer. HPV infection is a necessary factor in the development of nearly all cases of cervical cancer.

The HPV lifecycle begins from infection of epithelial tissues through micro-abrasions. At this point, the viral genome is transported to the nucleus and establishes itself at a copy number between 10-200 viral genomes per cell. A sophisticated transcriptional cascade then occurs as the host keratinocyte begins to divide and become increasingly differentiated in the upper layers of the epithelium. The viral oncogenes, E6 and E7, are thought to modify the cell cycle so as to make them amiable to the amplification of viral genome replication and consequent late gene expression. In the upper layers of the host epithelium, the late genes L1 and L2 are transcribed/translated and serve as structural proteins which encapsidate the amplified viral genomes.

HyTest offers a wide spectrum of monoclonal antibodies specific to oncoprotein E7 of “high-risk” HPV types 16 and 18 as well as of less oncogenic HPV type 11. MAbs can be used in routine immunoassays (direct or indirect ELISA, sandwich immunodetection systems, Western blotting). Some MAbs display high specificity to definite type of HPV while others can be used for determination of E7 proteins for all four types of viruses.

References:
Search Human papilloma virus references from PubMed
See other References

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Influenza Virus Types A and B

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Influenza Virus Types A and B TechNotes

Influenza viruses are unique in their ability to cause sudden, pervasive illnesses in all age groups of human population on a global scale. Three “pandemics” associated with “shift” of surface viral glycoproteins (HA, NA) have occurred in the past century. One of them, “Spanish flu”, in 1918 was responsible for more than 20 million deaths worldwide, primarily in young adults. Annual influenza epidemics associated with different virus types or subtypes caused excess morbidity and mortality especially in groups of high risk. In spite of special clinical signs of influenza (such as sudden fever (>38 °C), pronounced intoxication (headache, myalgias), dry cough, shortness of breath and sternal pain) common clinical picture varied in dependence of age, individual immunity condition, accompanied person pathology and pathogenicity of virus strain. Diagnosis during pre- and inter-epidemic periods became available as a result of laboratory test applications. Laboratory diagnosis for influenza is based on direct examination and paired serum specimens serological assay. Immunofluorescence (IF) testing performed directly on nasopharyngeal secretions. EIA tests are often more objective and more sensitive than IF for direct detection. These diagnoses are necessary for etiotropic chemotherapy prescription.

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Influenza A antigens

Influenza A monoclonal antibodies against:
1. Haemagglutinin (HA) H1, H2, H3, H5, H7, H9
2. Matrix protein M2
3. Nucleoprotein (NP)

Influenza B antigens

Influenza B monoclonal antibodies against:
1. Nucleoprotein (NP)
2. Haemagglutinin (HA)
3. Matrix protein M1

Influenza and Other Acute Respiratory Diseases (ARDs) TechNotes

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Influenza and other ARDs TechNotes

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1. Influenza A and B
Influenza viruses are unique in their ability to cause sudden, pervasive illnesses in all age groups of human population on a global scale. Three “pandemics” associated with “shift” of surface viral glycoproteins (HA, NA) have occurred in the past century. One of them, “Spanish flu”, in 1918 was responsible for more than 20 million deaths worldwide, primarily in young adults. Annual influenza epidemics associated with different virus types or subtypes caused excess morbidity and mortality especially in groups of high risk. In spite of special clinical signs of influenza (such as sudden fever (>38 °C), pronounced intoxication (headache, myalgias), dry cough, shortness of breath and sternal pain) common clinical picture varied in dependence of age, individual immunity condition, accompanied person pathology and pathogenicity of virus strain. Diagnosis during pre- and inter-epidemic periods became available as a result of laboratory test applications. Laboratory diagnosis for influenza is based on direct examination and paired serum specimens serological assay. Immunofluorescence (IF) testing performed directly on nasopharyngeal secretions. EIA tests are often more objective and more sensitive than IF for direct detection. These diagnoses are necessary for etiotropic chemotherapy prescription.

2. Respiratory Syncytial Virus (RSV)
Respiratory syncytial virus is one of the most important respiratory pathogens in infants and children provoking considerable morbidity, which often requires bed care. Severe diseases caused by Respiratory Syncytial virus are most common among infants during the first six months of life and patients with immunodeficiency. Serious lesions of the lower respiratory tract induced by Respiratory Syncytial virus (bronchitis, bronchiolitis, pneumonia) are one of the important causes of mortality in infants. 60-70 % of infants less than six months of age fail to induce detectable antibody response to natural infection. Repeated infections with Respiratory Syncytial virus are common and result in neutralizing antibody formation.

3. Adenovirus
Adenoviruses are a large group (more than 80 types) of agents, which induce respiratory infections among human beings, animals and birds. Clinical pattern of adenoviral infection is characterized by pronounced pharyngitis, conjunctivitis, general intoxication and pulmonary lesions with high fever in children. Adenovirus types 3, 4, 7, 14 and 21 often spread in military units and account for 72 % of ARDs among recruits. A considerable part of these diseases results in hospitalization. Adenovirus types 3, 4, 7, 8 and 19 are known as causative agents of epidemic keratoconjunctivitis. Some types of adenoviruses provoke outbreaks of gastroenteritis with long (more than 2 months) carriage of viruses.

4. Parainfluenza
Parainfluenza virus types 1-3 are common agents of acute respiratory infections predominating among children less than 5 years old. They induce about 15 % of acute respiratory infections. They are mostly causative agents of severe croup, bronchitis, bronchiolitis and pneumonia (Parainfluenza virus type 3) in infants. Children can be infected with Parainfluenza virus several times during one year.

5. Avian influenza
Avian influenza viruses occurring naturally among birds cause avian influenza infection. Usually “avian influenza virus” refers to influenza A viruses found mainly in birds, but infections with these viruses can occur also in humans. Avian influenza was first identified over 100 years ago during an outbreak in Italy. Since then, the disease has cropped up at irregular intervals in all world regions.
There are many different subtypes of type A influenza viruses and they differ because of changes in certain proteins on the surface of the influenza A virus (hemagglutinin [HA] and neuraminidase [NA] proteins). Many different combinations of HA and NA proteins are possible and each combination represents a different subtype. Of the 16 different hemagglutinin types only strains within the H5 and H7 subtypes cause highly pathogenic avian influenza, which is highly contagious and rapidly fatal in susceptible avian species. When highly pathogenic influenza H5 viruses cause outbreaks, the mortality rate among poultry is usually between 90 %- 100 %.

6. Newcastle disease virus (NDV)
Newcastle disease (ND) is a highly contagious and sometimes fatal illness affecting many domestic and wild bird species. The causal agent, Newcastle disease virus (NDV), is a negative-sense single-stranded RNA virus. NDV affects the respiratory, nervous, and digestive systems. Clinical signs are extremely variable depending on the strain of virus, species and age of bird, concurrent disease, and pre-existing immunity. NDV is so virulent that many birds die without showing any clinical signs.
Transmission occurs by exposure to foecal and other excretions from infected birds, and through contact with contaminated food, water, equipment and clothing. Virus-bearing material can be picked up on shoes and clothing and carried from an infected flock to a healthy one. Exposure of humans to infected birds (for example in poultry processing plants) can cause mild conjunctivitis and influenza-like symptoms, but NDV otherwise poses no hazard to human health. MAbs are negative with parainfluenza type 3 and avian influenza hemagglutinins.

References:
Search Influenza references from PubMed
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Myeloperoxidase TechNotes

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MPO TechNotes.

Myeloperoxidase (MPO) is a hemoprotein that is abundantly expressed in polymorphonuclear leukocytes (neutrophils) and secreted during their activation. The presence of a peroxidase in the cytoplasmic granules of leukocytes was suggested at the beginning of 20th century but it was the early 1940s that it was purified for the first time. Native MPO is a covalently bound tetrameric complex of two glycosylated alpha chains (MW 59 – 64 kDa) and two unglycosylated beta chains (MW 14 kDa) with total MW about 150 kDa and theoretical pI 9.2 (1).

MPO plays an important role in neutrophil microbicidal action through catalyzing chloride ion oxidation to hypochlorous acid, which is a potent antimicrobial agent. On the other hand, it was demonstrated that MPO causes oxidative modification of low density lipoprotein (LDL) to a high uptake form that is considered to be a key event in the promotion of atherogenesis (2). That’s why MPO is believed to participate in the initiation and progression of cardiovascular diseases. MPO possesses potent proinflammatory properties and may contribute directly to tissue injury. In addition, MPO is shown to be involved in pathogenesis of lung cancer (3), Alzheimer’s disease (4) and multiple sclerosis (5).

Now MPO is believed to be one of the most promising cardiac markers. Recently it was demonstrated that an increased MPO level in patient’s blood serves as a risk marker for atherosclerosis (6) and coronary artery disease (7). It predicts the early risk of myocardial infarction, as well as the risk of other major adverse cardiac events in patients with chest pain in the ensuing 30-day and 6-month periods (8, 9). The value of MPO as a marker is in that MPO predicts these outcomes independently of other known laboratory tested risk factors, including troponins, creatine kinase MB isoform (CK-MB), C-reactive protein (CRP) and lipid profile. Moreover, unlike troponins I and T, CK-MB, and CRP, MPO makes it possible to identify patients at risk for cardiac events in the absence of myocardial necrosis (8). All these factors make MPO measurements in patients an indispensable procedure to reveal patients with chest pain that are at increased risk of cardiovascular complications.

There are some autoimmune diseases connected with development of autoanibodies against MPO. MPO is a main target of anti-neutrophil cytoplasm antibodies (ANCA) - serological markers for certain systemic vasculitides, e.g. periarteriitis nodosa, microscopic polyarteriitis and pulmonary eosinophilic granulomatosis (Churg-Strauss syndrome) (10). Low to moderate anti-MPO autoantibody levels are also reported in rheumatoid arthritis.

HyTest has been producing human MPO for many years. Today it is our pleasure to announce that new generation of anti-MPO monoclonal antibodies suitable for the development of quantitative MPO immunoassay is available from HyTest. Sandwich immunoassays utilizing these antibodies demonstrated great results in precise MPO immunodetection in patient’s blood samples.

Myoglobin TechNotes

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Myoglobin TechNote.

Myoglobin is a small heme-containing protein (153 amino acid residues, molecular weight (w/o heme) 17053 Da and theoretical pI=7.29), that is responsible for the oxygen deposition in muscle tissues. Only one form of myoglobin is expressed in cardiac. Myoglobin is known as a marker of myocardial damage and it has been used for more than three decades. Nowadays, it continues to be is very commonly used in clinical practice as an early marker of AMI. It appears in patients’ blood 1 – 3 hours following onset of the symptoms, reaching peak level within 8 – 12 hours. Myoglobin is not so cardiac specific as cTnI or cTnT. Due to high myoglobin concentration in skeletal muscle tissue, even minor skeletal muscle injury results in the significant increase of myoglobin concentration in blood. Therefore, myoglobin is used together with cTnI or cTnT in clinical practice for improved specificity in AMI diagnosis.

New Innovations for Cardiovascular Diseases

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Markers of Cardiovascular Diseases Catalog.

Cardiovascular diseases (CVD) are the most lethal diseases in the Western world. As the standard of living is growing, CVD are going to be the leading cause of mortality in the whole world in the near future. This means more demanding challenges to scientifi c communities to research and develop new and more specifi c diagnostic approaches to unstable angina, acute myocardial infarction (AMI) and other life threatening cardiovascular pathologies. In this fi eld, different cardiac markers have already today a major role, but the demand for more effective and sensitive cardiac markers that could be used not only for the diagnosis but also for the prognosis of different cardiac pathologies is widely recognized in the whole clinical world.

For more than 15 years, HyTest specialists have been involved in the development and production of high quality products applicable for research and in vitro diagnostic activities (IVD) in the cardiovascular fi eld. Today, we can proudly say that HyTest is the leading producer of different reagents for cardiac diagnostics. Our reagents are widely used for the development of sensitive and precise immunoassays, some of our cardiac protein preparations were selected as international standards. We are constantly widening the product range and encouraging contacts with our customers.

There is one feature distinguishing HyTest products from the similar products of other companies. The majority of antibodies described in this catalogue were tested not only with purifi ed antigen, but also with the antigen that could be found in human blood, urine and different tissues. For the development of Introduction sandwich immunoassays, we are recommending selected pairs of antibodies. Our task is to minimize customers’ efforts and time spent for the development of immunoassays. We are offering products that could be used by our customers without additional research work.

New generation of high sensitivity cTnI antibodies TechNotes

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New generation of high sensitivity cTnI antibodies TechNotes

HyTest presents a new generation of high sensitivity anti-cardiac troponin I (anti-cTnI) monoclonal antibodies (MAbs). These antibodies were specially designed for our customers who look for the antibodies specific to the fragment 13-36 (13PAPAPIRRRSSNYRAYATEPHAKK36) of the cTnI molecule to be used for the development of high sensitivity cTnI assays.
These new MAbs are specific to the different epitopes located within the fragment 13-36 of cTnI. This fragment has a sequence unique for the cardiac isoform of troponin I, thus the MAbs are not cross-reacting with the two skeletal isoforms of TnI (skTnI). Also this fragment is distant from the site of cTnI -TnC interaction and that is why these new antibodies recognize the free cTnI and cTnI complexed with TnC with the same efficiency.

References:
Search Troponin I references from PubMed.
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PAPP-A TechNotes

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PAPP-A TechNotes.

Pregnancy-associated plasma protein-A (PAPP-A) was first identified as a high-molecular weight constituent in human pregnancy serum. In the blood of pregnant women PAPP-A exists as a covalent (disulfide bridged) heterotetrameric complex, consisting of two 200 kDa PAPP-A subunits and two 50-90 kDa subunits of the proform of eosinophil major basic protein (proMBP). Such complex is denoted as heterotetrameric PAPP-A (PAPP-A/proMBP or htPAPP-A). It was shown that proMBP has inhibitory properties against protease activity of PAPP-A in htPAPP-A heteromeric complex.

htPAPP-A is widely recognized biochemical marker of Down syndrome (DS) used in the first trimester of pregnancy. htPAPP-A level in maternal serum increases with gestational age until term. In case of DS pregnancy, htPAPP-A concentration in the first trimester is markedly decreased. So, currently htPAPP-A is used as a biochemical marker of DS in the first trimester of pregnancy in combination with free β-subunit of chorionic gonadotropin (βhCG) and nuchal translucency. Before this combination of markers was established, biochemical screening for DS was performed only in the second trimester (16-18 gestational weeks). Thus new approach in the DS screening allows starting medical intervention significantly earlier comparing with the previously used methods.

Recently it was shown that another form of protein – homodimeric PAPP-A (dPAPP-A) with molecular mass about 400 kDa, is abundantly expressed in unstable coronary atherosclerotic plaques. It has also been demonstrated that blood level of dPAPP-A is significantly elevated in patients with unstable angina or acute myocardial infarction in comparison with patients with stable angina and control subjects. In addition, dPAPP-A has also been shown to be a strong independent marker of risk stratification for patients with acute coronary syndrome (ACS). The ACS-related form of PAPP-A, presumably originating from ruptured plaque, is not complexed with proMBP subunit, like it is htPAPP-A. Therefore it is supposed, that in atherosclerotic plaque dPAPP-A functions as is an active protease and can promote IGF release. Thus, it may be speculated that dPAPP-A influences to the transformation of a stable atherosclerotic plaque into an unstable one. Such involvement of dPAPP-A in the pathophysiology of ACS suggests that it may serve as a marker of plaque destabilization and PAPP-A measurements in patient’s blood could be very helpful in identifying patients at the very beginning of the process of plaque disruption.

Structural difference of dPAPP-A from pregnancy related htPAPP-A makes possible immunochemical discriminations of these two PAPP-A forms. Antibodies, specific to dPAPP-A and having no cross-reaction with htPAPP-A, could be used for the development of immunoassays for precise selective dPAPP-A measurements in human blood.

HyTest is the biggest world supplier of htPAPP-A antigen purified from retroplacental blood. Now HyTest offers a new product - recombinant dPAPP-A. We also offer PAPP-A and proMBP specific monoclonal antibodies suitable for the development of highly sensitive and rapid sandwich-type PAPP-A immunoassays for quantitative detection of htPAPP-A in maternal blood as well as for detection of dPAPP-A in the blood of ACS patients. We also present here new generation of dPAPP-A specific monoclonal antibodies recognizing only recombinant and atherosclerotic dPAPP-A and having no cross-reaction with htPAPP-A form of antigen.

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ProBNP and proBNP-derived peptides (BNP and NT-proBNP) TechNotes

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Human ProBNP and proBNP-derived peptides (BNP and NT-proBNP) TechNotes.

PreproBNP is composed of 134 amino acid residues (a.a.r.) and is synthesized in cardiac myocytes. Removal of signal peptide results in the appearance of proBNP molecule. Then proBNP is cleaved by unknown protease forming two peptides; BNP and N-terminal part of the proBNP. Both BNP (biologically active molecule) and NT-proBNP (physiological activity is not clarified) as well as unprocessed proBNP are secreted into the bloodstream and circulate in human blood.

It was established that proBNP synthesis increases in response to mechanic or neurohormonal stimulation of the heart that leads to the increase of BNP and NT-proBNP concentrations in blood. Elevated level of BNP and NT-proBNP is described for patients with different cardiovascular pathologies: heart failure (HF), left ventricular dysfunction, unstable angina and myocardial infarction. Blood concentration of both analytes in HF patients correlates with the severity of disease. It has been reported that peptides concentrations are already elevated in asymptomatic patients during the very early stage of heart failure (NYHA I stage according to the New York Heart Association classification). Cardiac patients with symptoms of severe HF (NYHA class III or IV) and myocardial dysfunction show significantly increased values of BNP. Therefore BNP and NT-proBNP measurements in human blood are used for evaluation of patients with suspected HF and assessment of severity of the disease.

BNP and NT-proBNP measurements are also useful for risk stratification of the patients with different cardiac pathologies. It was demonstrated that patients with possible complications are characterized by significantly higher BNP and NT-proBNP concentrations than patients without complications. At present both analytes are used in clinical practice and still there is no agreement between clinicians which one is preferable. There is a correlation between NT-proBNP and BNP concentrations and apparently their diagnostic and prognostic values are similar.

Recently HyTest’s specialists obtained new data that could significantly influence the current approach to both BNP and NT-proBNP measurements. First of all, it was shown that the central parts of NT-proBNP and proBNP molecules circulating in human blood are glycosylated. Schellenberger et al. also reported that recombinant proBNP in CHO cells is expressed as a glycoprotein. Several sites of glycosylation located in the central part of the molecule were documented for the recombinant proBNP expressed in the CHO cells. It was demonstrated also that proBNP in patients’ blood is glycosylated too. HyTest specialists have shown that polysaccharide residues prevent antibodies from interaction with some regions of endogenous NT-proBNP and proBNP molecules. We have also demonstrated that the degree of glycosylation varies from patient to patient significantly and in the case of the high level of glycosylation concentrations of NT-proBNP and proBNP in human blood can be seriously underestimated. Thus antibodies that are specific to the regions not affected by glycosylation should be selected for the development of proBNP and NT-proBNP immunoassays.

HyTest scientists demonstrated recently that proBNP is the major BNP-immunoreactive form in human blood. In other words BNP assays detect mostly proBNP in human blood samples. The ratio proBNP/BNP is not constant and varies from patient to patient. We suggested that proBNP measurements by assays, utilizing one antibody specific to the BNP and another to the NT-proBNP part of the molecule, might be of the same clinical value as BNP measurements.

HyTest offers a set of high-affinity monoclonal an¬tibodies, specific to different epitopes of BNP and NT-proBNP molecules. We also supply our customers with detailed additional information about different applications. Please download the TechNotes from the link above to find out more information!

Additional product information:
Antibodies for New Type of BNP immunoassay – “Single Epitope Sandwich” assay TechNotes

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Procalcitonin TechNotes

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Procalcitonin TechNotes.

Procalcitonin (PCT) is a small protein comprising 116 amino acid residues (a.a.r.) with molecular weight of about 13 kDa. For the first time amino acid sequence of PCT was described by Moullec et al. in 1984 (1). PCT belongs to a family of related proteins (CAPA peptides family), which includes calcitonin gene-related peptides I and II, amylin, adrenomodulin and calcitonin. Like other peptides of CAPA family PCT appears from the precursor molecule - preprocalcitonin comprising 141 a.a.r. by removal 25 a.a.r. from N-terminus.

PCT is produced normally in C-cells of the thyroid glands. It undergoes successive cleavages to form three molecules: N-terminal fragment (N-terminal PÑÒ) (57 a.a.r.), Calcitonin (32 a.a.r.) and Katacalcin (21 a.a.r.).
In 1993 elevated level of PCT in patients with system infection of bacterial origin was reported (2) and now PCT is considered to be the main marker of disorders accompanied by systemic inflammation and sepsis. The diagnostic value of PCT is important due to the close correlation between PCT concentration and the severity of inflammation (3). It was shown that “inflammatory” PCT is not produced in C-cells. Cells of neuroendocrine origin are presumably the source of PCT during inflammation (4).

In some cases raising PCT concentration may be induced by factors independent of sepsis and infection. Surgery, polytrauma, heat shock, burn injures, cardiogenic shock also lead to increase of PCT level (3). The mechanism of the increasing level of PCT in these cases is not defined clearly. The importance of monitoring of PCT levels' changes after cardiac surgery or heart transplantation for differentiating acute graft rejection from bacterial or fungal infections was confirmed in multiple studies (4).

Thus, the diagnostic value of PCT is very high and usefulness of PCT quantification in patients’ blood using specific monoclonal antibodies is very perspective and quite obvious (4, 5).

Rat C-peptide and proinsulin TechNotes

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Rat C-peptide and proinsulin TechNotes.

Rat C-peptide is a polypeptide molecule comprising 31 amino acid residues with molecular mass of about 3.2 kDa. C-peptide is originated from proinsulin, which is synthesized in the beta-cells of the Islets of Langerhans and cleaved enzymatically releasing insulin and C-peptide. Scheme of proinsulin processing is represented on Fig. 1. Proinsulin is cleaved by two Ca-dependent endopeptidases (prohormone convertases) PC2 and PC3. Endopeptidase PC2 (type II) cleaves at the A/C chain junction of proinsulin (between amino acid residues 65 and 66) and PC3 (type I) cleaves at the B/C junction (between amino acid residues 32 and 33)⁽¹⁾. Carboxypeptidase H removes basic amino acids from the C-terminus of proinsulin-derived peptides to generate insulin and C-peptide⁽²⁾. In contrast to PC3 that recognizes des-64,65 proinsulin and intact proinsulin as similar substrates, PC2 has a stronger preference for des-31,32 proinsulin compared to intact proinsulin. This mechanism provides the preferential route of proinsulin conversion via des-31,32-proinsulin (type I processing) (Fig. 1: Ia and Ib)⁽³⁾. Des- rather than split- forms prevail in the blood and the major circulating form of partially processed proinsulin is des-31,32 proinsulin (Fig. 1: Ib). The term “proinsulin” refers to non-processed or “intact proinsulin” whereas term “partially processed proinsulin” is used for split- and des- forms of proinsulin molecule (Fig. 1: Ia, Ib, IIa and IIb).

Insulin, one of the two products of proinsulin processing, regulates carbohydrate metabolism. Insulin has a highly conservative sequence over mammals, reptiles, birds and fish. On the contrary, C-peptide (physiological activity was not shown) demonstrates considerable interspecies variability. For the most of species only one form of proinsulin is described. Unlike others, rats and mice produce two proinsulin isoforms – I and II, which differ from each other in two (rat) or three (mouse) amino acid residues of the C-peptide part of proinsulin.

Analysis of proinsulin synthesis and processing, insulin and C-peptide clearance are very important for better understanding of carbohydrate metabolism abnormalities. Assays for insulin, proinsulin and C-peptide are widely used for monitoring of hypoglycemia, pathogenesis and treatment of diabetes mellitus. It was demonstrated that C-peptide measurements in blood or urine have several advantages over the direct insulin quantification. C-peptide measurements could be the only method to determine insulin production in case of diabetes treatment when endogenous insulin is mixed in blood with the exogenous molecule. Being released into the bloodstream insulin is utilized very fast by liver. Fast excretion and fast elimination results in considerable fluctuations of insulin concentrations in the blood. C-peptide is eliminated and degraded mainly by kidneys and this process is not so impetuous as insulin elimination. Also insulin in blood is less stable than C-peptide. As a result the half life of insulin in blood is significantly shorter (4 min) than that of C-peptide (33 min)⁽⁴⁾. Finally, hemolysis is known to reduce significantly measured insulin concentration⁽⁵⁾. Consequently C-peptide seems to be more reliable indicator of insulin production than insulin by itself.

HyTest offers monoclonal antibodies specific to different parts of rat C-peptides I and II. Thoroughly chosen epitopes and original approaches for selecting specific monoclonal antibodies allowed us to develop highly sensitive and specific antibodies which make C-peptide detection possible without cross-reactivity with native proinsulin or some forms of partially processed proinsulin. Moreover we also offer pairs of antibodies that are able to detect either both isoforms of rat C-peptide (C-peptides I and II) or one of two isoforms (C-peptide I or II). Finally, we have generated monoclonal antibodies that specifically detect intact and partially processed proinsulin and do not interact with free C-peptide.

References:
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Recombinant human Thyroid Peroxidase (rTPO) TechNotes

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Recombinant human Thyroid Peroxidase (rTPO) TechNotes.

Thyroid Peroxidase (TPO) is an integral apical membrane glycoprotein of thyroid follicular cells, which is responsible for the of tyrosine residues in thyroglobulin, leading to thyroid hormone generation (Ruf and Carayon, 2006). In membrane TPO is found as a homodimer with subunits of approximately 100 kDa molecular weight (Baker et al. 1994). The anti-thyroid peroxidase autoantibodies are the most frequently represented autoantibodies in the sera of patients suffering from autoimmune thyroid disease; they are present in 90% of Hashimoto’s thyroiditis and 75% of Graves’ disease patients (Mariotti et al. 1990). Thus immunoassays for quantification of anti-TPO autoantibodies are widely used in clinical practice. For many years human native TPO, purified from thyroid glands, was used as a key component - an antigen - in such assays. Studies in early 90s revealed that soluble extracellular domain of recombinant human thyroid peroxidase (rTPO) produced in insect cells has immunochemical properties similar to native human thyroid peroxidase (Haubruck et al. 1993). But technical limitations in the production of bulk amounts of rTPO imposed some restrictions on the wide rTPO utilization in assays.
Recombinant human TPO offered by HyTest has immunochemical properties similar to the native antigen, purified from human thyroid glands and can be utilized as an antigen in the assays for the detection of human TPO-specific autoantibodies in the blood of the patients with autoimmune
thyroid diseases.

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Retinol-binding protein (RBP4) TechNotes

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RBP4 TechNotes.

Retinol-binding protein (RBP4) belongs to a lipocalin protein family and functions as a carrier protein for vitamin A in serum. Human retinol-binding protein circulating in blood consists of 183 amino acid residues. Several truncated isoforms of RBP4 lacking 1, 2, 4 or 6 of the very C-terminal residues were also described in literature. In blood RBP4 carries retinol (vitamin A) which is bound to RBP4 in equimolar ratio. Besides, major part of circulating RBP4 forms complex with prealbumin (transthyretin) and only small fraction of free RBP4 can be found in serum.

RBP4 has been studied since 1960s mainly as a transporter of retinol, but recent data suggest that RBP4 may contribute to pathogenesis of type 2 diabetes. It has been demonstrated that serum RBP4 levels are elevated in patients with obesity and type 2 diabetes. Studies in mice showed that serum RBP4 may cause insulin resistance. So, on the one hand, there is growing body of evidence demonstrating that RBP4 is a promising marker of the risk of type 2 diabetes, but on the other hand there is conflicting situation in the literature regarding RBP4 clinical utility in predicting insulin resistance and type 2 diabetes. Some authors show strict correlation between circulating RBP4 and magnitude of insulin resistance in subjects with obesity and type 2 diabetes and non-obese subjects with family history of type 2 diabetes. On the contrary, others had not found any correlation between those variables. This confusing situation could be at least partially explained by the heterogeneity of the RBP4 in serum and by methodological shortcomings in determining level of circulating RBP4. If epitope of diagnostic antibody is influenced by RBP4 truncation or by complex formation with retinol or prealbumin, then level of RBP4 determined by the assay, utilizing such antibody, would be different from the results of measurements by the assays with antibodies not susceptible to such modifications.

HyTest offers set of mouse monoclonal anti-human RBP4 antibodies suitable for development of sandwich immunoassays for quantitative detection of circulating RBP4 in human plasma as well as for immunodetection of RBP4 in direct ELISA, Western blotting or to be used for immunoprecipitation of the antigen.

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Transferrin and Transferrin receptor TechNotes

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Transferrin and Transferrin receptor TechNotes.

Troponin I TechNotes: Concept of precise immunoassay

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Troponin I TechNotes.

Cardiac isoform of Troponin I (cTnI) is a golden marker of cardiac muscle cell damage and death today. Different diagnostic platforms were designed for quantitative measurements of cTnI in human blood and are used extensively in big hospitals and small diagnostic laboratories. But still there is no between assay agreement and it often happens that one and the same blood sample gives different values when being analyzed by different cTnI assays.

The most common reason for the discrepancy in the assay measurements is difference in the epitope specificity of the antibodies used in different assays. Now we know that multiple factors are influencing cTnI measurements. Among them are posttranslational modifications (proteolytic degradation (1), phosphorylation (2)), complexing with other molecules (troponin C (3), heparin (2)) and cTnI-specific autoantibodies circulating in patients’ blood (4). Different mono- and polyclonal antibodies, utilized in assays, are sensitive to these factors in different degree.

HyTest specialists have been involved in cTnI studies almost for 15 years. We generated and tested several thousands of monoclonal antibodies specific to different regions of cTnI molecule; we tried dozens of hundreds of different two-site MAb combinations in order to find the best one for precise cTnI immunoassay. Summarizing results of our studies we can conclude that on this moment it is impossible to have one antibody pair (one capture and one detection antibody), which would be absolutely insensitive to all known cTnI modifications and interferences. According to our opinion MAb combinations, which could be used for the development of precise cTnI immunoassay, should utilize two monoclonal antibodies as capture (plate or particle coating) and two MAbs for detection (conjugated with the specific label). We call such an approach as “2+2 concept”. In these assays monoclonal antibodies should be selected in such a way, that if one of the MAbs (capture or detection) is sensitive to the presence of some factor in the sample, then the other MAb should be insensitive to the same factor. Thus the effect of negative or positive interference is minimized. Also one important parameter should be considered: antibodies utilized in the assay should be specific to the cardiac isoform of the protein and should not have crossreaction with the two skeletal isoforms.

Today HyTest can suggest several combinations of monoclonal antibodies useful for the development of cTnI assays according to 2+2 concept. Such assays would be cardiac specific and almost insensitive to all known factors that could affect cTnI measurements. Moreover, while selecting antibodies we also considered the fact that new generation of cTnI assays should display high sensitivity and antibody combinations could be used in point-of-care diagnostics platforms. So, assays described in Table 1 have good kinetics and they recognize standard preparation of antigen (cTnI in troponin complex) with sensitivity better than 50 pg/ml.

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Veterinary TechNotes

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Veterinary TechNotes

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Avian
1. Avian influenza
2. Marek disease virus
3. Newcastle disease virus (NDV)

Canine
1. Canine distemper virus (CDV)
2. Canine parvovirus (CPV)
3. Canine Adenovirus (CAV)
4. Rabies virus
5. Echinococcus granulosis

Bovine
1. Brucella abortus (Brucellosis)
2. Rotavirus
3. Bovine coronavirus
4. Alpha-1 – Acid Glycoprotein (AGP)
5. Foot-and-mouth disease

Porcine
1. Transmissible Gastroenteritis (TGE) virus of Pigs

Equine
1. Burkholderia (Pseudomonas) mallei (Glanders)

Piscine
1. Infectious Salmon Anemia virus

hsCRP TechNotes

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hsCRP TechNotes.

C-reactive protein (CRP) was originally discovered by Tillett and Francis in 1930 as a substance in the serum of patients with acute inflammation that reacted with the C polysaccharide of pneumococcus. CRP is a 224 residue protein with a monomer molecular mass about 25 kDa and pI 6.4. Monomers are noncovalently associated into pentameric structure.

For decades CRP had been known as liver-derived protein. However recent data showed significant level of CRP expression in other tissues, such as blood vessel wall and coronary artery smooth muscle cells, where CRP is supposed to be presented as a monomer (mCRP), while native pentameric protein (nCRP) is predominantly found in plasma.

Exact function of CRP in human organism is still under discussion. This protein has been shown to participate in inflammatory as well as innate immunity processes. Important bioactivities of CRP are determined by its ability to bind a variety of ligands, such as damaged cell membranes, apoptotic cells, fibronectin, etc, with highest affinity to phosphocholine residues. When CRP is ligand-bound, it could be recognized by complement component C1q, whereby activates classical complement pathway. Via interaction with complement factor H, CRP regulates the alternative complement pathway.

C-reactive protein is accepted in clinical use as the major, although rather non-specific, marker of inflammation. In generally healthy subjects CRP levels are usually less than 5 mg/L. Highest levels of CRP are observed in bacterial infection, such as septic arthritis, meningitis and pneumonia. Mildly elevated CRP has been described after myocardial infarction and other types of tissue damage. In 2003, the Centers for Disease Control and Prevention (CDC) and the American Heart Association (AHA) issued a statement identifying CRP as the inflammatory marker best suited for use in current clinical practice to assess cardiovascular risk. Many epidemiologic studies have indicated that CRP is a strong independent predictor of future cardiovascular events, including myocardial infarction, ischemic stroke, peripheral vascular disease, and sudden cardiac death without known cardiovascular disease. The CDC/AHA guidelines support the use of CRP in primary prevention and set cutoff points according to relative risk categories. That’s why present day high sensitivity CRP (hsCRP) assays are aimed at nanogram per milliliter CRP level distinction, and abbreviation hsCRP now is accepted name of the detected protein in such assays. The significant relationship between plasma hsCRP and the risk of death in patients with the acute coronary syndrome has been shown. Furthermore, it has been reported that increased circulating hsCRP concentrations are associated with an increased risk of death from several widespread chronic disease.

In native CRP molecule each protomer has two coordinated Ca²⁺ ions. HyTest offers anti-CRP MAbs which are either sensitive or insensitive to the absence of Ca²⁺ in the solution. Some of our antibodies recognize antigen only in the presence of Ca²⁺ (MAbs C3, C4). The most part of HyTest MAbs do not depend of Ca²⁺ presence in sandwich immunoassay and are able to efficiently recognize antigen even in the presence of EDTA in the tested sample. Our best antibody pairs provide 10,000-fold linearity in experimental immunofluorometric assays. These antibody combinations could be used for the development of hsCRP assays for different diagnostic platforms. In conventional CRP assays turbidimetry and competitive assay techniques are often applied. In such assays relatively low affinity of utilized MAbs is usually preferable. For convenience of our customers we derived monoclonal antibodies with different affinity, allowing for their utilization in different types of immunoassays.

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