Eagle Biosciences proudly offers a broad range of products in our extensive catalog. In addition to the well known ELISA format, we offer several assay kits in a enzyme-linked immunospot (ELISpot) assay format.
ELISA vs. ELISpot
ELISAs use antibodies to detect the presence and concentration of a protein in a liquid sample. In the most common form, antigens are applied to a stable surface (usually a microtiter plate). Then the corresponding antibody, linked with an enzyme, is added to form a complex. Finally, the enzyme’s substrate is added, which produces a measurable reaction, usually in the form of a color change.
ELISpots also utilize antibody-enzyme reactions. However, instead of measuring the concentration of protein in a sample, they measure the number of cells secreting the cytokine of interest in a sample. Antigens are applied to the plate surface, and when samples are added, cells settle on the bottom of the wells. The cytokines secreted by the cells are captured by the surrounding antigens. After the appropriate incubation time, the cells are removed and the secreted cytokine is detected using a detection antibody linked with an enzyme. The enzyme’s substrate is added, producing a reaction. The end result is visible spots on the on the surface of the plate. Each spot corresponds to an individual cell that was secreting the target protein.
The information provided by ELISpot kits is applicable to various fields of study including: transplantation, vaccine development, T-cell regulation analysis, autoimmune disease, cancer, allergy, and viral infections. These kits are highly sensitive and easy to use.
Does Human Milk Fortifier Affect Intestinal Inflammation in Preterm Infants?
Eagle Biosciences’ Calprotectin ELISA Assay Kit was used in a recent study dealing with inflammation of preterm infants in response to human milk fortifier. The Calprotectin ELISA Assay is part of our line of Gastrointestinal Assays which is a line of highly sensitive and specific kits used to detect the concentration of a variety of samples in serum, plasma, tissue, urine, and saliva.
Abstract
Background: Fecal calprotectin, a recognized marker of intestinal inflammation, is derived from neutrophil migration to a site of inflammation. Introduction of bovine-based human milk fortifier containing intact protein in preterm infants is associated with an increase in fecal calprotectin suggestive of intestinal inflammation. Newer fortifiers contain protein hydrolysates in place of intact protein.
Objective: To measure fecal calprotectin in human milk-fed preterm infants before and after human milk fortification using a fortifier containing hydrolyzed protein.
Methods: Serial stool samples were collected from 24 infants beginning at the first week to 60 days postnatal age. To compare the effect of human milk fortification, samples collected before and after fortification were compared. Infant demographics, diet, postnatal morbidities, and maternal characteristics were recorded.
Results: A total of 401 stool samples were collected from 24 study infants who had a birth weight of 993 ± 277 g (mean ± standard deviation), gestational age 27.5 ± 2.8 weeks, and fortifier initiation at 14 days. Median fecal calprotectin before and after fortification were similar. Calprotectin levels were not correlated with birth weight or gestational age but were inversely correlated with postnatal age (p = 0.005), use of fortifier (p < 0.001), receipt of antibiotics antenatally (p = 0.007) and postnatally (p = 0.008). After adjusting for postnatal age, calprotectin levels were significantly lower following receipt of fortifier (p < 0.001) and postnatal antibiotics (p < 0.001).
Conclusions: The feeding of protein hydrolysate-containing human milk fortifiers does not appear to be associated with increases in a marker of intestinal inflammation.
The relationship between the nasopharyngeal virus load, IgA and IgG antibodies to both the S1-RBD-protein and the N-protein, as well as the neutralizing activity (NAbs) against SARS-CoV-2 in the blood of moderately afflicted COVID-19 patients, needs further longitudinal investigation. Several new serological methods to examine these parameters were developed, validated and applied in three patients of a family which underwent an ambulatory course of COVID-19 for six months. The virus load had almost completely disappeared after about four weeks. Serum IgA levels to the S1-RBD-protein and, to a lesser extent, to the N-protein, peaked about three weeks after clinical disease onset but declined soon thereafter. IgG levels rose continuously, reaching a plateau at approximately six weeks, and stayed elevated over the observation period. Virus-neutralizing activity reached a peak about 4 weeks after disease onset but dropped slowly. The longitudinal associations of virus neutralization and the serological immune response suggest immunity in patients even after a mild clinical course of COVID-19.
Studies show that Angiopoietin-2 is a strong predictor in COVID-19 patients
Two research teams find that increased levels of the proinflammatory cytokine Angiopoietin-2 predicts transfer to the ICU and is responsible for hypercoagulation observed in critically ill COVID-patients.
Studies:
Angiopoietin-2 as a marker of endothelial activation is a good predictor factor for intensive care unit admission of COVID-19 patients. Smadja DM et al., Angiogenesis, 2020;1-10. Full text
Elevated Angiopoietin-2 inhibits thrombomodulin-mediated anticoagulation in critically ill COVID-19 patients. Hultstrom M et al., MedRxiv preprint server, 2021. Full text
SARS-CoV-2 is the causative agent of the coronavirus respiratory disease COVID-19. It has a diverse range of symptoms and may cause severe illness, in particular in patients with cardiovascular risk factors (1).
Endothelial damage and inflammation in SARS-CoV-2 infection
The inflammatory cytokine storm occurring in COVID-19 patients, leads to the recruitment of leukocytes which damage the capillary endothelium. The endothelial damage and inflammation in several organs in SARS-CoV-2 infection is a direct consequence of viral involvement and of the host inflammatory response (2).
Despite the routine thrombosis prophylaxis as standard of care treatment, the major COVID-19 complication is the hyperactivation of the coagulation system indicating a poor prognosis among COVID-19 patients in intensive care (3).
Angiopoietin-2 (ANG2) is a soluble marker of endothelial activation
Angiopoietin-2 is an angiogenesis regulator that can be rapidly released by the activated endothelium upon thrombin or inflammatory cytokines. ANG2 induces inflammation and vascular hyperpermeability and correlates with adverse outcomes in several critical care syndromes (4, 5).
Angiopoietin-2 is a crucial factor to predict transfer to the ICU
In COVID-19 patients, ANG2 was recently reported by Smadja and colleagues to be a relevant factor to predict transfer to the ICU as it was associated with poor lung compliance (6). Thus, showing that endothelial activation reinforces the hypothesis of a COVID-19-associated microvascular dysfunction.
Angiopoietin-2 inhibits anticoagulation in critically ill COVID-19 patients
Hulstrom and colleagues recently demonstrated that ANG2 levels in critically ill COVID-19 patients correlate with disease severity, hypercoagulation, and mortality. In addition, the researchers provided novel in vivo evidence for a direct role for ANG2 in coagulation through binding to and inhibition of thrombomodulin-mediated anticoagulation. The scientists suggest that inhibition of ANG2 might be beneficial for treating critically ill COVID-19 patients, as well as other patients with hypercoagulation (7).
Highly specific epitope mapped capture and detection antibodies
The human Angiopoietin-2 ELISA kit was developed and manufactured by Biomedica
Literature
Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Zhou F et al., Lancet, 2020; 395(10229):1054-1062.
Endotheliitis in COVID-19. Varga S. Der Pathologe, 2020; 41(Suppl 2):99-102.
COVID-19 and coagulation: bleeding and thrombotic manifestations of SARS-CoV-2 infection. Al-Samkari H et al., Blood, 2020:136, 489-500.
Role of Angiopoietin-2 in Vascular Physiology and Pathophysiology. Akwii RG et al., Cells, 2019; 8(5): 471.
Circulating angiopoietin-2 and the risk of mortality in patients with acute respiratory distress syndrome: a systematic review and meta-analysis of 10 prospective cohort studies. Li F et al., Therapeutic advances in respiratory disease, 2020; 14, 1753466620905274.
Angiopoietin-2 as a marker of endothelial activation is a good predictor factor for intensive care unit admission of COVID-19 patients. Smadja DM et al., Angiogenesis, 2020;1-10.
Elevated Angiopoietin-2 inhibits thrombomodulin-mediated anticoagulation in critically ill COVID-19 patients. Hultstrom M et al., MedRxiv preprint server, 2021.
Bone tissues have trabecular bone with a high bone turnover rate and cortical bone with a low turnover. Expression of sclerostin by osteocytes works to inhibit bone formation. A study performed recently aimed to evaluate the relationship between the secretion of sclerostin by osteocytes and the secretion of leukemia inhibitory factor (LIF) by osteoclasts. It was found that LIF secretion effectively suppresses sclerostin expression and promotes bone formation.
Numerous ELISA Assay Kits from our endocrinology line were used in a recent study published out of the University of Nebraska Medical Center in Omaha, Nebraska. These kits were used to analyze blood plasma for biomarkers that could implicate environmental triggers for the pathogenesis of Type 1 Diabetes. These kits included;
Multiple environmental triggers have been proposed to explain the increased incidence of type 1 diabetes (T1D). These include viral infections, microbiome disturbances, metabolic disorders, and vitamin D deficiency. Here, we used ELISA to examine blood plasma from juvenile T1D subjects and age-matched controls for the abundance of several circulating factors relevant to these hypotheses. We screened plasma for sCD14, mannose binding lectin (MBL), lipopolysaccharide binding protein (LBP), c-reactive protein (CRP), fatty acid binding protein 2 (FABP2), human growth hormone, leptin, total adiponectin, high molecular weight (HMW) adiponectin, total IgG, total IgA, total IgM, endotoxin core antibodies (EndoCAbs), 25(OH) vitamin D, vitamin D binding protein, IL-7, IL-10, IFN-γ, TNF-α, IL-17A, IL-18, and IL-18BPa. Subjects also were tested for prevalence of antibodies targeting adenovirus, parainfluenza 1/2/3, Coxsackievirus, cytomegalovirus, Epstein-Barr virus viral capsid antigen (EBV VCA), herpes simplex virus 1, and Saccharomyces cerevisiae. Finally, all subjects were screened for presence and abundance of autoantibodies targeting islet cell cytoplasmic proteins (ICA), glutamate decarboxylase 2 (GAD65), zinc transporter 8 (ZNT8), insulinoma antigen 2 (IA-2), tissue transglutaminase, and thyroid peroxidase, while β cell function was gauged by measuring c-peptide levels. We observed few differences between control and T1D subjects. Of these, we found elevated sCD14, IL-18BPa, and FABP2, and reduced total IgM. Female T1D subjects were notably elevated in CRP levels compared to control, while males were similar. T1D subjects also had significantly lower prevalence of EBV VCA antibodies compared to control. Lastly, we observed that c-peptide levels were significantly correlated with leptin levels among controls, but this relationship was not significant among T1D subjects. Alternatively, adiponectin levels were significantly correlated with c-peptide levels among T1D subjects, while controls showed no relationship between these two factors. Among T1D subjects, the highest c-peptide levels were associated with the lowest adiponectin levels, an indication of insulin resistance. In total, from our examination we found limited data that strongly support any of the hypotheses investigated. Rather, we observed an indication of unexplained monocyte/macrophage activation in T1D subjects judging from elevated levels of sCD14 and IL-18BPa. These observations were partnered with unique associations between adipokines and c-peptide levels among T1D subjects.
To learn more and read the full publication, click here.
For a full list of Eagle Bioscience’s Endocrinology line of ELISA kits click here.
Biomarker of urinary 5-HIAA as a valuable predictor of acute appendicitis
Science Direct
Acute appendicitis is one of the most common causes of severe acute abdominal pain globally. Increased levels of 5-Hydroxyindole acetic acid (5-HIAA), a metabolite of serotonin, in urine has been associated with acute appendicitis due to the densely packed serotonin-containing cells in the appendix. A study performed recently aimed to evaluate and determine the significance of 5-HIAA as a diagnostic marker.
Two Independent Research Teams Find Possible Link Between Neuropilin-1 and SARS-CoV-2 Transmission
Neuropilin-1 (NRP1) is an essential transmembrane cell surface receptor acting primarily as a co-receptor for various ligands (i.e. VEGF, Semaphorins). Due to alternative splicing or shedding, the extracellular region can be released into circulation as soluble Neuropilin-1. NRP1 functions in many key biological processes including the neuronal, cardiovascular and the immune system. The virus SARS-CoV-2 is the causative agent of the coronavirus disease COVID-19 NRP1 is expressed in multiple cell types in the body but occurs primarily on cells in the lung, nose and brain (i.e. respiratory and olfactory epithelium as well as in the CNS). SARS CoV 2 enters the host cells by its spike proteins mainly through its binding to the angiotensin converting enzyme 2 (ACE2).
Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system. Cantuti-Castelvetri L et al., Science 13 Nov, 2020; Vol. 370, Issue 6518, pp. 856-860. Full publication.
Neuropilin-1 is a host factor for SARS-CoV-2 infection. Daly JL et al., Science, 13 Nov 2020; Vol. 370, Issue 6518, pp. 861-865. Full publication.
Eagle Biosciences is now offering a new Human VEGF ELISA Kit from Biomedica. The VEGF ELISA Assay Kit is intended for the quantitative determination of human VEGF in serum, EDTA plasma, and citrate plasma.
VEGF ELISA Highlights:
DAY Test – results in 4.5 h
High sensitivity – measurable values in both serum and plasma
RELIABLE- rigorously validated according to FDA/ICH/EMA guidelines
READY to use – calibrators and controls included
EXCELLENT correlation to existing methods
SMALL sample size – only 10µl sample / well required
Areas of interest:
Cancer
Metabolic disease (diabetes and diabetic kidney disease, diabetic retinopathy, obesity)
Vascular endothelial growth factor (VEGF or VEGF-A), is a growth hormone secreted by endothelial cells, fi broblasts, smooth muscle cells, platelets, macrophages, and many other cell types. It belongs to the cysteine-knot growth factor superfamily (1) and has a molecular weight of about 40 kDa. Currently, 17 different VEGF isoforms have been described to be expressed from one single gene. They are produced by alternative promoter usage/initiation or alternative splicing/proteolysis after protein translation. The N-terminal region is responsible for receptor binding and conserved among all VEGF isoforms. In contrast, residues of the C-terminus differ between isoforms and determine protein length and properties: binding to co-receptor Neuropilin-1 (NRP1) or to extracellular matrix (ECM), agonist/antagonist of angiogenesis. Most isoforms result from the common transcripts: VEGF111, VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206. Additionally, a third VEGF variant (VEGFAx), that demonstrates pro- and anti-apoptotic properties, was described. Thus, vascularization is tightly controlled by the balance of various splice variants, their availability and concentration, whereas isoforms linked to the ECM constitute a reservoir of VEGF that can quickly be shed to circulating forms. One of the most potent pro-angiogenic isoforms is VEGF165a. After secretion, 50-70% of VEGF165a is attached to the extracellular matrix (via heparin binding site), the rest is freely diffusible. It is the most abundant isoform and enhances signaling over the VEGFR2 receptor by additionally binding to its co-receptor Neuropilin-1. VEGF A isoforms are glycosylated, homodimeric proteins. Two anti-parallel monomers are linked by intermolecular disulfide bonds whereas eight cysteine residues form a knot-like structure at one end of each monomer. However, heterodimerization with PLGF has been described as well.
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LEUCINE-RICH ALPHA-2-GLYCOPROTEIN (LRG) ELISA Now Available!
Austrian supplier Biomedica has released a new ELISA, the Leucine-Rich Alpha-2-Glycoprotein or LRG for short. The LRG ELISA Assay kit is intended for the quantitative determination of human LRG in serum, EDTA plasma, heparin plasma, and citrate plasma.
LRG (leucine-rich alpha-2-glycoprotein) is a glycoprotein with a molecular mass of 38.2 kDa. It is encoded by the human gene LRG-1 which is mapped on chromosome 19 at the cytogenetic band 19p13.3. The protein LRG (or also named LRG1) runs at approximately 50 kDa under reducing conditions, as it contains a carbohydrate content of 23%. LRG is the founding member of the family of leucine-rich repeat proteins. The mature protein consists of 312 amino acids, from Val36 to Gln347, with a leucine content of 66 amino acids. LRG is folded to eight leucine-rich repeat (LRR) domains of 22 amino acid length, and a C-terminal LRRCT domain with 49 amino acid length. Human LRG shows 62.5% sequence identity with mouse LRG, and 60.7% with rat LRG.
LRG binds to the TGFβ accessory receptor endoglin, and in the presence of TGFβ1 this leads to the induction of the TβRII-ALK1-Smad1/5/8 signaling pathway. TGFβ1 therefore promotes binding of LRG to the proangiogenic ALK1 but inhibits the interaction with angiostatic ALK5. Induced signaling leads to endothelial cell proliferation and blood vessel outgrowth.
Like many other family members of the leucine-rich repeat (LRR) family, LRG has multiple binding partners. LRG directly interacts with the mitochondrial electron transfer protein cytochrome c, whereas the physiological relevance of this interaction is not yet known. LRG further binds to TGFβ1, the most frequently expressed TGFβ isoform.
The tissue distribution of LRG varies, with high-level expression in the liver, lower expression in the heart, and minimal expression in spleen and lung. LRG is expressed during hematopoiesis. It plays a role in the innate immune system as it is upregulated during neutrophil differentiation; LRG is packed into peroxidase-negative granules of human neutrophils and then secreted upon activation to modulate the microenvironment. Differential expression of LRG is further associated with certain carcinomas, neurodegenerative disease, aging or autoimmune disease. In addition, studies have demonstrated an association between cardiac remodeling (hypertrophy, fibrosis, abnormal vasculature, heart failure) and reduced expression of LRG.
LRG is involved in cell proliferation and immune response, in cell migration, neovascularization and apoptosis. It is a proangiogenic factor which is involved in the regulation of the TGFβ signaling pathway. Up-regulation of LRG is described in response to acute phase response in hepatocytes.
LRG is potentially a biomarker for a variety of diseases e.g. as inflammatory biomarker for autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease. Numerous groups have shown that LRG is increased in various immune-related diseases such as psoriasis, juvenile idiopathic arthritis, Kawasaki disease, appendicitis, and cancers, indicating that LRG elevation is not only limited to autoimmune diseases. In addition, LRG may serve as a biomarker for several other disease conditions such as heart failure, and diabetes-related complications. Plasma Leucine-Rich α-2-Glycoprotein has also been demonstrated to predict cardiovascular disease risk in end-stage renal disease. Leucine-rich α-2-glycoprotein is highly expressed in the brain and it is possible to distinguish idiopathic normal pressure hydrocephalus (iNPH) from other neurodegenerative diseases such as Alzheimer disease by measuring LRG in cerebrospinal fluid.
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