HTRF: a technology tailored for biomarker determination-novel analytical detection system suitable for detection of specific autoimmune antibodies as biomarkers in nanogram level in different body fluids.
BACKGROUNDClassical methods of gene product analysis such as binding assays (e.g., ELISA, protein chip technology) are usually time-consuming, lab-intensive, a lot much less delicate, and lack high-throughput functionality.
In addition, all present methods used to measure proteins necessitate a quantity of divisions of the distinctive sample and specific particular person assessments carried out for each substance, with an associated worth for each take a take a look at.
METHODSTogether with a small biotech agency, we developed a new and fashionable analytical detection system primarily based totally on homogenous time-resolved fluorescence (HTRF) technology.
Our system facilitates the occasion of immune assays that measure selective different analytes such as chosen biomarkers in a small sample amount at decrease than 20 min with a a lot better sensitivity in comparability with frequent binding assay applications such as enzyme-linked immunosorbent assay (ELISA).
Recent advances of the equipment of this novel detection system combine the power of miniaturization, microfluidics, larger linear range, and faster quantification.
HTRF: a technology tailored for biomarker determination-novel analytical detection system suitable for detection of specific autoimmune antibodies as biomarkers in nanogram level in different body fluids.
RESULTSThe vitality of the HTRF technology offers good promise for point-of-care scientific testing and monitoring of many crucial analytes such as disease-specific biomarkers in the nanogram level in different human body fluids such as CSF, blood, serum, plasma, and saliva. The linear dynamical range of our HTRF assay was determined between 2.5 and 100 ng/mL.
Description: Elastases form a subfamily of serine proteases that hydrolyze many proteins in addition to elastin. Humans have six elastase genes which encode the structurally similar proteins. The product of this gene hydrolyzes proteins within specialized neutrophil lysosomes, called azurophil granules, as well as proteins of the extracellular matrix following the protein's release from activated neutrophils. The enzyme may play a role in degenerative and inflammatory diseases by its proteolysis of collagen-IV and elastin of the extracellular matrix. This protein degrades the outer membrane protein A (OmpA) of E. coli as well as the virulence factors of such bacteria as Shigella, Salmonella and Yersinia. Mutations in this gene are associated with cyclic neutropenia and severe congenital neutropenia (SCN). This gene is clustered with other serine protease gene family members, azurocidin 1 and proteinase 3 genes, at chromosome 19pter. All 3 genes are expressed coordinately and their protein products are packaged together into azurophil granules during neutrophil differentiation.
Description: Elastases form a subfamily of serine proteases that hydrolyze many proteins in addition to elastin. Humans have six elastase genes which encode the structurally similar proteins. The product of this gene hydrolyzes proteins within specialized neutrophil lysosomes, called azurophil granules, as well as proteins of the extracellular matrix following the protein's release from activated neutrophils. The enzyme may play a role in degenerative and inflammatory diseases by its proteolysis of collagen-IV and elastin of the extracellular matrix. This protein degrades the outer membrane protein A (OmpA) of E. coli as well as the virulence factors of such bacteria as Shigella, Salmonella and Yersinia. Mutations in this gene are associated with cyclic neutropenia and severe congenital neutropenia (SCN). This gene is clustered with other serine protease gene family members, azurocidin 1 and proteinase 3 genes, at chromosome 19pter. All 3 genes are expressed coordinately and their protein products are packaged together into azurophil granules during neutrophil differentiation.
Description: Neutrophil elastase, also called Elane, is a serine protease of neutrophil and monocyte granules. Its key physiologic role is in innate host defense, but it can also participate in tissue remodeling and possesses secretagogue actions important to local inflammatory responses. Elastases form a subfamily of serine proteases that hydrolyze many proteins in addition to elastin. Humans have six elastase genes which encode structurally similar proteins. The encoded preproprotein is proteolytically processed to generate the active protease. Following activation, this protease hydrolyzes proteins within specialized neutrophil lysosomes, called azurophil granules, as well as proteins of the extracellular matrix. This protein also degrades the outer membrane protein A (OmpA) of E. coli as well as the virulence factors of such bacteria as Shigella, Salmonella and Yersinia. Mutations in this gene are associated with cyclic neutropenia and severe congenital neutropenia (SCN). This gene is present in a gene cluster on chromosome 19.
Description: Elastases form a subfamily of serine proteases that hydrolyze many proteins in addition to elastin. Humans have six elastase genes which encode structurally similar proteins. The encoded preproprotein is proteolytically processed to generate the active protease. Following activation, this protease hydrolyzes proteins within specialized neutrophil lysosomes, called azurophil granules, as well as proteins of the extracellular matrix. The enzyme may play a role in degenerative and inflammatory diseases through proteolysis of collagen-IV and elastin. This protein also degrades the outer membrane protein A (OmpA) of E. coli as well as the virulence factors of such bacteria as Shigella, Salmonella and Yersinia. [RefSeq]
Precision and accuracy calculated for inter- as successfully as intra-assays was decrease than ± 10 %. Intra-assay and inter-assay precision for extreme, medium, and low analyte concentrations current indicate CV values decrease than ± 10 %.
Intra- and inter-assay accuracy for all three concentrations current indicate restoration values of 80-120 %.
CONCLUSIONSThe purpose of this work is to clarify the occasion and establishment of this novel HTRF system that permits the very fast detection and quantification of biomarkers in different human body fluids. Furthermore, a specific antibody combination that assures a specific binding of the correct refolded autoimmune IgG is evaluated.
