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Knowledge Bases
ELISA, Biochemistry Assay, Enzyme immunoassay, Polyclonal Antibodies (pAbs), Recombinant DNA (rDNA), Peptide Synthesis, Hemocyanins, Vector, Fluorophore, Biotin, Streptavidin, Alkaline_Phosphatase, Horseradish_Peroxidase, Ezymatic  Markers, Western_Blot, Fibrinogen , Mouse Fibrinogen, Glucose Regulated Protein 94 (GRP94) , C Reactive Protein (CRP) , Tivantinib Biochemical, CD44, VEGFR1-Flt1, Ku-70, IL1RA, Retroviral-like aspartic prote


n ELISA being developed with TMB
Enzyme-Linked Immunosorbent Assay (ELISA)
The enzyme-linked immunosorbent assay (ELISA) (/ɪˈlaɪzə//ˌiːˈlaɪzə/) is a commonly used analytical biochemistry assay, first described by Engvall and Perlmann in 1971.[1] The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.
In the most simple form of an ELISA, 
antigens from the sample to be tested are attached to a surface. Then, a matching antibody is applied over the surface so it can bind the antigen. This antibody is linked to an enzyme and then any unbound antibodies are removed. In the final step, a substance containing the enzyme's substrate
 is added. If there was binding the subsequent reaction produces a detectable signal, most commonly a color change.
https://en.wikipedia.org/wiki/ELISA

Polyclonal antibodies (pAbs)

Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body (whereas monoclonal antibodies come from a single cell lineage). They are a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope.
The general procedure to produce polyclonal antibodies is as follows:
1.
Antigen preparation

2.
Adjuvant selection and preparation
3. Animal selection
4. Injection process
5. Blood serum extraction

An antigen/adjuvant conjugate is injected into an animal of choice to initiate an amplified immune response. After a series of injections over a specific length of time, the animal is expected to have created antibodies against the conjugate. Blood is then extracted from the animal and then purified to obtain the antibody of interest.
Inoculation is performed on a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is purified from the mammal's serum.
https://en.wikipedia.org/wiki/Polyclonal_antibodies
 


 
Elderly woman with osteoporosis showing a curved back from compression fractures of her back bones.

Construction of recombinant DNA, in which a foreign DNA fragment is inserted into a plasmid vector. In this example, the gene indicated by the white color is inactivated upon insertion of the foreign DNA fragment.

Recombinant DNA (rDNA)
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

Recombinant DNA is the general name for a piece of DNA that has been created by combining at least two fragments from two different sources. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure, and differ only in the nucleotide sequence within that identical overall structure. Recombinant DNA molecules are sometimes called chimeric DNA, because they can be made of material from two different species, like the mythical chimera. R-DNA technology uses palindromic sequences and leads to the production of sticky and blunt ends.
The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. For example, plant DNA may be joined to bacterial DNA, or human DNA may be joined with fungal DNA. In addition, DNA sequences that do not occur anywhere in nature may be created by the chemical synthesis of DNA, and incorporated into recombinant molecules. Using recombinant DNA technology and synthetic DNA, literally any DNA sequence may be created and introduced into any of a very wide range of living organisms.
Proteins that can result from the expression of recombinant DNA within living cells are termed recombinant proteins. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein is not necessarily produced.
[1] Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring by foreign coding sequences.
https://en.wikipedia.org/wiki/Recombinant_DNA

Hemocyanins / Carriers / Vector
Hemocyanins
 (also spelled haemocyanins and abbreviated Hc) are proteins that transport oxygen throughout the bodies of some invertebrate animals. These metalloproteins contain two copper atoms that reversibly bind a single oxygen molecule (O2). They are second only to hemoglobin in frequency of use as an oxygen transport molecule. Unlike the hemoglobin in red blood cells found in vertebrates, hemocyanins are not bound to blood cells but are instead suspended directly in the hemolymph. Oxygenation causes a color change between the colorless Cu(I) deoxygenated form and the blue Cu(II) oxygenated form.

https://en.wikipedia.org/wiki/Hemocyanin
 


 

Single oxygenated functional unit from the hemocyanin of an octopus


Chromogenic immunohistochemistry: The cell is exposed to a primary antibody (red) that binds to a specific antigen (purple square). The primary antibody binds a secondary (green) antibody that is chemically coupled to an enzyme (blue). The enzyme changes the color of the substrate to a more pigmented one (brown star).

 

Reporters / Chromogenic / Fluorophore 

Traditional ELISA typically involves chromogenic reporters and substrates that produce some kind of observable color change to indicate the presence of antigen or analyte. Newer ELISA-like techniques use fluorogenicelectrochemiluminescent, and quantitaoppositiontive PCR reporters to create quantifiable signals. These new reporters can have various advantages, including higher sensitivities and multiplexing.[13][14] In technical terms, newer assays of this type are not strictly ELISAs, as they are not "enzyme-linked", but are instead linked to some nonenzymatic reporter. However, given that the general principles in these assays are largely similar, they are often grouped in the same category as ELISAs.
Reporter molecules vary based on the nature of the detection method, the most popular being chromogenic and fluorescence detection mediated by an enzyme or a fluorophore, respectively. With chromogenic reporters, an enzyme label reacts with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. While the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, which produce a brown or purple staining, respectively, wherever the enzymes are bound. Reaction with DAB can be enhanced using nickel,[7] producing a deep purple/black staining.
Fluorescent reporters are small, organic molecules used for IHC detection and traditionally include 
FITCTRITC and AMCA, while commercial derivatives, including the Alexa Fluors and Dylight Fluors, show similar enhanced performance but vary in price. For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semi- and fully quantitative data, respectively, to correlate the level of reporter signal to the level of protein expression or localization.
https://en.wikipedia.org/wiki/Immunohistochemistry#IHC_reporters

fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.[1]

Fluorophores are sometimes used alone, as a tracer in fluids, as a dye for staining of certain structures, as a substrate of enzymes, or as a probe or indicator (when its fluorescence is affected by environmental aspects such as polarity or ions). More generally they are covalently bonded to a macromolecule, serving as a marker (or dye, or tag, or reporter) for affine or bioactive reagents (antibodies, peptides, nucleic acids). Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, i.e., fluorescent imaging and spectroscopy.
Fluorescein, via its amine-reactive isothiocyanate derivative fluorescein isothiocyanate (FITC), has been one of the most popular fluorophores. From antibody labeling, the applications have spread to nucleic acids thanks to carboxyfluorescein (FAM), TET, ...). Other historically common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine.[2] Newer generations of fluorophores, many of which are proprietary, often perform better, being more photostable, brighter, and/or less pH
-sensitive than traditional dyes with comparable excitation and emission.
https://en.wikipedia.org/wiki/Fluorophore
 


 

A fluorophore-labeled human cell.


Other names
Vitamin B7; Vitamin H; Coenzyme R; Biopeiderm

Biotin, also called vitamin B7, is one of the B vitamins.[1][2][3] It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids.[4] The name biotin derives from the Greek word “bios” (to live) and the suffix “-in” (a general chemical suffix used in organic chemistry).
Chemically modified versions of biotin are widely used throughout the 
biotechnology industry to isolate proteins and non-protein compounds for biochemical assays.[30] Because egg-derived avidin binds strongly to biotin with a dissociation constant Kd of ≈10−15 M,[31] biotinylated compounds of interest can be isolated from a sample by exploiting this highly stable interaction. First, the chemically modified biotin reagents are bound to the targeted compounds in a solution via a process called biotinylation. The choice of which chemical modification to use is responsible for the biotin reagent binding to a specific protein.[30] Second, the sample is incubated with avidin bound to beads, then rinsed, removing all unbound proteins while leaving only the biotinylated protein bound to avidin. Last, the biotinylated protein can be eluted from the beads with excess free biotin.[32] The process can also utilize bacteria-derived streptavidin bound to beads, but because it has a higher dissociation constant than avidin, very harsh conditions are needed to elute the biotinylated protein from the beads, which often will denature the protein of interest.

https://en.wikipedia.org/wiki/Biotin#Use_in_biotechnology

Streptavidin /ˌstrɛpˈtævɪdɪn/ is a 66.0 (tetramer) kDa protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation constant (Kd) on the order of ≈10−14 mol/L,[1] the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Streptavidin is used extensively in molecular biology and bionanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants (e.g. guanidinium chloride), detergents (e.g. SDSTriton X-100), proteolytic enzymes, and extremes of temperature and pH.
Among the most common uses of streptavidin are the purification or detection of various biomolecules. The strong streptavidin-biotin interaction can be used to attach various biomolecules to one another or onto a solid support. Harsh conditions are needed to break the streptavidin-biotin interaction, which often denatures the protein of interest being purified. However, it has been shown that a short incubation in water above 70 °C will reversibly break the interaction (at least for biotinylated DNA) without denaturing streptavidin, allowing re-use of the streptavidin solid support.
[7] A further application of streptavidin is for purification and detection of proteins genetically modified with the Strep-tag peptide. Streptavidin is widely used in Western blotting and immunoassays conjugated to some reporter molecule, such as horseradish peroxidase. Streptavidin has also been used in the developing field of Nanobiotechnology, the use of biological molecules such as proteins or lipids to create nanoscale devices/structures. In this context streptavidin can be used as a building block to link biotinylated DNA molecules to create single walled carbon nanotube scaffolds[8] or even complex DNA polyhedra.[9] The tetrameric streptavidin has also been used as a hub around which other proteins may be arranged, either by an affinity tag such as Strep-tag or AviTag or by genetic fusion to SpyTag.[10] Fusion to SpyTag allowed generation of assemblies with 8 or 20 streptavidin subunits. As well as a molecular force probe for atomic force microscopy studies,[11] novel materials such as 3D crystalline lattices[12] have also been created. Streptavidin has a mildly acidic isoelectric point (pI) of ~5, but a recombinant form of streptavidin with a near-neutral pI is also commercially available.
https://en.wikipedia.org/wiki/Streptavidin

 


 

Monomeric streptavidin (ribbon diagram) with bound biotin (spheres); 

Ribbon diagram (rainbow-color, 
N-terminus = blue, C-terminus = red) of the dimeric structure of bacterial alkaline phosphatase.

Alkaline phosphatase (ALPALKPALPaseAlk Phos) (EC 3.1.3.1), or basic phosphatase,[2] is a homodimeric protein enzyme of 86 kilodaltons. Each monomer contains five cysteine residues, two zinc atoms and one magnesium atom crucial to its catalytic function, and it is optimally active at alkaline pH environments.

Another important use of alkaline phosphatase is as a label for enzyme immunoassays.

https://en.wikipedia.org/wiki/Alkaline_phosphatase

The enzyme horseradish peroxidase (HRP), found in the roots of horseradish, is used extensively in biochemistry applications. It is a metalloenzyme with many isoforms, of which the most studied type is C. It catalyzes the oxidation of various organic substrates by hydrogen peroxide.
Horseradish peroxidase is a 44,173.9-dalton glycoprotein with 6 
lysine residues which can be conjugated to a labeled molecule. It produces a coloured, fluorimetric,[6] or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified. HRP is often used in conjugates (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. For example, an antibody conjugated to HRP may be used to detect a small amount of a specific protein in a western blot. Here, the antibody provides the specificity to locate the protein of interest, and the HRP enzyme, in the presence of a substrate, produces a detectable signal.[7] Horseradish peroxidase is also commonly used in techniques such as ELISA and Immunohistochemistry due to its monomeric nature and the ease with which it produces coloured products. Peroxidase, a heme-containing oxidoreductase, is a commercially important enzyme which catalyses the reductive cleavage of hydrogen peroxide by an electron donor.
Horseradish peroxidase is ideal in many respects for these applications because it is smaller, more stable, and less expensive than other popular alternatives such as 
alkaline phosphatase. It also has a high turnover rate that allows generation of strong signals in a relatively short time span.[8] High concentrations of phosphate severely decrease stability of horseradish peroxidase. In addition to biomedical applications, horseradish peroxidase is one of the enzymes with important environmental applications. This enzyme is suitable for the removal of hydroxylated aromatic compounds (HACs) that are considered to be primary pollutants in a wide variety of industrial wastewater.

https://en.wikipedia.org/wiki/Horseradish_peroxidase

 


 

Horseradish peroxidase C1


The following table lists the enzymatic markers commonly used in ELISA assays, which allow the results of the assay to be measured upon completion.

l   OPD (o-phenylenediamine dihydrochloride) turns amber to detect HRP (Horseradish Peroxidase), which is often used to as a conjugated protein.[25]

l   TMB (3,3',5,5'-tetramethylbenzidine) turns blue when detecting HRP and turns yellow after the addition of sulfuric or phosphoric acid.[25]

l   ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) turns green when detecting HRP.[25]

l   PNPP (p-Nitrophenyl Phosphate, Disodium Salt) turns yellow when detecting alkaline phosphatase.

https://en.wikipedia.org/wiki/ELISA#Commonly_used_enzymatic_markers

Western Blot

The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract.[1]
Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize.
[1] A synthetic or animal-derived antibody (known as the primary antibody) is created that recognizes and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognizes and binds to the primary antibody. The secondary antibody is visualized through various methods such as stainingimmunofluorescence, and radioactivity, allowing indirect detection of the specific target protein.

Other related techniques include dot blot analysis, quantitative dot blotimmunohistochemistry and immunocytochemistry, where antibodies are used to detect proteins in tissues and cells by immunostaining, and enzyme-linked immunosorbent assay (ELISA).
https://en.wikipedia.org/wiki/Western_blot

 

 


 


crystal structure of native chicken fibrinogen with two different bound ligands

Fibrinogen (factor I)
Fibrinogen (factor I) is a glycoprotein complex, made in the liver,[1] that circulates in the blood of all vertebrates.[2] During tissue and vascular injury, it is converted enzymatically by thrombin to fibrin and then to a fibrin-based blood clot. Fibrin clots function primarily to occlude blood vessels to stop bleeding. Fibrin also binds and reduces the activity of thrombin. This activity, sometimes referred to as antithrombin I, limits clotting.[1] Fibrin also mediates blood platelet and endothelial cell spreading, tissue fibroblast proliferation, capillary tube formation, and angiogenesis and thereby promotes revascularization and wound healing.[3]
Reduced and/or dysfunctional fibrinogens occur in various congenital and acquired human fibrinogen-related disorders. These disorders represent a group of rare conditions in which individuals may present with severe episodes of pathological bleeding and thrombosis; these conditions are treated by supplementing blood fibrinogen levels and inhibiting blood clotting, respectively.[4][5] These disorders may also be the cause of certain liver and kidney diseases.[1]
Fibrinogen is a "positive" acute-phase protein, i.e. its blood levels rise in response to systemic inflammation, tissue injury, and certain other events. It is also elevated in various cancers. Elevated levels of fibrinogen in inflammation as well as cancer and other conditions have been suggested to be the cause of thrombosis and vascular injury that accompanies these conditions.[6][7]
https://en.wikipedia.org/wiki/Fibrinogen

Mouse Fibrinogen ELISA Kit
https://www.mybiosource.com/fb-mouse-elisa-kits/fibrinogen/721901

Pathways associated with Fb Elisa kit
Common Pathways
Complement And Coagulation Cascades

Complement And Coagulation Cascades
Extracellular Matrix Organization
Formation Of Fibrin Clot (Clotting Cascade)

GRB2:SOS Provides Linkage To MAPK Signaling For Integrins
Hemostasis
Integrin AlphaIIb Beta3 Signaling
Integrin Cell Surface Interactions
Platelet Aggregation (Plug Formation)

Diseases associated with Fb Elisa kit
Cardiovascular
Brain
Heart
Lung
Disease Models, Animal
kin
Inflammation
Liver
Myocardial Infarction

Thrombosis

Organs/Tissues associated with Fb Elisa kit
Brain
Muscle

Liver
Embryonic Tissue
Testis
Reticulum

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