Enzyme-linked fluorescence activation

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa has proven its worth as an enzyme label for many years. It is stable, has a moderate size (140 kDa), a high turnover number, and can be assayed using a variety of different substrates. Its activity is easily detected by eye in, for example, immunoblots, and it can be quantified by changes in absorbance, fluorescence, or luminescence for use in enzyme-linked immunosorbent assays. 

Pharmacokinetic data analysis requires determination of the analyte in various body fluids. In the case of therapeutic antibodies, serum is the most common matrix to be analyzed. For a critical interpretation of pharmacokinetic data the chosen bioanalytical methods must be considered. The most frequently used for mAbs include enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE)/polyacrylamide gel electrophoresis (PAGE), fluorescence-activated cell sorting (FACS), and surface plasmon resonance (SPR). The challenges and limitations of bioanalytical methods used for the analysis of mAb concentrations are discussed in detail in Chapter 6. 

DOTMA E. coli E EBV ECFP ECV EGFP ELISA EYFP FACS FdG FH2 FH4 FK506 FLP propane-aminium-trifluoracetate 7V-[2,3-(dioleyloxy) propyl]-/V,/V,/V-trimethyl ammonium chloride Escherichia coli erythromycine operon/repressor Epstein-Barr virus enhanced cyan fluorescence protein extracellular viral particles enhanced green fluorescence protein enzyme-linked immunosorbent assay enhanced yellow fluorescence protein fluorescence-activated cell sorter fluorescein di- 3-D-galactopyranoside dihydrofolate tetrahydrofolate human immunophilins native recombinase isolated from the 2pm plasmid from Saccharomyces cerevisiae... 

Note. ELISA = enzyme-linked immunosorbent assay, FACS = fluorescence-activated cell sorting, HPLC = high-performance liquid chromatography, IHC = immunohistochemistry, LC/MS = liquid chromatography/MS = mass spectrometry, LPS = lipopolysaccharide (endotoxin), NA = not applicable, PAHA = (nonhuman) primate anti-human antibodies. 

Note CHO = Chinese hamster ovary cells, ELISA = enzyme-linked immunosorbent assay, FACS = fluorescent-activated cell sorting or flow cytometry IHC = immunohistochemistry, LBI = ligand-binding inhibition, rec = recovery, RMN = rat micronucleus. 

Enzyme active sites and receptors rarely interact with hgands without some attendant change in conformation, and the ability to detect and quantify a conformational change hes at the heart of contemporary biochemical kinetics. See Induced Fit Model Fluorescence Spectroscopy Linked Functions Flemoglobin Cooperativity... 

Several general approaches have been used to measure the activities of extracellular enzymes in aquatic systems. These methods typically measure a potential activity, inasmuch as a substrate added to a sample to measure enzyme activity is in competition with naturally occurring substrates (whose concentration is usually unknown) for enzyme active sites. The most commonly applied method involves a small substrate proxy, typically consisting of a monosaccharide or an amino acid covalently linked to a small fluorophore substrates frequently used include methyumbellifery- (MUF-) monosaccharides and 4-methyl-coumainylamide (MCA)- amino acids. Upon hydrolysis of the bond between the monomer and the fluorophore, the fluorophore becomes fluorescent, and hydrolysis is measured as an increase in fluorescence signal with time (Hoppe, 1983 Somville and Billen, 1983). 

Protein cross-links may be also produced in reaction of 4-hydroxynonenal with lysine, histidine, serine, and cysteine residues, primarily via Michael addition (J5, R7, U8). These reactions occur spontaneously, but also may be catalyzed by certain glutatione 5-transferases. The glutathione transferase A4-4, which unlike other alpha-class glutathione transferases, shows high catalytic activity toward lipid peroxidation products such as 4-hydroxynon-2-enal, is the key enzyme for these reactions (B31). Products of protein coupling with aldehydes secondary to lipid peroxidation have a specific fluorescence, which can herald the protein oxidative modification process (CIO). 

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