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17-426: Enzyme multiplied immunoassay technique ( EMIT ) is a common method for qualitative and quantitative determination of therapeutic and recreational drugs and certain proteins in serum and urine . It is an immunoassay in which a drug or metabolite in the sample competes with a drug/metabolite labelled with an enzyme, to bind to an antibody. The more drug there is in the sample, the more free enzyme there will be, and

34-551: A comparable resolution, that can be used for scientific questions, where semi-preparative and preparative amounts of samples are needed. It is used to quantitatively separate samples according to differences in charge or isoelectric point by forming a pH gradient. Because of the versatility of the technique, a wide range of protocols for the separation of samples like rare metal ions, protein isoforms, multiprotein complexes , peptides , organelles , cells , DNA origami , blood serum and nanoparticles exist. The advantage of FFE

51-583: A unique population of antibodies), physicians sometimes speak of sera (the Latin plural, as opposed to serums ). Etymologically serum is derived from the Proto-Indo-European *ser- ("to flow, run"). Free-flow electrophoresis Free-flow electrophoresis ( FFE ), also known as carrier-free electrophoresis , is a matrix-free, high-voltage electrophoretic separation technique. FFE is an analogous technique to capillary electrophoresis , with

68-407: Is serology . Serum is used in numerous diagnostic tests as well as blood typing . Measuring the concentration of various molecules can be useful for many applications, such as determining the therapeutic index of a drug candidate in a clinical trial . To obtain serum, a blood sample is allowed to clot ( coagulation ). The sample is then centrifuged to remove the clot and blood cells, and

85-478: Is also used in protein electrophoresis , due to the lack of fibrinogen which can cause false results. Fetal bovine serum (FBS) is rich in growth factors and is frequently added to growth media used for eukaryotic cell culture . A combination of FBS and the cytokine leukemia inhibitory factor was originally used to maintain embryonic stem cells , but concerns about batch-to-batch variations in FBS have led to

102-462: Is possible to do a pre-separation by free-flow electrophoresis that usually consists of a depletion of serum albumin protein. This method enables greater penetration of the proteome via separation of a wide variety of charged or chargeable analytes, ranging from small molecules to cells. Like many other mass nouns , the word serum can be pluralized when used in certain senses . To speak of multiple serum specimens from multiple people (each with

119-434: Is the fast and gentle separation of samples dissolved in a liquid solvent without any need of a matrix, like polyacrylamide in gel electrophoresis . This ensures a very high recovery rate since analytes do not adhere to any carrier or matrix structure. Because of its continuous nature and high volume throughput, this technique allows a fast separation of preparative amounts of samples with a very high resolution. Furthermore,

136-470: The development of serum substitutes. Blood serum and plasma are some of the largest sources of biomarkers , whether for diagnostics or therapeutics. Its vast dynamic range, further complicated by the presence of lipids, salts, and post-translational modifications, as well as multiple mechanisms of degradation, presents challenges in analytical reproducibility, sensitivity, resolution, and potential efficacy. For analysis of biomarkers in blood serum samples, it

153-533: The drug itself or a metabolite of the drug present in a concentration above a pre-specified minimum detection cutoff limit. In the U.S., the cutoff limits must be set in accordance with Mandatory Guidelines for Federal Workplace Drug Testing Programs that were developed by SAMHSA (The Substance Abuse and Mental Health Services Administration is a branch of the U.S. Department of Health and Human Services). The setting of reasonable cutoff limits help reduce false positive results that occur from assay limitations. Because of

170-622: The end of the separation cell, the separated sample is split up at the fractionation tubes and collected in microtiter plates. Afterwards the samples can be characterized by all major techniques like HPLC , LC-MS , mass spectrometry ( ESI / MALDI , depending on the protocol used) or electrophoresis (IEF / SDS PAGE , 2D-PAGE ). Standard application include the high-resolution separation of protein complexes, membrane proteins, protein and antibody isoforms, cells, subcellular compartments (like organelles, ribosomes) and liposomes. By making use of very flat pH-gradients, generated by ampholytes , it

187-407: The front- and the backplate can be adjusted by spacers and is usually between 0.1 and 0.5 mm. The front plate also contains the inlets for the separation buffers and the sample, the outlets for the fractionation tubes and the platinum wires as electrodes. By applying different buffers over the multiple buffer inlets the operator is able to change pH, salt concentrations or composition and therefore

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204-529: The increased enzyme activity causes a change in color. Determination of drug levels in serum is particularly important when the difference in the concentrations needed to produce a therapeutic effect and adverse side reactions (the therapeutic window ) is small. EMIT therapeutic drug monitoring tests provide accurate information about the concentration of such drugs such as immunosuppressant drugs and some antibiotics. EMIT urine assays for drugs such as cannabinoids, morphine, and amphetamine are designed to detect

221-429: The resulting liquid supernatant is serum. The serum of convalescent patients successfully recovering (or already recovered) from an infectious disease can be used as a biopharmaceutical in the treatment of other people with that disease, because the antibodies generated by the successful recovery are potent fighters of the pathogen . Such convalescent serum ( antiserum ) is a form of immunotherapy . Serum

238-535: The separation conditions over the width of the chamber. The separation buffers and the sample are applied by peristaltic pumps , to ensure a laminar flow . A high voltage electric field is applied perpendicular to the laminar flow. Analytes in the laminar flow can be separated by charge density and isoelectric point . Because of its highly versatile nature, this technique can make use of different modes of electrophoresis, like for example isotachophoresis, isoelectric focusing or (interval) zone electrophoresis. At

255-409: The separation of proteins and charged particles. Some groups are also working on miniaturized versions of FFE systems or micro FFEs. The separation chamber consists of a backplate and a front plate. The backplate usually consists of a cooled aluminum block, covered with a plastic covered glass mirror. The front plate is nowadays made of PMMA , in earlier times glass has been used. The distance between

272-569: The separations can be conducted under native or denaturing conditions. FFE was developed in the 1960s by Kurt Hannig at the Max-Planck-Institute in Germany. Until the 1980s, it was a standardized technology for the separation of cells and organelles , and FFE was even tested in space to minimize the sedimentation under zero gravity . As flow cytometry became the standard method for cell sorting, FFE developments focused on

289-544: The social and legal consequences, a positive test result must be confirmed by an alternative method, usually Gas Chromatography/Mass spectrometry. As an example the SAMHSA cutoffs for cannabinoids are 50 ng/ml for the immunoassay and 15 ng/ml as confirmed by GC/MS. Immunoassays that do not conform with SAMHSA, featuring a cutoff of 20 ng/ml, have been shown to produce false positives from passive inhalation of marijuana smoke. Serum (blood) The study of serum

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