APOENZYME REACTIVATION IMMUNOASSAY PDF

reactivation. immunoassay system), strips are impregnated with the dry conjugate , antibody, apo-enzyme, glucose and reagents for detecting hydrogen peroxide. The use of antiserum to glucose oxidase in the apoenzyme reactivation immunoassay system (ARIS) is described. Formation of an immune complex between. Apoenzyme reactivation immunoassay; Cofaetor-labeled; Inhibitor-labeled. Introduction. Homo~’:icous immunoassay is defined as an immunoassay system in.

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After the apoenzyme or inactive electrochemical enzyme has been converted to an active form by interacting with the enzyme activation factor portion such as a prosthetic group of the test analyte detection moiety conjugates, various means may be used to transfer electrons produced by the catalytic activity of the newly reactivated electrochemical enzyme back to a reference electrode.

This combination creates a novel combination test technology capable of detecting a wide range of different analytes, and operating in a wide variety of wet or dry, in vivo or in vitro environments. Note that although use of genetic antibody-enzyme hybrids has certain advantages, the above techniques may also be used with conventional antibody based immunochemical diagnostic devices, such as those discussed in FIGS. Apoenzyme reactivation microassay for zinc II ions with flow-through transducers.

The amplification substrate for the electrode electrochemically active enzyme may either be incorporated into the reagent itself, or else can be a normal component of the liquid sample.

Using this technique, multiple single-use detectors can be mounted onto a biosensor or surgical tool, and progressively used to monitor analytes on an as-needed basis. The inactive electrochemical sensor is turned into an analyte specific sensor through the aid of ARIS like enzyme activation factor cofactor —test analyte detection moiety conjugates. Porous electrode 1 also contains a population of protease proteolytic enzyme peptide test substrate conjugated microspheres microbeads 6 and apoenzyme conjugated microspheres 20 within multiple voids 3.

In addition to monitoring for diabetes and blood circulation disorders, such implantable sensors would be well suited for monitoring sepsis or septic shock in at-risk patients, cardiovascular or kidney failure, and many other common, intensive care, medical antigen analytes. In this example, prosthetic group 6 is bound to molecule or surface 7 by way of a peptide 8 that contains a region that serves as a peptide test substrate to a coagulation factor proteolytic enzyme.

The detection device of claim 1mounted on an invasive device selected from the group consisting of surgical tools, catheters, implantable electrodes, implantable chips, and implantable biosensors, in which the detection device is initially protected from body fluids by a removable covering.

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Homogeneous apoenzyme reactivation immunoassay for thyroxin-binding globulin in serum.

The activated form kmmunoassay factor VII 12 in turn converts the inactive form of Factor X 13 to the active form Apoenzyme 1 additionally contains a substrate-binding site 3 for enzyme substrate 4. Similarly, if the test substrate 818 is a long chain carbohydrate such as glycogen, and the analyte enzyme 919 is a glycogen cleaving enzyme such as amylase, the amylase will also act to cleave the link and separate the prosthetic group 616 from the blocking group or surface 717again liberating the prosthetic group, reactivating apoenzyme 2131and producing a detectable signal.

The reactivatjon of the enzyme thus obtained is proportional to the concentration of the cobamide coenzyme providing the presence of the excess of the apoenzyme in the assayed sample. In a third scheme, the resctivation or inactive form of the electrochemical enzyme is chemically modified by binding an electron transport mediator to the surface of the apoenzyme.

It can simply be a larger soluble and relatively inert protein, such as an antibody or albumin immnuoassay, chosen solely on its ability to serve as a steric blocking group, and not otherwise interfere with the assay.

Typically, apoenzyme 21 will contain prosthetic binding site 22 and the enzyme active site 23 which, in the absence of the prosthetic group 8 will be in an inactive state. An additional advantage is that because the hybrid antibody-apoenzyme and test antigen are tethered to the solid support 313and thus have a limited ability to diffuse to other areas of the device, many such detection elements may be placed onto a single support in close proximity, creating a microarray reactivatioon multiple immmunoassay detector capable of analyzing a large number of different test antigens at the same time.

Monoclonal antibodies against the target analyte of interest can usually be obtained from many different commercial immunoasay. Other apoenzymes and cofactor requiring enzymes suitable for the present invention require other cofactor molecules. The resulting finished FAD- peptide substrate -Anchor will then be: Depending on the sensitivity of enzyme assay In environmental studies, it is often important to rapidly identify trace levels of pollutants.

Homogeneous apoenzyme reactivation immunoassay for thyroxin-binding globulin in serum.

In other cases, it may be desirable to incorporate these tethered immunochemical detectors into invasive diagnostic elements designed to be incorporated into the body of a human or animal. This porous electrode 1 contains regions of electron transporting material 2such as thin metal or carbon fibers, typically arranged in a open but connected three dimensional meshwork rdactivation that allows electrical transport over the length or width of the electrode, as well as multiple voids 3 of various sizes, at least some of which are in fluid communication with the outside surface of the electrode.

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In agricultural areas, it is often important to identify trace levels apoensyme contaminants or pathogens, such as harmful bacteria, adulterants, or other undesirable contaminants.

Electron Transport Mediator Discussion: One exemplary grade TGP-H is 0. The substrate amplification substrate for the electrically active enzyme is different from the cleavable aopenzyme substrate for the test or analyte enzyme analyte substrate previously discussed.

Here, the mediator molecule may be an electrode bound artificial electron transport mediator, such as R – and S Methylferrocene carboxylic acid bound to a silver electrode. Similarly a negative low control can be obtained by using free FAD or reacfivation prosthetic group.

In this diagram, apoenzymes 131 are mounted or otherwise associated with the surface of two different electrodes 5051 mounted on solid support 5. Sepsis markers include calcitonin, procalcitonin, C-reactive protein, endogenous activated protein C, tumor necrosis factor, interleukin-6 and 10, endotoxin, lipopolysacharide binding protein, and pro-atrial natriuretic peptide.

Although complex automated chemical analyzers exist, using liquid chemical reagents, which can rapidly analyze many different types of test ligand, such devices tend to be expensive, delicate, and often require skilled users.

Reference to other US reavtivation applications: The various plastic support layers, containing the transfer electrodes, can then be laminated together with an additional 10 mil 0. Discover all that is hidden in the words on. At present, all coagulation tests are significantly more expensive than electrochemical blood glucose tests, and all require more complex and sophisticated metering systems. When operation inside the body is desired, immuniassay detectors should preferably be mounted on a biocompatible surface, and may additionally contain additional elements such as a porous covering e.

The test will also contain excess amplification substrate 40 for the electrochemically active enzyme, as well as other chemicals and cofactors needed to stimulate the desired coagulation protease reaction such as thromboplastin and calcium for a prothrombin time test, etc. Prior art for electrochemically based prothrombin time assays may be found in U.

This in turn reactivates the apoglucose oxidase, and the resulting electrochemical reaction can be detected. To treat these disorders, physicians commonly prescribe oral anticoagulants, such as warfarin.

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