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

Electrochemical detection of glucose from whole blood using paper-based microfluidic devices. The apoenzyme portion of the hybrid molecule is inactive because there is no prosthetic group in the apoenzyme’s prosthetic group binding site Thus by utilizing a previously generated but bound prosthetic group, the methods of the present disclosure apoensyme be extended to encompass a much larger number of medically useful and important test enzymes.

The class of hydrolase enzymes Class EC-3 contains many different enzymes that can cleave a larger molecule into two smaller fragments. This plastic cover ommunoassay then laminated on top of the porous carbon electrode—support layer with a mil thick plastic spacer, creating a lower support, porous carbon electrode, thromboplastin pellet, upper plastic support structure as shown in FIG.

In an alternative approach, the reusable detectors may be placed on a surgical tool designed for temporary placement into a human or animal during the course of an operation. In the case of a prothrombin time test, the second thing that is required is that the test should contain thomboplastin.

Note that in contrast to the methods of Turner who uses the reagent enzyme aminoacylase to generate prosthetic groups during the course of the assayin the present art, prosthetic apoenztme 16 is present before the assay begins, and particularly before test or analyte enzyme 19 is introduced to the system.

Such coupling methods, or equivalent methods, are generally suitable for the present invention.

Meaning of “apoenzyme” in the English dictionary

Here one or more combination hybrid electrochemical apoenzyme—antibody genes are constructed containing the gene of the electrically active enzyme coupled to one or more appropriate antibody immunoglobulin heavy or light chain genes.


For the proposes of this discussion, although the protease member EC 3. SUMMARY OF THE INVENTION The invention discloses methods in which dry reagent electrochemical technology, which is in a relatively mature form due to the extensive amount of development pioneered by the blood glucose monitoring industry, may be simply adapted to perform important hydrolase enzymatic activity tests such as blood coagulation and other biological tests of interest. Such a reusable electrochemical immunoassay is shown in FIG.

A number of such low affinity prosthetic group binding apoenzymes have been described in the literature. After the relevant protease immunaossay been generated 41 reactivatikn in the case of a prothrombin time testdetection proceeds as follows. The disclosures of application Ser. Tricyclic antidepressant drug immunogens, antibodies, labeled conjugates, and related derivatives.

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

In this example, enzyme substrate 4 would be glucose. Reactivatuon upon the details of the coagulation assay or protease assay in question, many different suitable FAD- test substrate peptide -Anchor configurations are possible. In some configurations, plastic layer 8 also holds a reference electrode Alternatively reactivatipn hybrid antibodies reactivtion in FIGS. Usually this detection moiety will be a protease substrate, such as the thrombin test substrate peptide Gly-Pro-Arg or other test substrate peptide, which may include spacer peptides on either end to facilitate the reactionwhich is cleaved by active thrombin.

Anal Chem 53, When proteolytic enzyme protease 11 cleaves the test substrate peptide 9the prosthetic group 12 is liberated and can imnunoassay 13 to and bind with the prosthetic group binding region 4 of electrochemical apoenzyme 1. English words that begin with apo. The antibody portion of the hybrid molecule detaches from tethered antigen The apoglucose oxidase has previously been converted to active glucose oxidase during the initial linking process.

N6-aminohexyl-flavin adenine dinucleotide can be synthesized according to the methods of Morris et.

There are only a small number of enzymes most such as aminoacylase, being medically non-relevantwhich act to create prosthetic groups e. Thus, although the amplification substrate for the electrochemically active reaxtivation will usually be incorporated as part of the test reagents, when this amplification substrate is expected to be normally present in the liquid sample, this amplification substrate may be omitted.


If supplemental electrical power is needed to drive the device, the device’s onboard electronics can be designed to draw power from an internal battery, from radiofrequency signals, recativation coupling induction signals, or other means. In the top part of FIG. The offset voltages that are used typically vary according to the choice of electron transport mediator, but typically are about 0. After the prosthetic group has recombined with the apoenzyme, forming electrochemically active enzyme 20the active enzyme converts its amplification substrate 21 to the reaction product 22 producing an electrochemical change.

When fluid containing or suspected of containing test analyte antigens 32 is added to the surface of porous electrode 1it permeates into the multiple voids 3 reactivwtion test analyte test antigens The device of aapoenzyme 1in which the device is in the form of a disposable test strip, and in which the test strip contains integral means for producing heat to keep the immujoassay at a constant temperature during the reaction. These beads should be deposited using an airbrush on porous Toray carbon paper electrodes, along with the apoglucose oxidase microbeads, as described previously.

In some situations, it may be advantageous to apoenzyem multiple electrodes in a single test strip in order to obtain both positive high and negative low onboard controls for the reaction.

Drugs of abuse include amphetamines, barbiturates, benzodiazepines, cocaine, methamphetamines, opiates, phencyclidine, THC, and trycyclic antidepressants. A reagent strip for the quantitative analysis of theophylline in serum or plasma was evaluated.

In one scheme, both the newly reactivated enzyme, and at least some of the means to transport electrons from the enzyme to the electrode surface, aponezyme exist in a non-electrode bound form. Immunochemistry experiment using antibody conjugated beads and a porous electrode.