Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-12
2001-05-08
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007700, C435S007920, C435S007100, C435S007200, C435S007330, C435S007370, C435S188000, C435S028000, C435S004000, C436S536000, C436S073000, C436S074000, C436S066000
Reexamination Certificate
active
06228602
ABSTRACT:
Peroxidase is an enzyme that catalyzes the oxidation of various compounds, such as phenols and amines, by peroxides. Various compounds are referred to as pseudoperoxidases because they behave in a manner similar to the peroxidase enzyme by liberating an electron from hydroperoxides to create an oxidant capable of accepting an electron from a donor species. Accordingly, the pseudoperoxidases are enzyme like in that they catalyze, or otherwise participate in, reactions between peroxides or otherwise oxidizable compounds. The pseudoperoxidases, which include hemoglobin and its derivatives, are collectively referred to as peroxidatively active substances. For example, a peroxidatively active substance, such as hemoglobin and its derivatives, catalyzes the interaction between a hydroperoxide and an oxidizable dye. In such interactions, the peroxidatively active substance imitates the peroxidase enzyme and catalyzes or otherwise participates in an interaction between the oxidizable dye and the peroxide. The oxygen transferred from a peroxide to a peroxidatively active substance creates an oxidant capable of accepting an electron from an oxidizable dye. The resulting interaction provides a detectable response, such as a color transition, wherein the intensity of the response is indicative of the presence or the concentration of the peroxidatively active substance. Suitable oxidizable dyes for use in such an assay include benzidine; o-tolidine, 3,3′5,5;-tetraalkylbenzidine wherein the alkyl groups contain from one to six carbon atoms; 9-dianisidine; 2,7-diaminofluorene; bis-(N-ethyl-quinol-2-one)-azine; (N-methylbenzthiazol-2-one)-(1-ethyl-3-phenyl-5-methyl-triazol-2-one)-azine or a combination thereof. Useful peroxides include hydrogen peroxide, cumene hydroperoxide; 5-butyl hydroperoxide; diisopropylbenzene hydroperoxide; 1-hydroxycyclohexane-1-hydroperoxide; 2,5-dimethyl-hexane-2,5-dihydroperoxide; paramenthane hydroperoxide; 1,4-diisopropylbenzene hydroperoxide; p-5-butyl-isopropylbenzene hydroperoxide; 2-(&agr;-hydroperoxy-isopropyl)-6-isopropylnaphthalene; tetralin hydroperoxide or a combination thereof.
In U.S. Pat. No. 4,493,890 it is disclosed that glucose oxidase is a conjugated enzyme composed of an enzymatically inactive, high molecular weight protein component (apoenzyme) and FAD (a low molecular weight, nonproteinaceous prosthetic group). Unlike transition metal porphyrins, the flavin adenine dinucleotide (FAD) portion of glucose oxidase does not contain a transition metal. The FAD moiety is sometimes referred to as a prosthetic group because the protein portion of the enzyme forms a complex with this small ligand through high affinity binding. As a result, the prosthetic group becomes an integral part of the protein which is required for the enzyme to function. Apoglucose oxidase and FAD have a high binding affinity (binding constant of about 10
10
molar
−1
) but can be effectively separated by treatment with acidified ammonium sulfate. In U.S. Pat. No. 4,238,565 there is described a specific binding assay wherein FAD is employed as a label and is monitored by its ability to combine with apoglucose oxidase to form active glucose oxidase. In a homogenous assay for determining an antigen in a liquid medium, a test sample of the liquid medium is combined with antibody to the antigen and a labeled conjugate comprising the antigen (or analog thereof) coupled to FAD so that antigen contained in the sample competes with antigen-FAD for binding with antibody. Apoglucose oxidase is also present and is capable of combining with antigen-FAD which is not associated with antibody to yield active glucose oxidase. However, since antibody bound antigen-FAD is not capable of such combination with apoglucose oxidase, the concentration of antigen in the test sample dictates the amount of measurable glucose oxidase which results from the recombination by known methods such as a calorimetric assay. This sort of assay, sometimes referred to as apoenzyme reactivation immunoassay system (ARIS) involves attachment of hapten to the FAD prosthetic group of glucose oxidase which still permits reactivation of the apoglucose oxidase by its interaction with the prosthetic group. However, binding of the anti-hapten antibody to the hapten-FAD conjugate can prevent its association with the apo-glucose oxidase, so that no glucose oxidase activity is observed. Glucose oxidase has been found to be an excellent enzyme for this system since i) the FAD can be dissociated from the intact enzyme to provide a stable apoglucose oxidase, ii) the apoglucose oxidase cannot express enzyme activity but can be easily reconstituted with FAD to the holoenzyme and iii) the parent holoenzyme has a high turnover rate and the H
2
O
2
reaction product can be determined with high sensitivity by a variety of methods. However, the apoglucose oxidase ARIS method has not been successfully applied in urinalysis due to the presence of FAD derivatives in urine resulting in reconstitution with the apoglucose oxidase to form the active enzyme which can cause false positive assay results. The ARIS systems are not limited by molecular weight of the prosthetic-conjugate:binding partner, i.e. reconstitution occurs regardless of the molecular weight of the binding system. This type of assay is based on binding partner interaction with the prosthetic-conjugate occurring in such an orientation that it causes steric hindrance to reconstitution. This steric hindrance leads to an inhibition that causes the rates of reconstitution to be different between prosthetic conjugate and prosthetic-conjugate binding partner. The difference in reconstitution rates of the two species is used to measure the analyte. Peroxidases also contain a prosthetic group in the form of a transition metal porphyrin which can be separated from the protein to form an apo-peroxidase. The apo-peroxidase system of the present invention is limited by the molecular weight of the analyte/analyte specific binding partner conjugate attached to the metal porphyrin since it was discovered that when this moiety has a total molecular weight of greater than that which will permit the apo-peroxidase and metal porphyrin to recombine, reconstitution with the peroxidase is prevented. Conjugates with a combined molecular weight of greater than about 180 K Dalton have been found to be suitable for preventing reconstitution. The present assay is based on the binding partner:prosthetic conjugate being so large that it prevents reconstitution. Since the binding partner typically has a high molecular weight (antibodies are normally in the 165 K Dalton range) the presence of peroxidase activity can only result from one species, the unbound prosthetic-conjugate, and the activity of this single species is used to measure the analyte.
Steric hindrance to reconstitution requires a specific orientation of the antibody:antigen binding, which orientation is easily developed for small antigens but not for large antigens. Accordingly, the ARIS method is normally limited to small molecular weight antigens of 100 to 1000 g/mol. The present method does not rely on steric hindrance but rather on the molecular weight of the prosthetic-conjugate:binding partner. This technique results in the advantage that the assay is able to detect any size of analyte and eliminates the need to prepare antibodies with a specific binding orientation. Additionally, the present invention, which is based upon the discovery that the size effect can be used to provide an apo-peroxidase system, can be used without measuring rates. This is especially advantageous in urinalysis using test strips since urine strips are usually read visually and visual readings are not readily applicable to rate determination.
The pseudoperoxidases also have prosthetic groups which, when removed, result in an inactive apo- form of the pseudoperoxidase which lacks the peroxidase activity. For example, when iron hematin is separated from hemoglobin, such as by subjecting the hemoglobin to low pH to dissociate the iron hematin followed by f
Bayer Corporation
Devi S.
Jeffers Jerome L.
Smith Lynette R. F.
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