Hydrogen peroxide stabilization in assays

Abstract: Hydrogen peroxide stabilized in the presence of blood components by the addition of a metal oxide oxidant, an anionic chelating agent, particularly hydroxylic carboxylate and nitroprusside, and a catalase inhibitor. The composition finds particular application for stabilizing hydrogen peroxide in a diagnostic assay on a bibulous support.

Description: The technical field of the subject invention concerns diagnostic assays or other systems requiring stabilization of hydrogen peroxide. In many assays, hydrogen peroxide is employed as a reagent. When the assays are quantitative or semi-quantitative, it is necessary that the hydrogen peroxide react in the manner intended to produce a detectable signal, usually chromogen formation. In many assays, blood, or blood derivatives such as serum or plasma are present, where the blood sample contains a complex mixture of components. These components vary from individual to individual and, it is found can have an effect on the quantitation of hydrogen peroxide. It is therefore important to provide systems which prevent the reaction of hydrogen peroxide by pathways other than the desired assay pathway. Co-pending Application EP-A-0 296 826 , describes a solid phase diagnostic assay employing hydrogen peroxide as a reagent. And, co-pending Application EP-A-0 342 447 which describes a solid phase diagnostic assay where serum components are enzymatically converted to hydrogen peroxide which is subsequently used to generate a detectable signal. In systems employing blood, or red blood cell free blood, e.g. plasma or serum, where hydrogen peroxide is employed as a reagent to quantitate a component in the blood sample, a formulation is provided for stabilizing the hydrogen peroxide to prevent its reaction by pathways other than the assay pathway. Particularly, the formulation comprises a chelating agent, comprising a hydroxylated carboxylate, a metal oxide oxidant, comprising stannate, nitroprusside, and optionally a catalase inhibitor, such as an azide, particularly sodium azide. The additives may be combined, so that the chelating agent, stannate and nitroprusside have a molar ratio of about 0.2-5:0.2-5:0.2-5, preferably about 0.5-2:0.5-2:0.5-2. Desirably, the various additives are equimolar. Sodium azide would be present primarily as a catalase inhibitor but also as a preservative, and is generally present in minor amount, when present, generally from about 1 x 10⁻⁴ to 0.1 weight percent of the wet or dry formulation. The formulation may take many forms. The formulation may be added to the assay medium during the assay. Alternatively, the formulation may be absorbed by a solid substrate, so that it dissolves into the assay medium upon addition of the assay medium to the substrate. The concentration of the various essential components in the assay medium will generally range from about .0001 to 0.5, more usually from about 0.001 to 0.1 M. When impregnating a bibulous substrate, the solution employed to impregnate the bibulous substrate will generally range from about 0.005 to 0.1, more usually from about 0.01 to 0.05 M for each of the components. The azide will generally range from about 0.005 to 0.1%. The compositions will usually be buffered at a pH of about 6 to 8, usually about 7 with a conventional buffer, e.g. phosphate at a concentration generally in the range of about 0.05 to 0.75 M. The subject compositions find particular use with diagnostic assays which employ an oxidase as a reagent, where the reaction of the oxidase is to produce hydrogen peroxide. Various enzymes may be employed, such as glucose oxidase, cholesterol oxidase, uric oxidase, alcohol oxidase, xanthine oxidase and the like. These enzymes will be used in conventional ways and in conventional amounts, in accordance with the particular assay involved. Where the stabilizing formulation is applied to a bibulous support, various supports may be employed, such as cellulosic supports, e.g. paper, cellulose acetate, cellulose nitrate, glass fibers, or the like. Particularly, the bibulous support may serve as a reagent pad, where other reagents are also bound to the pad, particularly the enzyme reagent which results in formation of hydrogen peroxide. Exemplary of the subject invention is the use of a reagent pad, where plasma is added to the pad for initiation of the assay. To the reagent pad is bound the oxidase, for example, for a glucose assay, glucose oxidase, for a cholesterol assay, cholesterol esterase and cholesterol oxidase, and a mixture of detergents, the subject formulation, and a mixture of enzyme stabilizers. Particularly, the detergents will be about a total of 0.10 to 10 weight percent of the solution applied to the pad and will be a mixture of non-ionic and anionic detergents. The enzyme stabilizers may include various additives, such as sugars in from about 0.1 to 20 dry weight percent, proteins, gum arabic, non-ionic detergents, where the total amount of the enzyme stabilizers will generally range from about 0.5 to 10, more usually from about 1 to 5 weight percent of the solution. The amount of enzyme will vary, depending upon the assay, and will generally be present in an amount of from about 1 to 500 units per ml. In the case of the cholesterol assay as illustrative of other assays, the impregnating solution will have from about 2 to 100 units/ml of the two enzymes, cholesterol esterase and cholesterol oxidase. The detergents will be in total weight from about 0.1 to 5 weight percent of the medium, while in the case of mixtures the weight of the non-ionic detergents may be from about 10 to 90%, usually from about 25 to 75 weight percent of the total detergent mixture. The binding agents will generally be in the range of about 0.2 to 10, more usually from about 1 to 5 weight percent of the medium. A preservative or hydrogen bonding agent may be present in from about 1 to 20 weight percent, more usually from about 2 to 10 weight percent. The remaining additives will generally be present in total amount in less than about 10 weight percent, more usually in less than about 5 weight percent. The remaining composition may be water, non-reactive ingredients, excipients, extenders, and the like. After allowing the reagent pad to absorb the solution, the solution may be dried and may then be used as appropriate. In carrying out the assay, the plasma is applied to the reagent pad, so as to be absorbed by the reagent pad, whereby hydrogen peroxide is produced. Any technique may then be used for determining the hydrogen peroxide, usually employing horseradish peroxidase and a chromogen, where the chromogen may produce a detectable signal, usually light absorption. An assay may be carried out by impregnating a sample pad which serves as a bridge between two bibulous members. A first bibulous member serves to receive the transport solution, which may or may not have reaction components, depending upon the assay. The first bibulous member transfers the fluid to the sample pad. The second bibulous member receives the transport fluid from the sample pad and serves as a bridge to transfer the transport fluid from the sample pad to the assay measurement region. The sample is prevented from interacting with the two bibulous members when sample is transferred to the pad by a separation means, usually an inert non-porous film, which blocks transfer from the sample pad to the bibulous members. The amount of sample accepted by the sample pad and involved in the assay medium may be controlled by providing for transfer of fluid beyond the amount saturating the pad through a non-wetting screen into an absorbant layer. After addition of the sample to the sample pad, and an incubation of from 1 to 30 minutes, the porous non-wetting material and absorbant layer are removed, leaving the sample pad as the sole repository of sample for the assay. The invention also provides compositions comprising stannate, nitroprusside, tartrate and, optionally, an azide. The stannate and tartrate may be provided as sodium and/or potassium salts. The composition may be provided as a powder, an assay solution or may be impregnated on a bibulous support. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL The Dry Reagent Pad contains: REAGENT: Cholesterol Esterase 0.18u Enzymes Cholesterol Oxidase 0.50u Sodium Cholate 1% Detergents Nonidet PL 40™ 1% Mega -8™ 1% NaK Tartrate 0.025 M Additives Nitroprusside 0.025 M Na Stannate 0.025 M Na Azide 0.01% Gum Arabic 2% Enzyme Stabilizers Gelatin 0.5% Gantrez 0.5% Sucrose 5% The immobilization reagent for the enzymatic conversion pad contains one of the following formulations. FORMULATION FOR CONVERSION PAD Formulation #1: 1. 0.43 M Sodium Phosphate Buffer pH 7.0 462 µL 2. 12.5% Sucrose 100 µL 3. 10% Nonidet P-40 100 µL 4. 10% Sodium Cholate 100 µL 5. 10% Mega-8 83 µL 6. 1 M NaK Tartrate 25 µL 7. 1 M Na Nitroprusside 25 µL 8. 1 M Na Stannate 25 µL 9. 2.5% Na Azide 4 µL 10. 500 u/ml Cholesterol Esterase 36 µL 11. 1250 u/ml Cholesterol Esterase 40 µL Total: 1.0 mL Formulation #2: 1. 20% Gum Arabic 100 µL 2. 5% Gelatin 100 µL 3. 5% Gantrez AN-149 100 µL 4. 50% Sucrose 100 µL 5. 10% Nonidet P-40 100 µL 6. 10% Sodium Cholate 100 µL 7. 10% Mega-8 83 µL 8. 1 M NaK Tartrate (Sigma) 25 mM 25 µL 9. 1 M Na Nitroprusside (Sigma) 25 µL 10. 1 M Na Stannate (Alfa) 25 µL 11. 2.5% Na Aside (Sigma) 4 µL 12. 500 u/mL Cholesterol Esterase 36 µL 13. 1250 u/mL Cholesterol Oxidase 40 µL 14. 1M Sodium Phosphate pH 7.0 162 µL Total: 1.0 mL It is evident from the above results, that the subject composition provides for stabilization of hydrogen peroxide, so as to allow for quantitation of the hydrogen peroxide in the presence of components of blood. In this manner, assays can be carried out either in the liquid phase or on a bibulous support, where the amount of hydrogen peroxide produced in relation to an analyte may be quantitated. The composition does not interfere with the other aspects of the assay, so as to allow for an accurate and simple method for determining a wide variety of analytes.

Claims: An assay method for determining hydrogen peroxide in an assay for determining an analyte in a blood sample, wherein hydrogen peroxide is produced in an amount in relation to the amount of analyte present in an assay medium, characterised in that a composition including stannate, nitroprusside and a hydroxylated carboxylate chelating agent is used to stabilize said hydrogen peroxide. A method according to claim 1, wherein an enzyme which produces hydrogen peroxide is included in said assay. A method according to claim 2, wherein said enzyme is cholesterol oxidase, glucose oxidase, uric oxidase, alcohol oxidase or xanthine oxidase. A method according to any one of the preceding claims, wherein said composition comprises a catalase inhibitor. A method according to claim 4, wherein said analyte is cholesterol, glucose or alcohol. A method according to any one of the preceding claims, wherein said chelating agent comprises tartrate. A composition comprising stannate, nitroprusside and tartrate. A composition according to claim 7, comprising said stannate, nitroprusside, and tartrate in mole ratios of stannate, nitroprusside and tartrate of about 0.2-5:0.2-5:0.2-5. A composition according to claim 7 or claim 8, impregnated on a bibulous support. A composition according to any one of claims 7 to 9, further comprising detergents and enzyme stabilizers.

Inventors: ALLEN MICHAEL PATRICK, LI SHENG-FEN, ALLEN, MICHAEL PATRICK, LI, SHENG-FEN
Applicants: CHEMTRAK INC, CHEMTRAK, INC.
Application year: 1990
Main classification: C12Q1
IPC codes: C12Q1