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The invention provides an analytical element and a method for preparing an analytical element for evaluation of a liquid sample with respect to a particular analyte by (a) incorporating a carrier with a composition in a liquid, the composition comprising at least one reagent of an analyte-detecting

The invention provides an analytical element and a method for preparing an analytical element for evaluation of a liquid sample with respect to a particular analyte by (a) incorporating a carrier with a composition in a liquid, the composition comprising at least one reagent of an analyte-detecting system, under conditions effective to ionize a reagent migration inhibiting substance, which substance is insoluble in said liquid when in its nonionized form and soluble in said liquid when ionized, and drying the carrier; and then (b) incorporating the carrier of (a) with a second composition in a liquid different from that of (a), the composition comprising the remaining reagents of the analyte-detecting system which are reactive with the at least one reagent of (a) and a reagent migration inhibiting substance which is soluble in said liquid when in its nonionized form, which liquid is effective to prevent reaction of the second composition with the at least one reagent of (a) prior to contact of the element with the sample and to prevent the migration inhibiting substance from ionizing prior to contact with the sample, and drying the carrier. Preferably, conditions which ionize the reagent migration inhibiting substance are the use of an aqueous liquid at a pH of at least about 5. Preferably, the reagent migration inhibiting substance is a monoester maleic acid polymer such as a copolymer of methyl vinyl ether and the monomethylester of maleic acid. The liquid of (b) is preferably an organic liquid, such as acetone.

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1. A method for preparing an analytical element useful for evaluation of a liquid sample for the presence of an analyte, comprising the steps of incorporating a single analyte detection layer with an aqueous solution of at least one reagent of an analyte detecting system, and a buffer capable of

1. A method for preparing an analytical element useful for evaluation of a liquid sample for the presence of an analyte, comprising the steps of incorporating a single analyte detection layer with an aqueous solution of at least one reagent of an analyte detecting system, and a buffer capable of providing a pH of at least 5, drying the single analyte detection layer, and incorporating the dried single analyte detection layer with a solution comprising a solvent, any reagent or reagents other than said at least one reagent contained in said analyte detecting system, and an unionized ionizable reagent migration inhibiting substance selected from the group consisting of carboxylic acid polymers and sulfonic acid polymers, wherein said solvent is one in which the substance is insoluble in its ionized state, but soluble in its unionized state. 2. The method of claim 1 wherein the reagent migration inhibiting substance is a carboxylic acid polymer. 3. The method of claim 2 wherein the carboxylic acid polymer is selected from the group consisting of copolymers of (1) methyl vinyl ether and the monomethylester of maleic acid, (2) methyl vinyl ether and the monoethylester of maleic acid, (3) methyl vinyl ether and the monopropylester of maleic acid, (4) methyl vinyl ether and a monobutylester of maleic acid, (5) vinyl acetate and the monomethylester of maleic acid, (6) vinyl acetate and the monoethylester of maleic acid, (7) ethylene and the monomethylester of maleic acid, (8) ethylene and the monoethylester of maleic acid, (9) octadecyl vinyl ether and the monomethylester of maleic acid, and (10) octadecylvinyl ether and the monoethylester of maleic acid. 4. The method of claim 1 wherein the reagent migration inhibiting substance is a sulfonic acid polymer. 5. The method of claim 1 wherein the sulfonic acid polymer is selected from the group consisting of polystyrene sulfonate, the copolymer of 2-acrylamido-2-methylpropanesulfonic acid and styrene, and polyvinylsulfonic acid. 6. The method of claims 4 or 5 wherein the conditions effective to ionize the sulfonic acid polymer are achieved by use of an aqueous liquid, the pH of which is maintained in a range related to the pKa of the particular sulfonic acid polymer being used such that the sulfonic acid groups are ionized. 7. The method of claim 1 wherein the solvent is an organc solvent. 8. The method of claim 1 wherein the solvent is selected from toluene, acetone, chloroform, n-propane, methylene chloride and ethylene dichloride. 9. The method of claim 1 wherein the reactive reagent comprises a specific binding partner for the analyte. 10. The method of claim 1 wherein the at least one reagent comprises a specific binding partner for the analyte and a component which is reactive with a label conjugate, comprising a label component coupled to an analyte moiety or a specific binding analog thereof, to cleave the label component. 11. The method of claim 1 which, prior to (a) and (b) comprises the additional step of incorporating the single analyte detection layer with an indicator reagent in a liquid effective to prevent reaction of the indicator reagent with the at least one reagent prior to contact of the element with the sample and drying the single analyte detection layer. 12. The method of claim 11 wherein the indicator reagent comprises a tetraalkylbenzidine. 13. A method for preparing an assay element for determining an analyte in a liquid sample which method comprises: impregnating a single analyte detection layer with an aqueous composition comprising β-galactosidase and antibody to the analyte, under conditions effective to ionize the carboxylic acid group of a nonionized, ionizable monoester maleic acid polymer, and drying the single analyte detection layer; and then impregnating the dried carrier with a composition in an organic liquid, the composition comprising β-galactosyl-umbelliferone-analyte or analyte analog conjugate and a nonionized ionizable monoester maleic acid polymer, and drying the single analyte detection layer. 14. A method for preparing an assay element for determining gentamicin in a liquid sample which method comprises: impregnating a single analyte detection layer with an aqueous composition comprising β-galactosidase and antibody to gentamicin, under conditions effective to ionize the carboxylic acid group of a nonionized, ionizable monoester maleic acid polymer, and drying the single analyte detection layer; and then impregnating the dried single analyte detection with a composition in an acetone containing liquid, the composition comprising β-galactosyl-umbelliferonesisomicin conjugate and a nonionized, ionizable monoester maleic acid polymer, and drying the single analyte detection layer. 15. A method for preparing assay element for determining theophylline in a liquid sample which method comprises: impregnating a single analyte detection layer with a composition in an acetone-containing liquid, the composition comprising 3,3',5,5'-tetramethylbenzidine, and drying the single analyte detection layer; then impregnating the single analyte detection layer with a composition in an aqueous liquid, the composition comprising glucose, peroxidase, glucose oxidase apoenzyme and antibody to theophylline, under conditions effective to ionize the carboxylic acid group of a nonionized, ionizable monoester maleic acid polymer, and drying the single analyte detection layer; and then impregnating the single analyte detection layer with a composition in an acetone-containing liquid, the composition comprising flavin adenine dinucleotide-theophylline conjugate and a nonionized, ionizable monoester maleic acid polymer, and drying the single analyte detection layer. 16. A method for preparing an assay element for determining an analyte in a liquid sample which method comprises: impregnating the single analyte detection layer with a composition in an aqueous liquid, the composition comprising glucose, peroxidase, glucose oxidase apoenzyme and antibody to the analyte, under conditions effective to ionize the carboxylic acid group of a nonionized, ionizable monester maleic acid polymer, and drying the single analyte detection layer; and then impregnating the single analyte detection layer with a composition in an acetone-containing liquid, the composition comprising flavin adenine dinucleotide-analyte or analyte analog conjugate, 3,3',5,5'-tetramethylbenzidine and a nonionized, ionizable monoester maleic acid polymer, and drying the single analyte detection layer. 17. A test device for evaluation of a liquid sample for the presence of an analyte which comprises a single analyte detection layer incorporated with an analyte detecting system, buffer capable of providing a pH of at least 5, and a nonionized, ionizable reagent migration inhibiting substance selected from the group consisting of carboxylic acid polymers and sulfonic acid polymers. 18. The test device of claim 17 in which the reagent migration inhibiting substance is a copolymer of methyl vinyl ether and the monomethylester of maleic acid, methyl vinyl ether and the monoethylester of maleic acid, methyl vinyl ether and the monopropylester of maleic acid, methyl vinyl ether and a monobutylester of maleic acid, vinyl acetate and the monomethylester of maleic acid, vinyl acetate and the monoethylester of maleic acid, ethylene and the monomethylester of maleic acid, ethylene and the monoethylester of maleic acid, octadecyl vinyl ether and the monomethylester of maleic acid, or octadecylvinyl ether and the monoethylester of maleic acid, or mixtures thereof. 19. The test device of claim 17 in which the reagent migration inhibiting substance is polystyrene sulfonate, a copolymer of 2-acrylamido-2-methylpropanesulfonic acid and styrene, polyvinylsulfonic acid, or mixtures thereof.