Dry analytical element and method for the detection of an aminopeptidase or transpeptidase

A multilayer analytical element for the determination of leucine aminopeptidase comprising an inert polymeric support having thereon, in order and in fluid contact:

• a first reagent layer containing an oxidizing compound and a non-diffusible color-forming coupler,
• a porous spreading layer containing an aromatic substrate which upon reaction with an aminopeptidase or transpeptidase provides a reactant having a primary amino group on a phenyl ring and a hydroxy, amino or substituted amino group in the ortho or para position to said primary amino group,
• said element further comprising in one or more of said layers, a buffer which is present in an amount effective to provide a pH of from about 6.5 to about 11 during an assay of a biological fluid for an aminopeptidase or transpeptidase.
• said non-diffusible color-forming coupler having the properties of:
• a) being capable of undergoing electrophilic substitution,
• b) comprising a ballasting group, BALL, which has a molecular weight of at least about 150,
• c) solubility in organic solvents having a molecular weight of at least about 150 and a boiling point of at least about 150.degree. C., and
• d) when coupled with the oxidized form of said reactant provided by said aromatic substrate, said coupler will provide a dye having an absorbance in the range of from about 400 to about 800 nm, and
• said non-diffusible color-forming coupler having the structure:
• ##STR6## wherein R.sup.6 and R.sup.6' are independently hydrogen, halo, --CONR.sup.9 R.sup.10, --NR.sup.9 COR.sup.10, --SO.sub.2 NR.sup.9 R.sup.10, alkyl or alkoxy,
• R.sup.7 is phenyl or phenyl substituted with halo, alkyl or alkoxy,
• R.sup.8 is an organomercapto,
• R.sup.9 is hydrogen, alkyl, phenyl or phenyl substituted with halo or alkyl,
• R.sup.10 is hydrogen or BALL,
• BALL is said ballasting group having 8 to 32 carbon atoms, and
• said oxidizing compound in said first reagent layer being a compound which oxidizes the primary amine group of said reactant to render the oxidized form of said reactant suitable for reaction with said color-forming coupler to form a dye.

A method for the determination of an aminopeptidase or transpeptidase comprising the steps of:

• A. contacting a fluid sample suspected of containing an aminopeptidase or transpeptidase with an analytical element for the determination of an aminopeptidase or transpeptidase comprising, in fluid contact, a plurality of zones,
• a first zone being a porous spreading zone, and
• a second zone containing an oxidizing compound and a non-diffusible color-forming coupler,
• said element containing in a zone other than said second zone, an aromatic substrate which upon reaction with an aminopeptidase or transpeptidase provides a reactant having a primary amino group on a phenyl ring and a hydroxy, amino or substituted amino group in the ortho or para position to said primary amino group, and
• said element further containing in one or more of the zones, a buffer which is present in an amount effective to provide a pH of from about 6.5 to about 11 during an assay of a biological fluid for an aminopeptidase or transpeptidase,
• said non-diffusible color-forming coupler having the properties of:
• a) being capable of undergoing electrophilic substitution,
• b) comprising a ballasting group, BALL, which has a molecular weight of at least about 150,
• c) solubility in organic solvents having a molecular weight of at least about 150 and a boiling point of at least about 150.degree. C., and
• d) when coupled with the oxidized form of said reactant provided by said aromatic substrate, said coupler will provide a dye having an absorbance in the range of from about 400 to about 800 nm,
• said non-diffusible color-forming coupler having the structure:
• ##STR8## wherein R.sup.6 and R.sup.6' are independently hydrogen, halo, --CONR.sup.9 R.sup.10, --NR.sup.9 COR.sup.10, --SO.sub.2 NR.sup.9 R.sup.10, alkyl or alkoxy,
• R.sup.7 is phenyl or phenyl substituted with halo, alkyl or alkoxy,
• R.sup.8 is an organomercapto,
• R.sup.9 is hydrogen, alkyl, phenyl or phenyl substituted with halo or alkyl,
• R.sup.10 is hydrogen or BALL, and
• BALL is said ballasting group having 8 to 32 carbon atoms, and
• said oxidizing compound in said second zone being a compound which oxidizes said primary amino group of said reactant to render the oxidized form of said reactant suitable for reaction with said color-forming coupler to form a dye, and
• B. detecting the formation of said dye at an absorbance in the range of from about 400 to about 800 nm as an indication of the presence of the aminopeptidase or transpeptidase in said fluid sample.