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

A dry analytical element has been prepared for the assay of leucine aminopeptidase at a pH of 6.5 to 11. The element zone contains an aromatic substrate for the enzyme. This substrate provides an aromatic reactant which has a primary amino group on the aromatic ring in the ortho or para position to an electron donor group. The aromatic reactant is oxidized with an oxidizing compound (such as an oxidase) and the oxidized compound reacts with a ballasted color-forming coupler to provide a dye.

FIELD OF THE INVENTION 
The present invention relates to clinical chemistry. In particular, it 
relates to an analytical element and method for the determination of an 
aminopeptidase or transpeptidase in aqueous liquids, such as biological 
fluids. 
BACKGROUND OF THE INVENTION 
Peptidase is known as a general term for an enzyme which acts on a peptide 
bond in an L-peptide, splitting at the N-terminal to liberate amino acids 
or lower peptides. Such enzymes include, but are not limited to, leucine 
aminopeptidase, cysteine aminopeptidase, proline aminopeptidase, arginine 
aminopeptidase, alanine aminopeptidase and gamma-glutamyl transpeptidase. 
Leucine aminopeptidase and gamma-glutamyl transpeptidase are widely 
distributed in human tissues and certain body fluids, such as serum. These 
enzymes increase in concentration in certain disease states, and are thus 
important clinical indicators for clinical diagnosis and treatment. 
In particular, leucine aminopeptidase is an enzyme capable of liberating 
leucine from L-peptides, and particularly from those peptides having 
amino-terminal leucine groups. Its concentration has been known to vary 
greatly with certain health states. For example, it increases in the serum 
of people suffering from acute hepatitis, hepatoma, metastic hepatoma, 
liver cirrhosis or cholangia. 
Gamma-glutamyl transpeptidase is clinically important in the diagnosis of 
cholestatic hepatitis, obstructive jaundice and primary metastatic 
hepatoma, active chronic hepatitis and non-active chronic hepatitis. 
Known assays for leucine aminopeptidase or gamma-glutamyl transpeptidase 
activity generally involve the release of amine compounds from substrates 
by the enzyme to provide a colorimetric signal. Various substrates have 
been developed and described in the art for this very purpose, such as 
those described in U.S. Pat. No. 4,209,459 (Nagasawa et al), U.S. Pat. No. 
4,588,836 (Matsumoto et al) and U.S. Pat. No. 4,681,841 (Matsumoto et al), 
and by Shimamoto et al, Clin. Chem,, 31, pp. 1636-1639, 1985. The assays 
described in these references and in many others not cited here are 
carried out in solution to yield a water-soluble dye if the enzyme is 
present in the serum specimen. Urine has also recently been tested to 
yield information about leucine aminopeptidase. 
In recent years, analytes have been detected to great advantage using dry 
analytical elements which contain all of the appropriate reagents for the 
assay. In preferred dry elements, a topmost porous layer is used for 
spreading a specimen uniformly for contact with reagents in the element. 
Such spreading layers are prepared from a number of materials including a 
structure of adhered particles as described, for example, in U.S. Pat. No. 
4,258,001 (Pierce et al) and pigmented layers such as those described in 
U.S. Pat. No. 3,992,158 (Przybylowicz et al). Pigmented layers containing 
titanium dioxide are often preferred for reduction of interferences, and 
ease of coating and finishing of the element. 
It would be desirable to carry out the known assays for leucine 
aminopeptidase in a dry analytical element. However, there are a number of 
problems with doing so. Most of the dyes produced in known assays provide 
a signal at 400 nm or below which increases the risk of interference from 
bilirubin or hemoglobin. Moreover, some known substrates for the enzyme 
are toxic and require special handling and disposal. 
In U.S. Pat. No. 4,681,841 (noted above), a useful dye is generated for 
detection at higher wavelength, but the color couplers used to generate 
the dye are water-soluble. This property presents additional problems for 
their use in dry analytical elements because the color couplers can 
migrate throughout the element, thereby reducing the observable dye 
signal. Moreover, the pH of the assay is critical for obtaining desired 
enzyme activity and sensitivity. Assay pH is easily controlled in solution 
assays, but it is difficult to control the pH when using dry analytical 
elements. 
It would be highly desirable to be able to detect an aminopeptidase or 
transpeptidase in a dry analytical element whereby the reagents are kept 
separate and the pH is strictly controlled for optimum results. The known 
technology does not suggest how to solve these problems. 
SUMMARY OF THE INVENTION 
The problems noted above have been solved 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, 
the element containing in a zone other than the 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 
the primary amino group, and 
the 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, 
the non-diffusible color-forming coupler having the properties of: 
a) being capable of undergoing electrophilic substitution, 
b) comprising a ballasting group 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 the primary amino-containing 
reactant provided by the aromatic substrate, it will provide a dye having 
an absorbance in the range of from about 400 to about 800 nm, 
the oxidizing compound in the second zone being a compound which oxidizes 
the primary amino group of the primary amino-containing reactant to render 
it suitable for reaction with the color-forming coupler to form a dye. 
This invention also provides 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 the analytical element described above to form a dye, 
and 
B. detecting the formation of the 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 the fluid sample. 
This invention provides a simple, relatively rapid and sensitive means for 
assay of an aminopeptidase or transpeptidase, such as leucine 
aminopeptidase, gamma-glutamyl transpeptidase, cysteine aminopeptidase and 
others, which has all the known advantages of dry analytical systems. The 
dry element is readily adapted to automated assay equipment and is dry to 
the touch. In addition, the present invention effectively incorporates a 
color-forming coupler in the element which will not migrate and cause loss 
of dye signal. Moreover, the color-forming coupler is not toxic and 
provides high sensitivity. Use of a spreading layer advantageously reduces 
interferences arising from unwanted substances in biological specimens. 
Assay pH is carefully controlled in the element by use of a buffer in one 
or more of the zones so that sensitivity is optimized. Interference from 
hemoglobin or bilirubin is avoided. 
These advantages are particularly achieved by use of certain color-forming 
couplers which are located in a zone of the element separate from that 
containing the aromatic substrate for the analyte. The color-forming 
coupler has the following properties: 
a) it is capable of undergoing electrophilic substitution, 
b) it comprises a ballasting group which has a molecular weight of at least 
about 150, 
c) it is soluble 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 the primary amino-containing 
reactant provided by the aromatic substrate, it will provide a dye having 
an absorbance in the range of from about 400 to about 800 nm. 
Because the coupler is ballasted, it cannot migrate into other layers. Yet, 
it is available for reaction with other reagents because they can readily 
move throughout the zones of the element.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a determination (either quantitative or 
qualitative) of an aminopeptidase or transpeptidase including, but not 
limited to, leucine aminopeptidase, gamma transpeptidase, cysteine 
aminopeptidase, proline aminopeptidase, arginine aminopeptidase and 
alanine aminopeptidase. In particular, the invention can be used to assay 
any aqueous fluid suspected of containing the enzyme, and particularly 
biological fluids including, but not limited to, sera, urine, lymph, 
plasma, whole blood and cerebral spinal fluid. It is also possible to 
assay fluid preparations of tissue such as preparations of liver or 
intestinal tissue. Preferably, human serum or urine is assayed with this 
invention. 
The dry element of this invention has two or more contiguous zones (or 
layers) which are fluid permeable and contain all of the reagents needed 
for the detection of the enzyme analyte. The elements are known as test 
strips, test slides or diagnostic devices. The zones can be 
"self-supporting", which means that the zones can be composed of materials 
which maintain their integrity when exposed to aqueous fluids, such as 
filter papers or microporous membranes. Preferably, however, such zones 
are disposed on a separate, nonporous support which is dimensionally 
stable, inert to chemical reaction and preferably transparent (that is, 
radiation transmissive for wavelengths between about 200 and 900 nm). 
However, non-transparent supports can be used if the mode of detection is 
reflectance spectroscopy instead of transmission spectroscopy. Useful 
supports are well known in the art, including but not limited to 
polyesters, papers, metal foils and polystyrene, polycarbonates and 
cellulose esters. 
At least one zone of the element (and preferably, the outermost zone), is a 
porous spreading zone prepared from any of the known materials used for 
such zones as described, for example in U.S. Pat. No. 4,292,272 (Kitajima 
et al), U.S. Pat. No. 3,992,158 (noted above), U.S. Pat. No. 4,258,001 
(noted above) U.S. Pat. No. 4,430,436 (Koyama et al) and related U.S. 
patents, and JP 57(1982)-101760 (published Jun. 24, 1982). It is desired 
that the spreading zone be isotropically porous, meaning that the porosity 
is the same in each direction in the zone as caused by interconnected 
spaces or pores between particles, fibers or polymeric strands. 
Preferred spreading zones are those described in U.S. Pat. No. 3,992,158 as 
"blush polymer" zones. Such zones can be formed on a supporting material 
by dissolving a polymer in a mixture of two organic liquids, one of which 
is a lower boiling, good solvent for the polymer and other being a high 
boiling, non-solvent or poor solvent for the polymer. The resulting 
polymer solution is coated on the supporting material and dried under 
controlled conditions to leave an isotropically porous zone. Various 
polymers are known to be useful in this context including, but not limited 
to, polycarbonates, polyamides, polyurethanes and cellulose esters such as 
cellulose acetate (which is preferred). 
Within the porous zone can be incorporated particulate materials of various 
sizes to enhance the void volume. Useful particulate materials include, 
but are not limited to, inorganic pigments such as titanium dioxide, 
barium sulfate, zinc oxide, lead oxide with titanium dioxide being 
preferred. Further details of the preparation of "blush polymers" are 
described in U.S. Pat. No. 3,992,158. 
The elements contain at least one other zone which contains one or more 
reagents needed for the assay. Such a zone is often known in the art as a 
reagent or registration zone, but it can also include a second porous 
spreading zone if desired or printed layers located on other zones. The 
zones are generally in fluid contact with each other, meaning that fluids, 
reagents and reagent products can pass or be transported between 
superposed regions of adjacent zones, unless of course, a reagent is 
immobilized in some manner so it will not migrate within or without a zone 
(see below with regard to color-forming couplers). Preferably, the zones 
are separately coated and superposed layers on an inert support (see 
Example 1 below). The reagent zones or layers can be composed of one or 
more binder materials (such as gelatin and other colloidal materials, 
hydrophilic polymers such as polyvinyl alcohol, polyacrylamide and others 
known in the art) in which reagents are incorporated. 
The methods of preparing such elements are well known in the art and 
involve application of wet formulations of the zone composition onto a 
support and drying under suitable conditions. Coating procedures are well 
described in the art cited above for describing the spreading zones. 
The assay of this invention is carried out with the following sequence of 
reactions, illustrated for leucine aminopeptidase: 
a) leucine aminopeptidase catalyzes the conversion of an aromatic leucine 
aminopeptidase substrate into L-leucine and an aromatic reactant having a 
primary amino group which is ortho or para to a hydroxy, amino or 
substituted amino group on the ring, 
b) the one or more primary amino groups of the aromatic reactant are 
oxidized to one or more reactive imine groups with an oxidizing compound 
(the resulting oxidized compound can be a quinonimine or a quinondiimine), 
and 
c) the resulting imine (or diimine)-containing aromatic compound is reacted 
with a non-diffusible color-forming coupler to provide a dye. 
A substrate for the enzyme analyte is provided in one of the zones of the 
element other than the zone which contains the color-forming coupler. In 
one embodiment, it can be included in the porous spreading zone described 
above. In another embodiment, it can be located in yet a different (or 
third) zone (or layer). 
The substrate is aromatic and capable, upon reaction with the enzyme, of 
providing a reactant having a primary amino group on the aromatic ring 
(such as a phenyl ring), and also has a hydroxy, amino or substituted 
amino group in the ortho or para position to the primary amino group. 
Substitution in the para position is preferred. Substituted amino groups 
include, but are not limited to, methylamino, dimethylamino, 
isopropylamino and diethylamino. 
In a preferred embodiment, the aromatic substrate is represented by the 
structure (I): 
##STR1## 
wherein R is an amino acid or peptide amido group which is the 
condensation product of a carboxylic acid group of an amino acid or 
peptide with a primary amino group appended to the aromatic ring. Examples 
of R include, but are not limited to, L-leucylamido, cysteinylamido, 
prolylamido, arginylamido, alanylamido, gamma-glutamylamido and others 
which would be apparent to one skilled in the art. 
More preferably, R is --NH--CO--R.sup.5 wherein R.sup.5 is linear or 
branched alkyl having 1 to 6 carbon atoms (such as methyl, ethyl, 
isopropyl, t-butyl, pentyl and hexyl) which is substituted with at least 
one primary amino group. R.sup.5 can also be substituted with one or more 
phenyl or substituted phenyl (such as hydroxyphenyl), p-aminophenyl, 
imidazolyl, indolyl, hydroxy, methylthio or other groups readily apparent 
to one skilled in the art. Representative R.sup.5 groups include, but are 
not limited to 1-amino-3-methylbutyl, 1-amino-2-mercaptoethyl, 
1-amino-4-guanidinobutyl, 1-aminoethyl, 1-aminophenethyl, 
1-amino-3-carboxypropyl, 1-amino-2-carboxyethyl, 
1-amino-2-(5-imidazolyl)ethyl, 1-amino-2-(4-hydroxyphenyl)ethyl, 
1-amino-2-carbamoylethyl, 1-amino-2-carbamoylpropyl, 
1-amino-2-hydroxyethyl, 1,5-diaminopentyl, 1-amino-2-hydroxypropyl, 
aminomethyl, 1-aminoethyl, 1-amino-2-methylpropyl, 1-amino-2-methylbutyl, 
1-amino-3-methylbutyl, 1-amino-3-methylthiopropyl and 
1-amino-2-(3-indolyl)ethyl. Most preferably, R is L-leucylamido (that is, 
R.sup.5 is 1-amino-3-methylbutyl). 
In structure (I) noted above, R' is hydroxy, or primary, secondary or 
tertiary amino (substituted with one or more lower alkyl groups of 1 to 6 
carbon atoms, such as methyl, ethyl, isopropyl and t-butyl). Preferably, 
R' is hydroxy. 
Also, each of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 can independently be 
hydrogen, halo (for example fluoro, chloro or bromo), alkyl of 1 to 6 
carbon atoms (such as methyl, ethyl, isopropyl, chloromethyl and 
methoxymethyl), alkoxy of 1 to 6 carbon atoms (such as methoxy, ethoxy, 
isopropoxy and t-butoxy), hydromy or amino (primary, secondary or 
tertiary). Preferably, R.sup.1 and R.sup.4 are independently halo (such as 
chloro or bromo) and each of R.sup.2 and R.sup.4 is hydrogen or methyl. 
The substrates can optionally be used in the form of a salt, such as a 
formate, acetate, propionate, maleate, citrate, tartrate or oxalate. 
Examples of useful substrates are described in U.S. Pat. Nos. 4,209,459, 
4,588,836, and 4,681,841. 
A most preferred substrate is L-leucine-3,5-dibromo-4-hydroxyanilide useful 
for the determination of leucine aminopeptidase. 
The aromatic substrates described above can be prepared using known 
procedures or purchased from various commercial sources, including Toyo 
Jozo (now part of Asahi Chemical Industry Co., Ltd.) of Japan. Preparatory 
procedures are described, for example, in U.S. Pat. Nos. 4,209,459 and 
4,588,836. 
In a second zone of the element is an oxidizing compound (or oxidant) which 
converts the primary amino-containing aromatic reactant to a compound 
having an imine group which can then react with the color-forming coupler 
(defined below). Useful oxidizing compounds include, but are not limited 
to, oxidases which consume oxygen when oxidizing the primary amino group. 
Other oxidizing compounds include ferric ethylenediaminetetraacetic acid 
(or equivalent salts) and cuprous chloride (or equivalent salts). Examples 
of useful oxidases include ascorbic acid oxidase, lactase, tyrosinase, 
aminophenol oxidase, phenol oxidase and polyphenol oxidase. Ascorbic acid 
oxidase, from any suitable source, is preferred as the oxidizing compound. 
In the same zone as the oxidizing compound is a non-diffusible 
color-forming coupler which is used to provide a suitable dye in the 
element if the aminopeptidase or transpeptidase is present. The 
color-forming coupler must be capable of undergoing electrophilic 
substitution which means that the coupler has a "leaving" group (also 
known as an electrofuge) that is cleaved from the compound without its 
electron pair when displaced by an electrophile, that is a "positive 
attacking" group (a positive ion or positive end of a dipole or induced 
dipole). The "leaving" group can be, for example, hydrogen, mercapto, 
substituted mercapto (such as an organomercapto, for example methylthio, 
ethylthio, phenylthio, benzylthio and 2-pivalamidophenylthio), hydroxy, 
substituted or unsubstituted alkoxy of 1 to 12 carbon atoms (such as 
methoxy, ethoxy, isopropoxy, butoxy and octoxy), N-heterocyclic groups 
(such as pyrazolyl, imidazolyl and pyrrolyl) or halo (such as chloro and 
bromo). Preferably, the "leaving" group is hydrogen or substituted 
mercapto and 2-pivalaminophenylthio is most preferred. Electrophilic 
substitution is described in the art, for example, by March, Advanced 
Organic Chemistry--Reaction Mechanisms and Structures, 3rd Ed., John Wiley 
and Sons, New York, pages 447, 512 and 570. 
The color-forming coupler comprises a ballasting group which has a 
molecular weight of at least about 150. That is, the ballasting group is 
of such size and configuration as to render the coupler non-diffusible. 
These groups can be substituted or unsubstituted with groups which enhance 
the non-diffusibility of the coupler, or modify the reactivity of the 
coupler. The ballasting group can contain a linking group through which it 
is joined to the coupler moiety. Such linking groups include, but are not 
limited to, oxy, thio, imino, carbonyloxy, sulfamoyl, sulfonamido, amido, 
carbamoyl and azo. Preferred ballasting groups include alkyl, aryl 
(substituted or unsubstituted), alkoxy, aryloxy, alkylthio and arylthio 
groups, each containing 8 to 32 carbon atoms as long as the molecular 
weight requirement is also met. Useful ballasting groups include, but are 
not limited to, dodecyl, tridecyl, tetradecanamido, 
2-chloro-5-tetradecanamidoanilino, 4-(4-benzyloxyphenylsulfonyl)phenoxy, 
3-tetradecylphenoxy, 4-butylsulfonamidophenoxy, 
4-(2,5-di-t-pentylphenoxy)butylcarbamoyl, 
2-ethyl-2-(3-tetradecylphenoxy)acetamido, 
2-[4-(4-benzyloxyphenylsulfonyl)phenoxy]-2-decylacetamido, and substituted 
aryl such as trichlorophenyl. Other representative ballasting groups are 
described, for example, in U.S. Pat. No. 4,420,556 (Booms). 
More preferably, the ballasting groups are alkyl (branched or linear), aryl 
or alkoxy (branched or linear) groups having 10 to 24 carbon atoms and 
substituted with one or more amino, amido, carbamoyl, sulfonamido or 
sulfamoyl groups. These groups can also be substituted with alkyl (1 to 25 
carbon atoms), alkoxy (1 to 25 carbon atoms), halo, phenyl or phenyl 
substituted with alkyl (1 to 10 carbon atoms) or halo. Such preferred 
ballasting groups include alkyl, alkylamido, alkylamino, alkylcarbamoyl, 
alkylsulfonamido, alkylsulfamoyl, alkylamidoarylamino, aryloxy, 
aryloxyalkylamido, arylamino, arylamido, arylcarbamoyl, arylsulfonamido 
and arylsulfamoyl. 
The color-forming coupler also has the property of being soluble in organic 
solvents, each having a molecular weight of at least about 150 and a 
boiling point of at least about 150.degree. C. Examples of useful organic 
solvents include, but are not limited to, dibutyl phthalate, 
2,4-di-n-pentylphenol, N,N-diethyllauramide, di-n-octyl phthalate, 
di-2-ethylhexyl phthalate and mixtures thereof. 
Particularly useful color-forming couplers are those represented by the 
structures (II)-(V): 
##STR2## 
(VI) BALL--CO--CHR.sub.8 --CO--NHR.sup.11 
In structure (II), R.sup.6 and R.sup.6' are independently hydrogen, halo 
(for example, fluoro, chloro or bromo), --CONR.sup.9 R.sup.10, --NR.sup.9 
COR.sup.10, --SO.sub.2 NR.sup.9 R.sup.10, alkyl of 1 to 6 carbon atoms 
(such as methyl, isopropyl, hexyl and t-butyl) or alkoxy of 1 to 6 carbon 
atoms (such as methoxy, ethoxy, isopropoxy and hexoxy). Preferably, 
R.sup.6 and R.sup.6' are independently hydrogen or halo and more 
preferably, R.sup.6 is hydrogen and R.sup.6' is halo (such as chloro). 
R.sup.7 in structure (II) is phenyl or phenyl substituted with one or more 
halo (for example fluoro, chloro or bromo), alkyl of 1 to 5 carbon atoms 
(such as methyl, ethyl, isopropyl and chloromethyl) or alkoxy of 1 to 5 
carbon atoms (such as methoxy, ethoxy and isopropoxy). Preferably, R.sup.7 
is trichlorophenyl. 
Moreover, R.sup.8 is hydrogen or a "leaving" group as defined above. Useful 
leaving groups are also defined above. Preferably, in structure II, 
R.sup.8 is pivalamidophenylthio, and in structure VI, it is hydrogen. 
R.sup.9 is hydrogen, alkyl of 1 to 24 carbon atoms (such as methyl, ethyl, 
t-butyl, hexyl, dodecyl, pentadecyl and 3-methyloctyl), phenyl or phenyl 
substituted with one or more halo or alkyl groups as defined above for 
R.sup.7. Preferably, R.sup.9 is hydrogen. 
R.sup.10 is hydrogen or BALL wherein BALL is a ballast group as defined 
above. 
R.sup.11 is substituted or unsubstituted alkyl of 1 to 20 carbon atoms 
(such as methyl, ethyl, isopropyl, t-butyl, n-hexyl, octyl, nonyl, 
isononyl, decyl, dodecyl and hexadecyl), substituted or unsubstituted 
carbocyclic aryl of 6 to 14 carbon atoms in the ring (such as phenyl, 
naphthyl, anthryl, tolyl, xylyl, carbamoylphenyl, 3,5-dichlorophenyl and 
4-cyanophenyl), heterocyclyl or BALL as defined above. Preferably, 
R.sup.11 is aryl or heterocyclyl. When R.sup.11 is heterocyclyl, it is a 
5- or 6-membered ring of carbon atoms and at least one nitrogen, sulfur or 
oxygen atom, and optionally has one or two fused aromatic groups (such as 
benzo or naphtho) attached thereto. Any of the foregoing radicals defining 
R.sup.11 can be substituted with one or more halo (such as fluoro, chloro, 
bromo or iodo), cyano, carboxy, substituted or unsubstituted alkyl as 
defined above, substituted or unsubstituted aryl as defined above, 
carbamoyl, sulfamoyl, alkylformamido or arylformamido (with alkyl and aryl 
defined as above), alkylsulfonamido or arylsulfonamido (with alkyl or aryl 
as defined above), alkoxy of 1 to 12 carbon atoms, aryloxy of 6 to 10 
carbon atoms, alkoxycarbonyl (with alkoxy as defined above), 
aryloxycarbonyl (with aryloxy as defined above), acyl of 1 to 12 carbon 
atoms, acyloxy or 1 to 12 carbon atoms, or a BALL group as defined above. 
In structure V, X is hydrogen, halo (such as chloro or bromo) or phenoxy 
(with or without substituents). Preferably, X is chloro or unsubstituted 
phenoxy. 
In structure II, BALL is preferably alkyl or aryl as defined above. 
The color-forming couplers having the structure (II) are preferred. 
Representative color-forming couplers are listed below with structure 
(VII) being most preferred. 
##STR3## 
The color-forming couplers as described herein can be prepared using 
conventional procedures and starting materials, as described for example, 
in U.S. Pat. No. 4,853,319 (Krishnamurthy). They are generally dissolved 
in organic solvents (described above) for coating purposes. 
The elements of this invention can also contain one or more other addenda 
commonly included for various manufacturing or operational advantages. 
Such addenda include surfactants, ion chelating agents, buffers, organic 
solvents (such as organic solvents for the color-forming couplers), 
hardeners for binders, antioxidants, and others known in the art. 
Representative elements and components are described below in the 
examples. One or more buffers which maintain the pH within the element at 
from about 6.5 to about 11 during the assay are particularly useful. The 
assay is preferably carried out at a pH of from about 6.5 to about 9 with 
a pH of from about 8 to about 8.5 being most preferred. The buffer can be 
in any of the zones of the element. Acceptable buffers include, but are 
not limited to, tris(hydroxymethyl)aminomethane, glycine, borate and 
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. 
The amounts of reagents which can be incorporated within the element are 
generally within the skill of a worker in the art. More specifically, the 
aminopeptidase or transpeptidase aromatic substrate is present in a range 
of from about 0.05 to about 1 g/m.sup.2, with an amount within the range 
of from about 0.2 to about 0.6 g/m.sup.2 being preferred. The 
color-forming coupler is generally present in an amount of from about 0.05 
to about 0.5 g/m.sup.2, with from about 0.2 to about 0.4 g/m.sup.2 being 
preferred. If the oxidizing compound is an oxidase, the generally useful 
amount is from about 1000 to about 50,000 I-U./m.sup.2 with from about 
10,000 to about 30,000 I-U./m.sup.2 being preferred. Where the oxidizing 
compound is not an enzyme, the useful amount can be readily calculated to 
be that which would provide oxidizing capacity comparable to that of an 
oxidase. For example, for ethylenediaminepentaacetic acid (or salt), the 
amount on a molar basis would be from about 0.1 to about 2 molar/m.sup.2. 
As used in this application, one I.U. represents the International Unit for 
enzyme activity and is defined as the amount of enzyme activity required 
to catalyze the conversion of 1 micromole of substrate per minute under 
standard pH and temperature conditions. For the oxidases (for example 
ascorbic acid oxidase) described herein, the standard conditions are 
37.degree. C. and a pH of about 7.8. 
The amounts of buffers and other addenda would be readily apparent to one 
skilled in the art given the teaching in the art and that provided in the 
examples below. 
A variety of different elements, depending upon the method of assay, can be 
prepared in accordance with this invention. Elements can be configured in 
a variety of forms, including elongated tapes of any desired width, 
sheets, slides or chips. 
In one embodiment of this invention, a multilayer element for the 
determination of an aminopeptidase comprises an inert polymeric support 
having thereon, in order and in fluid contact: 
a first reagent layer containing the non-diffusible color-forming coupler 
and oxidizing compound as described above, 
a second reagent layer containing the aromatic substrate for the 
aminopeptidase as described above, and 
a porous spreading layer as described above, 
the element further comprising a buffer in one or more of the layers as 
described above. 
In a preferred embodiment of this invention, a multilayer element comprises 
an inert polymeric support having thereon, in order and in fluid contact: 
a reagent layer containing the non-diffusible color-forming coupler and 
oxidizing compound as described above, 
optionally a subbing layer (such materials being well known in the art), 
and 
a porous spreading layer as described above which contains the aromatic 
substrate for the enzyme analyte, 
the element further comprising a buffer in one or more layers as described 
above. 
The assay of this invention can be manual or automated. In general, in 
using the dry elements, the enzyme analyte is determined by taking the 
element (for example from a supply roll, slide tray or packet) and 
physically contacting it on the porous spreading zone with a sample (for 
example from 1 to 200 .mu.l) to be tested. The sample and reagents within 
the element then become mixed in the various zones. Such contact can be 
accomplished in any suitable manner, for example by dipping or immersing 
the element into the sample or preferably, by spotting the sample onto the 
element by hand or machine with a suitable dispensing means. 
After sample application, the element is exposed to any conditioning, such 
as incubation, heating or otherwise, that may be desirable to quicken or 
otherwise facilitate obtaining a test result. 
Generally within about 2 minutes, a first spectrophotometric measurement is 
made of any dye formed in the element. Since the analyte is an enzyme 
which reacts over time, generally a second measurement is taken a few 
minutes later. The rate of dye formation can be measured with suitable 
reflection or transmission spectrophotometric equipment and procedures as 
a measure of the enzyme activity. 
The dye formed as a measure of the analyte is generally evaluated at a 
wavelength in the range of from about 400 to about 800 nm, with 
measurement at a wavelength of from about 500 to about 670 nm being 
preferred. In most cases, the wavelength measured is the maximum 
wavelength, although it is possible to evaluate many dyes off-peak (not at 
the maximum wavelength). The most preferred color-forming coupler 
identified as compound (VII) above provides a dye having a maximum 
wavelength at about 540 nm. 
In the following examples which are used to illustrate, but not limit, the 
present invention, the materials used were commercially obtained as 
follows: 
ESTANE.RTM. polyurethane resin from B. F. Goodrich, 
TRITON.RTM. X-100, TRITON.RTM. X-405 and TRITON.RTM. X-705 surfactants from 
Rohm and Haas, 
BRIJ.RTM. surfactant from ICI Americas, Inc. 
tris(hydroxymethyl)aminomethane buffer from Sigma Chemical Co., 
L-leucine-3,5-dibromo-4-hydroxyanilide from Toyo Jozo, and the remainder of 
materials from either Eastman Kodak Company or other commercial sources, 
or they were prepared using standard procedures, or procedures described 
herein, and readily available starting materials. 
EXAMPLE 1 
Multilayer Analytical Element for the Determination of Leucine 
Aminopeptidase 
The element illustrated below was prepared by formulating the materials of 
each layer into coating dispersions using conventional procedures and 
solvents and coating them in the order shown using standard coating 
procedures. 
______________________________________ 
Layer Coverage (g/m.sup.2) 
______________________________________ 
Titanium dioxide 
67 
Cellulose 9.8 
acetate 
Spreading Layer 
TRITON .TM. X-405 
1.85 
BRIJ .TM. 78 0.93 
surfactant 
ESTANE .TM. 1.8 
polyurethane 
resin 
Subbing Layer 
Poly(vinyl- 0.94 
pyrrolidone) 
or poly(N-iso- 
0.39 
propylacryl- 
amide) 
First Reagent 
Gelatin 5 
Layer (unhardened) 
tris(hydroxy- 1 
methyl)amino- 
methane buffer 
TRITON .TM. X-705 
0.5 
surfactant 
L-leucine-3,5- 
0.3 
dibromo-4- 
hydroxyanilide 
Gelatin 10 
(hardened) 
Second Reagent 
tris(hydroxy- 3 
Layer methyl)amino- 
methane buffer 
TRITON .TM. X-705 
0.5 
or TRITON .TM. X- 
100 surfactant 
2,4-di- -n- 2 
pentylphenol 
Color-forming 0.2 
coupler (VII) 
Ascorbic acid 25,000 I.U./m.sup.2 
oxidase 
Poly(ethylene- 
terephthalate) 
Support 
______________________________________ 
The element was used to determine leucine aminopeptidase in the following 
manner. 
Serum samples (10 .mu.l each) containing various amounts (50-1500 
I.U./liter) of leucine aminopeptidase were spotted on the porous spreading 
layer of individual elements. While the element was incubated at 
37.degree. C., reflectance density readings were recorded at 540 nm over a 
six minute time period. FIG. 1 shows the resulting dye signals over time 
for each sample, indicating that aminopeptidase can be acceptably 
determined using an element of this invention. 
EXAMPLE 2 
Preferred Analytical Element and Assay 
This example demonstrates a preferred embodiment of this invention for the 
determination of leucine aminopeptidase whereby the aromatic substrate is 
located in the spreading layer. 
The element of this example was like that shown in Example 1 except that 
the "First Reagent Layer" was eliminated. The substrate 
L-leucine-3,5-dibromo-4-hydroxyanilide (0.3 g/m.sup.2) was coated in the 
spreading layer. The assay was carried using the protocol described in 
Example 1. 
FIG. 2 shows the results of the dye signal (reflectance density) 
determinations (50-1500 I.U. analyte/liter)over the six minute time 
period. This embodiment is an improvement over that shown in Example 1 
because the curves become linear more quickly (generally within two 
minutes). Thus, the assay can be performed in less time when the substrate 
is put into the spreading layer. 
FIG. 3 is a calibration curve (activity vs. rate as measured by dye signal) 
generated using from 50 to 1500 I.U. analyte/liter. 
The invention has been described in detail with particular reference to 
preferred embodiments thereof, but it will be understood that variations 
and modifications can be effected within the spirit and scope of the 
invention.