Use of phenols and anilines to increase the rate of peroxidase catalyzed oxidation of leuco dyes

Certain phenols and anilines can be used as electron transfer agents to increase the rate of oxidation of leuco dyes by peroxidase. These phenols and anilines can react with hydrogen peroxide in the presence of peroxidase to provide intermediates which have higher oxidation potentials than the slowly oxidized substrates, e.g. the leuco dyes. These phenols and anilines can be used to advantage in both solution and dry assays of various analytes. They are particularly useful for the determination of an immunologically reactive ligand in an immunoassay.

FIELD OF THE INVENTION 
The present invention relates generally to clinical chemistry. In 
particular, it relates to analytical compositions, elements and methods 
useful for the determination of an analyte in biological fluids. For 
example, the present invention can be used in an immunoassay. 
BACKGROUND OF THE INVENTION 
It is well known to perform a quantitative or qualitative analysis of an 
aqueous liquid by contacting that liquid with a combination of reagents 
capable of yielding a detectable product in proportion to the 
concentration of the analyte in the liquid. As used herein, this 
combination of reagents is termed an interactive composition which is 
capable of chemical reactivity, catalytic activity, or any other form of 
chemical or physical interaction that can result in the ultimate 
production of a change that is detectable with suitable procedures and 
equipment. 
One type of useful assay utilizes enzymatic reactions wherein the analyte, 
upon contact with the appropriate reagents, reacts with oxygen in the 
presence of a suitable enzyme to produce hydrogen perozide in proportion 
to the concentration of the analyte. A detectable product is then produced 
by the reaction of hydrogen peroxide in proportion to the concentration of 
the analyte in the tested liquid. Peroxidase is generally used in such 
assays to catalyze the oxidation of interactive composition by hydrogen 
peroxide. 
Peroxidase can be used for diagnostic determinations of various analytes 
such as glucose, triglycerides, uric acid, cholesterol, creatine kinase, 
etc. and can be used as a label in ligand analogs used in the 
determination of immunologically reactive species (i.e. immunoassays). 
Such determinations can be carried out in solution or in dry analytical 
elements, such as those described in U.S. Pat. Nos. 3,992,158 (issued Nov. 
16, 1976 to Przybylowicz et al), 4,089,747 (issued May 16, 1978 to 
Bruschi) and 4,258,001 (issued Mar. 24, 1981 to Pierce et al). 
The rate of reaction of various substrates with peroxidase varies over many 
orders of magnitude. In some instances, where the reaction proceeds 
slowly, a large amount of peroxidase is used to increase the reaction 
rate. However, the use of large amounts of peroxidase to increase the rate 
of reaction cannot be used in certain assays. For example, enzyme 
immunoassays using a peroxidase-labeled ligand analog have become 
important for determining a drug, antigen or other immunologically 
reactive compound. In such assays, a large amount of peroxidase cannot be 
added to increase the enzymatic reaction rate. 
E.P. Publication No. 116,454 (published Aug. 22, 1984) describes an 
immunoassay using peroxidase, an oxidant (e.g., hydrogen peroxide), a 
chemiluminescent substrate and a phenol as a sensitivity enhancer. These 
enhancers allegedly increase the sensitivity of the chemiluminescent assay 
thereby providing a higher quantity of measurable light in the assay. No 
leuco dyes are used in a chemiluminescent assay. There is no suggestion in 
this reference that certain phenols or anilines could act as electron 
transfer agents to increase the rate of oxidation of leuco dyes. 
Chemiluminescent assays have the disadvantages of (1) requiring 
specialized equipment, (2) being excessively sensitive to sample volume 
changes, (3) often requiring relatively high pH, and (4) exhibiting poor 
assay precision. Hence, there are sufficient reasons for avoiding 
chemiluminescent assays if possible. 
Useful assays utilizing triarylimidazole leuco dyes are described in U.S. 
Pat. No. 4,089,747, noted above. However, it has been observed in such 
assays that the oxidation of the leuco dye by peroxidase is relatively 
slow. Large amounts of peroxidase are needed to increase the rate of 
reaction, but using increased amounts of peroxidase is often undesirable 
or impractical for economic reasons or because increased amounts adversely 
affect the assay, e.g. when peroxidase is used as a label in immunoassays. 
Hence, it would be desirable to be able to increase the reaction rate of 
peroxidase catalyzed oxidation of leuco dyes without having to increase 
the amount of peroxidase. 
SUMMARY OF THE INVENTION 
The problems noted above are overcome with an analytical composition 
comprising: peroxidase or a peroxidase-labeled analog of an 
immunologically reactive ligand, a leuco dye which is capable of providing 
a detectable dye in the presence of peroxidase and hydrogen peroxide, and 
a phenol or aniline electron transfer agent which is capable of reacting 
with hydrogen peroxide in the presence of peroxidase to provide an 
intermediate compound which has a higher oxidation potential than the 
leuco dye. 
This invention also provides an analytical element comprising an absorbent 
carrier material containing: peroxidase or a peroxidase-labeled analog of 
an immunologically reactive ligand, a leuco dye which is capable of 
providing a detectable dye in the presence of peroxidase and hydrogen 
peroxide, and a phenol or aniline electron transfer agent which is capable 
of reacting with hydrogen peroxide in the presence of peroxidase to 
provide an intermediate compound which has a higher oxidation potential 
than the leuco dye. 
In a preferred embodiment, an analytical element comprises a support having 
thereon a registration zone and a porous spreading zone, 
the element further comprising, independently in any of the zones, 
peroxidase or a peroxidase-labeled analog of an immunologically reactive 
ligand, a leuco dye which is capable of providing a detectable dye in the 
presence of peroxidase and hydrogen peroxide, and a phenol or aniline 
electron transfer agent which is capable of reacting with hydrogen 
peroxide in the presence of peroxidase to provide an intermediate compound 
which has a higher oxidation potential than the leuco dye. 
A method for the determination of an analyte comprises the steps of: 
A. contacting a sample of a liquid suspected of containing an analyte with: 
peroxidase or a peroxidase-labeled analog of an immunologically reactive 
ligand, 
a leuco dye which is capable of providing a detectable dye in the presence 
of peroxidase and hydrogen peroxide, and 
a phenol or aniline electron transfer agent which is capable of reacting 
with hydrogen peroxide in the presence of peroxidase to provide an 
intermediate compound which has a higher oxidation potential than the 
leuco dye, and 
B. determining the detectable dye as a result of the presence of the 
analyte. 
The present invention provides a means for increasing the reaction rate of 
leuco dye oxidation by peroxidase catalysis when used in combination with 
a leuco dye. This advantage is achieved by using certain phenols or 
anilines with peroxidase and the leuco dye in a solution or dry assay. 
These phenols and anilines must be capable of acting as electron transfer 
agents, i.e. capable of reacting with hydrogen peroxide in the presence of 
peroxidase to provide an intermediate compound which has a higher 
oxidation potential than the leuco dye. This invention combines the 
advantages obtained with use of leuco dyes which provide high extinction 
dyes with the advantage of very rapid reaction rates.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to the determination (qualitative or 
quantitative measurement) of a chemical or biological substance, termed an 
analyte herein, in aqueous liquids. In particular, the invention can be 
used to assay biological fluids of either animals or humans. Such fluids 
include, but are not limited to, whole blood, plasma, sera, lymph, bile, 
urine, spinal fluid, sputum, perspiration and the like as well as stool 
secretions. It is also possible to assay fluid preparations of human or 
animal tissue such as skeletal muscle, heart, kidney, lungs, brains, bone 
marrow, skin and the like. 
Hydrogen peroxide can be determined with this present invention. In 
addition, the invention can be used to determine analytes which are 
capable of producing hydrogen peroxide, i.e. they can participate in one 
or more reactions which produce hydrogen peroxide in the presence of 
suitable interactive compositions. Analytes which can be determined in 
this manner include glucose, triglycerides, uric acid, cholesterol, 
galactose, amino acids, creatine kinase, pyruvate, and others known to one 
skilled in the art. This invention is particularly useful for the 
determination of glucose, triglycerides, uric acid, cholesterol and 
creatine kinase. 
In a preferred embodiment, the present invention is useful for the 
determination of an immunologically reactive ligand which is a substance 
that will complex specifically with a corresponding receptor. Such ligands 
include, but are not limited to, antigens, haptens, antibodies, toxins, 
hormones, therapeutic drugs, natural and synthetic steroids, proteins, 
viruses, bacteria, peptides, nucleotides, etc. In this embodiment, the 
ligand to be determined and the corresponding labeled ligand analog 
compete for a fixed amount of common reactant. This reactant which 
specifically recognizes the ligand and ligand analog and reacts to form 
complexes with them is referred to herein as the receptor. 
The analytical composition of this invention comprises peroxidase or a 
peroxidase-labeled ligand analog. Either synthetic or naturally occurring 
(i.e. obtained from plant, milk, bacteria and other known sources) 
peroxidases can be used. These materials are generally available 
commercially or readily extracted from available sources. The 
peroxidase-labeled ligand analogs useful in this invention are prepared 
using any suitable technique known to one skilled in the art. Generally, 
they are prepared by covalently binding (with or without a linking group) 
the peroxidase label to the ligand molecule which may be modified in any 
suitable way to achieve the binding. 
Any suitable leuco dye can be used in the practice of this invention as 
long as it is capable of providing a detectable dye when oxidized in the 
presence of peroxidase and hydrogen peroxide. 
Examples of useful leuco dyes include, but are not limited to, imidazole 
derivatives such as those described in U.S. Pat. No. 4,089,747 (noted 
above) and references noted therein, E.P. Application No. 122,641 
(published Oct. 24, 1984) and Jap. Patent Publication No. 
58(1983)-045,557, and the triarylmethanes described, for example, in U.S. 
Pat. No. 4,670,385 (issued Jun. 2, 1987 to Babb et al). Now U.S. Pat. No. 
4,670,385. 
The triarylimidiazoles of U.S. Pat. No. 4,089,747 are preferred in the 
practice of this invention. These leuco dyes generally have the formula 
##STR1## 
wherein R.sup.1, R.sup.2 and R.sup.3 are independently an organic group 
such that at least one of them is an ortho or para hydroxy-substituted 
aryl group of up to 18 carbon atoms, and the other two groups being chosen 
such that the oxidation potential of the compound is within the range of 
from about -70 mV to about +100 mV as measured by cyclic voltometry 
against a standard calomel electrode using a carbon based electrode. 
Further details of these preferred leuco dyes and the technique of 
measuring the oxidation potential are provided in U.S. Pat. No. 4,089,747, 
noted above. 
Phenols and anilines useful in the practice of this invention are those 
which act as electron transfer agents in the oxidation of the leuco dye to 
provide a detectable dye. These phenols and anilines react with hydrogen 
peroxide in the presence of peroxidase to provide intermediate compounds 
having a higher oxidation potential than the leuco dye used in the assay. 
The reaction of the phenol or aniline, hydrogen peroxide and peroxidase 
must be faster than the reaction of the leuco dye, hydrogen peroxide and 
peroxidase, and the intermediate formed thereby must react rapidly with 
the leuco dye or other slowly oxidized substrates. Preferably, the first 
reaction is at least 2 times faster than the second reaction. 
Therefore, a simple test can be carried out to determine if a given phenol 
or aniline is useful in this invention: the rates of oxidation of leuco 
dye are compared in the presence or absence of a phenol or aniline. If the 
rate in the presence of a phenol or aniline is faster by at least 2 times, 
the phenol or aniline is useful in the practice of this invention. It 
should be understood that a phenol or aniline useful in this invention may 
not be useful with every leuco dye, but a skilled worker in the art can 
readily determine which leuco dye and phenol or aniline are useful in the 
practice of this invention. 
Representative phenols useful in this invention include: p, p'-biphenol 
4'-hydroxyacetanilide, p-methoxyphenol, chlorphenol red, p-cresol, 
m-methoxphenol, vanillin, 4-chloro-3,5-dimethylamino-phenol, homovanillic 
acid, p-hydroxybenzoic acid, p-hydroxyphenylacetic acid, o-methoxyphenol, 
phenol, resorcinol, and methyl-p-hydroxybenzoate. Useful anilines include: 
p-anisidine, 4'-aminoacetanilide, p-hydroxy-N, N-dimethylaniline and 
o-phenylenedi-amine. Phenols and anilines selected from the group 
consisting of p, p'-biphenol 4'-hydroxyacetanilide, p-methoxyphenol, 
p-anisidine and 4'-aminoacetanilide are preferred, and 
4'-hydroxyacetanilide is most preferred. 
As noted above, an interactive composition can be used with the analytical 
composition of this invention in order to determine an analyte other than 
hydrogen peroxide. An interactive composition comprises one or more 
reagents which react with the analyte to produce hydrogen peroxide. 
Suitable interactive compositions are known to one skilled in the art. For 
example, an interactive composition for the determination of uric acid 
includes uricase, and an interactive composition for the determination of 
glucose includes glucose oxidase. An interactive composition for the 
determination of cholesterol includes cholesterol oxidase and cholesterol 
ester hydrolase. 
The analytical composition of this invention can also be used to determine 
an immunologically reactive ligand using a ligand analog which comprises a 
ligand covalently bound to a suitable label. As noted above, in one 
embodiment, that label is peroxidase. In other embodiments, the label is 
an enzyme other than peroxidase e.g. glucose oxidase, galactose oxidase, 
etc. that participates in the conversion of the analyte to hydrogen 
peroxide and the leuco dye to a detectable dye. These ligand analogs can 
be prepared by techniques known to one skilled in the art. Preferably, the 
ligand analog comprises glucose oxidase or peroxidase. 
The analytical composition of this invention can also include other addenda 
commonly included for assays, e.g. buffers, surfactants, etc. in amounts 
known in the art. 
The analytical composition and method of this invention are adaptable to 
both solution and dry assays. In a solution assay for analytes other than 
immunologically reactive ligands, the analytical composition and the 
interactive composition (if included) are contacted and mixed with a 
liquid sample suspected of containing the analyte in a suitable container 
(e.g. test tube, petri dish, beaker, cuvette, etc.). The resulting 
solution may be incubated for a short period of time at a temperature up 
to 40.degree. C., and the detectable dye resulting from the presence of 
the analyte is measured using suitable detection equipment and procedures. 
In an immunoassay of this invention, either the bound (complexed) or 
unbound (uncomplexed) fraction of the labeled ligand analog can be 
measured. Physical separation of bound and unbound ligand analog, if 
desired, can be accomplished with any suitable technique. In a solution 
immunoassay, the ligand analog, appropriate receptor and the liquid sample 
suspected of containing the ligand are mixed in a container as described 
above. After complexation, the sample is evaluated by measuring the bound 
or unbound ligand analog using suitable equipment and procedures. 
In a solution assay, the amount of the leuco dye used will depend upon the 
extinction coefficient of the resulting dye. The appropriate amounts can 
be readily determined. Generally, the leuco dye is present in a 
concentration of at least about 4.times.10.sup.-7, and preferably from 
about 2.times.10.sup.-6 to about 1.times.10.sup.-4, molar. Similarly, 
peroxidase is present in an amount sufficient to oxidize the leuco dye to 
provide a detectable signal. Generally, peroxidase is present in an amount 
of at least about 10.sup.-13 molar. The advantage of this invention is 
that less peroxidase can be used in many assays because the phenol or 
aniline described herein enhances the reaction rate of the enzyme. The 
amount of phenol or aniline used in the assay can be varied depending upon 
the leuco dye used and the rate of its oxidation to a dye. Generally, 
however, it is present in an amount of at least about 10.sup.-6, and 
preferably from about 10.sup.-4 to about 10.sup.-2, molar. 
In a solution immunoassay, the ligand analog is generally present in a 
concentration of at least about 10.sup.-11, and preferably from about 
10.sup.-10 to about 10.sup.-7, molar. The corresponding receptor (e.g. 
antibodies) are generally present in an amount of at least about 
10.sup.-8, and preferably from about 10.sup.-8 to about 10.sup.-3, molar. 
The method of this invention can also be practiced with a dry analytical 
element. The simplest element can be composed of an absorbent carrier 
material, e.g. a thin sheet of a self-supporting absorbent or bibulous 
material, such as filter paper or strips, which contains the analytical 
composition of this invention. The element can be divided into two or more 
discrete zones with different components of the composition incorporated 
into individual zones of the carrier material. Such elements are known in 
the art as test strips, diagnostic elements, dip sticks, diagnostic agents 
and the like. 
Useful absorbent carrier materials are insoluble and maintain their 
structural integrity when exposed to water or biological fluids such as 
whole blood or serum. Useful elements can be prepared from paper, porous 
particulate structures, porus polymeric films, cellulose, glass fibers, 
woven and nonwoven fabrics (synthetic and nonsynthetic) and the like. 
Useful materials and procedures for making such elements are well known in 
the art as exemplified in U.S. Pat. Nos. 3,092,465 (issued June 4, 1963 to 
Adams et al), 3,802,842 (issued Apr. 9, 1974 to Lange et al), 3,915,647 
(issued Oct. 28, 1975 to Wright), 3,917,453 (issued Nov. 4, 1975 to 
Milligan et al), 3,936,357 (issued Feb. 3, 1976 to Milligan et al), 
4,248,829 (issued Feb. 3, 1981 to Kitajima et al), 4,255,384 (issued Mar. 
10, 1981 Kitajima et al), 4,270,920 (issued June 2, 1981 to Kondo et al) 
and 4,312,834 (issued Jan. 26, 1982 to Vogel et al). 
Preferably, the absorbent carrier material of the dry analytical element of 
this invention is a porous spreading zone. This zone can be 
self-supporting (i.e. composed of a material rigid enough to maintain its 
integrity), but preferably it is carried on a separate support. Such a 
support can be any suitable dimensionally stable, and preferably, 
nonporous and transparent (i.e. radiation transmissive) material which 
transmits electromagnetic radiation of a wavelength between about 200 and 
about 900 nm. A support of choice for a particular element should be 
compatible with the intended mode of detection (transmission or 
reflectance spectroscopy). Useful supports can be prepared from paper, 
metal foils, polystyrene, polyesters [e.g. poly(ethylene terephthalate)], 
polycarbonates, cellulose esters (e.g. cellulose acetate), etc. 
The porous spreading zone can be prepared from any suitable fibrous or 
non-fibrous material or mixtures of either or both as described in U.S. 
Pat. Nos. 4,292,272 (issued Sept. 29, 1981 to Kitajima et al), 3,992,158 
(noted above), 4,258,001 (noted above) and 4,430,436 (issued Feb. 7, 1984 
to Koyama et al) and Japanese Patent Publication 57(1982)-101760 
(published June 24, 1982). It is desirable 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, polymeric strands, etc. 
The elements can have two or more discrete zones, either in the same layer 
or as superimposed layers. At least one of the zones is preferably a 
porous spreading zone. The other zones can be reagent zones or 
registration zones as those zones are known in the art, additional 
spreading zones, radiation-blocking or filter zones, subbing zones, 
barrier zones, etc. The zones are generally in fluid contact with each 
other, meaning that fluids, reagents and reaction products (e.g. color 
dyes) can pass or be transported between superposed regions of adjacent 
zones. Preferably, each zone is a separately coated layer. 
In the elements of this invention, the components of the analytical 
composition are present in amounts which can be varied depending upon the 
same factors mentioned above in relation to solution assays. Generally, 
the leuco dye is present in an amount of at least about 20 mg/m.sup.2. 
Peroxidase is present in an amount of at least about 10 I.U./m.sup.2, and 
the phenol or aniline is present in an amount of at least about 2.5 
mg/m.sup.2. Optimal levels can be readily determined by one skilled in the 
art. The elements of this invention can also contain one or more other 
addenda commonly put in the elements for various manufacturing or 
operational advantages. Such addenda include or surfactants, buffers, 
solvents. hardeners and the like. These materials can be independently 
located in one or more of the zones of the element described above. In 
some embodiments, the phenol or aniline and leuco dye are in the same 
zone. In other embodiments, the phenol or aniline and peroxidase are in 
the same zone. 
In using the element of this invention in an immunoassay, the labeled 
ligand analog and corresponding receptor can be incorporated into the 
element prior to use, or added at the time of the assay. In either case, 
they are generally present in the amounts noted above for the solution 
immunassay. Preferably, both are incorporated into the element prior to 
use. The labeled ligand analog may be incorporated into a separate 
water-soluble zone or layer in order to isolate it from the receptor. Such 
zones are described in copending and commonly assigned U.S. patent 
application Ser. Nos. 884,249 of Eikenberry filed on even date herewith 
and entitled BINDER COMPOSITION AND ANALYTICAL ELEMENT HAVING STABILIZED 
PEROXIDASE IN LAYER CONTAINING THE COMPOSITION, and 884,237 of Columbus et 
al filed on even date herewith and entitled ANALYTICAL ELEMENT HAVING 
WATER-SOLUBLE POLYMERS AND DETERMINATIONS USING SAME. 
The receptor is preferably immobilized within the element prior to use, 
e.g. during manufacture. For example, it can be immobilized within the 
absorbent carrier material of the element. More particularly, it is 
immobilized within the porous spreading zone on a carrier material, such 
as glass or polymeric beads or other particles, resins, fibers and the 
like. One useful carrier material is a microorganism, such as 
Saphylococcus aureus. Alternatively, the porous absorbent carrier material 
can serve as the carrier material for immobilization. 
A variety of different elements, depending on the method of assay, can be 
prepared in accordance with the present invention. Elements can be 
configured in a variety of forms, including elongated tapes of any desired 
width, sheets, slides or chips. 
The assay of this invention can be manual or automated. In general, in 
using the dry elements, analyte determination is made by taking the 
element from a supply roll, chip packet or other source and physically 
contacting it with a sample (e.g. 1-200 .mu.l) of the liquid suspected of 
containing the analyte so that the sample and reagents within the element 
become mixed. Such contact can be accomplished in any suitable manner, 
e.g. dipping or immersing the element into the sample or, preferably, by 
spotting the element by hand or machine with a drop of the sample with a 
suitable dispensing means. 
After sample application, the element is exposed to any conditioning, such 
as incubation, heating or the like, that may be desirable to quicken or 
otherwise facilitate obtaining any test result. 
The immunoassay of this invention is carried out in an element in such a 
manner that a complex is formed between the ligand and ligand analog and 
the receptor. Once complexation has taken place, any suitable separation 
technique can be used to vertically or horizontally separate complexed 
ligand analog from uncomplexed ligand analog. 
In one embodiment, contact of the sample can be accomplished in such a 
manner that complexation of receptor and ligand and substantial horizontal 
separation of uncomplexed and complexed ligand occur during sample 
introduction. This contact can be carried out by hand or with a machine 
using a pipette or other suitable dispensing means to dispense the test 
sample. The sample of liquid can be applied to the element in a number of 
ways to effect horizontal separation. For example, a relatively large 
liquid sample (e.g. up to 200 .mu.l) can be applied slowly (e.g. over at 
least about 5 seconds) in a continuous manner using a suitable dispensing 
means. Alternatively, the sample can be applied in small portions, e.g. as 
a series of two or more droplets (e.g. 0.1 to 1 .mu.l) over a period of 
time (e.g. over at least about 5 seconds). 
In another embodiment, separation can be accomplished by slowly adding a 
wash fluid after the liquid sample has been applied to the element. This 
wash causes uncomplexed materials to move away from the complexed 
materials. 
The amount of ligand in the test sample is then determined by detecting the 
dye formed from oxidation of the leuco dye as a result of the reaction of 
peroxidase and substrate (e.g. change in reflection or transmission 
density). Either complexed or uncomplexed ligand can be determined in this 
manner. 
In one embodiment noted above involving horizontal separation, the 
complexed ligand analog is measured in a finite area in the center of the 
contacted area. The amount of the ligand in the test sample is inversely 
proportional to the amount of ligand analog measured in that finite area. 
Generally, ligand analog measurement is carried out after from about 5 to 
about 500 seconds subsequent to applying the test sample to the element. 
In the examples which follow, illustrating the practice of this invention, 
the materials used were obtained as follows: SURFACTANT 10G surfactant 
from Olin Corporation (Stamford, Conn., U.S.A.), TRITON X-100, X-165 and 
X-200E surfactants from Rohm and Haas (Philadelphia, Pa., U.S.A.), ZONYL 
FSN surfactant from DuPont (Wilmington, Del., U.S.A.), cholesterol oxidase 
from Upjohn Corp. (Kalamazoo, Mich., U.S.A.), cholesterol ester hydrolase 
from Enzyme Development Corp. (New York, N.Y. , U.S.A.), peroxidase from 
Sigma Chemical Co. (St. Louis, Mo., U.S.A.) or Miles Laboratories 
(Elkhart, Ind., U.S.A.), and the remainder either from Eastman Kodak 
Company (Rochester, N.Y., U.S.A.) or prepared using standard procedures 
and readily available starting materials. 
As used in the context of this disclosure and the claims, I.U. represents 
the International Unit for enzyme activity defined as one I.U. being 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 enzyme. 
EXAMPLE 1 
Use of p-Methoxyphenol as Electron Transfer Agent to Increase Peroxidase 
Reaction Rate 
Potassium phosphate buffer (KP) solution (50.mu.molar, pH 6.5) was prepared 
from K.sub.2 HPO.sub.4 (4.36 g/500 ml water) and KH.sub.2 PO.sub.4 (3.41 
g/500 ml water). A surfactant solution was prepared from TRITON X-165 (10% 
by weight) in 50 mmolar KP buffer. 
A solution of 2-(3,5-dimethoxy-4-hydroxy-phenyl) 
-4,5bis(4-dimethylaminophenyl) imidazole leuco dye was prepared as 
follows: TRITON X-165 solution from above (28.6 ml), 50 mmolar KP buffer 
from above (171.4 ml) were purged with nitrogen and 0.2 g of leuco dye was 
added. The resulting mixture was stirred at 25.degree. C. until solution 
was obtained (about 2 hours). This solution was diluted 1:19 with 200 
mmolar KP buffer. 
Test solutions were prepared by adding the reagents to a cuvette in the 
following order: 3 ml of leuco dye solution, 10 .mu.l of 10 mmolar 
hydrogen peroxide in 200 mmolar KP buffer, p-methoxyphenol (various 
concentrations shown in Table I below) and 10 .mu.l of peroxidase solution 
(7.58 mg enzyme having 165 purpurogallin units of activity/mg in 25 ml of 
200 mmolar KP buffer). 
Absorbances were determined at 670 nm using a standard spectrophotometer at 
30.degree. C. The resulting data, shown in Table I below as the change in 
absorbance (.DELTA.A) after 1 minute, indicate an increased rate of leuco 
dye oxidation as catalyzed by peroxidase in the presence of 
p-methoxyphenol (PMP). 
TABLE I 
______________________________________ 
PMP Concentration (Molar) 
.DELTA.A 
______________________________________ 
0 0.344 
3.3 .times. 10.sup.-8 
0.369 
8.3 .times. 10.sup.-8 
0.368 
1.7 .times. 10.sup.-7 
0.400 
3.3 .times. 10.sup.-7 
0.398 
8.3 .times. 10.sup.-7 
0.467 
1.7 .times. 10.sup.-6 
0.560 
3.3 .times. 10.sup.-6 
0.652 
8.3 .times. 10.sup.-6 
1.004 
1.7 .times. 10.sup.-5 
1.240 
3.3 .times. 10.sup.-5 
1.675 
6.6 .times. 10.sup.-5 
2.314 
9.9 .times. 10.sup.-5 
2.516 
______________________________________ 
EXAMPLE 2 
Use of 4'-Hydroxyacetanilide as Electron Transfer Agent in Increasing 
Peroxidase Reaction Rate 
The leuco dye solution used in Example 1 above was also used in this 
example. Test solutions were prepared by adding the reagents to a cuvette 
in the following order: leuco dye solution (3.0 ml), 10 .mu.l of 10 mmolar 
hydrogen peroxide in 200 mmolar KP buffer, 4'-hydroxacetanilide (various 
concentrations shown in Table II below), and 10 ml of peroxidase (3.03 mg 
of enzyme having 165 purpurogallin units/mg in 50 ml of 200 mmolar KP 
buffer). 
Absorbances were determined at 670 nm using a standard spectrophotometer at 
30.degree. C. The resulting data, shown in Table II below as the change in 
absorbance (.DELTA.A) after 1 minute, indicate an increased rate of leuco 
dye oxidation as catalyzed by peroxidase in the presence of 
4'-hydroxyacetanilide (4-HA). 
TABLE II 
______________________________________ 
4-HA Concentration (Molar) 
.DELTA.A 
______________________________________ 
0 0.167 
8.3 .times. 10.sup.-8 
0.178 
1.7 .times. 10.sup.-7 
0.200 
3.3 .times. 10.sup.-7 
0.226 
8.3 .times. 10.sup.-7 
0.265 
1.7 .times. 10.sup.-6 
0.356 
3.3 .times. 10.sup.-6 
0.535 
8.3 .times. 10.sup.-6 
0.831 
1.7 .times. 10.sup.-5 
1.108 
3.3 .times. 10.sup.-5 
1.544 
8.3 .times. 10.sup.-5 
1.900 
1.7 .times. 10.sup.-4 
2.078 
3.3 .times. 10.sup.-4 
2.104 
6.6 .times. 10.sup.-4 
1.743 
1.7 .times. 10.sup.-3 
1.461 
______________________________________ 
EXAMPLE 3 
Use of p, p'-Biphenol as Electron Transfer Agent to Increase the Reaction 
Rate of Peroxidase 
A solution of leuco dye was prepared from the following: 200 mmolar sodium 
phosphate buffer (pH 6.5), 100 mmolar glucose solution, sodium 
dodecylsulfate (5 g in 50 ml buffer), 0.05 g leuco dye in 50 ml of KP 
buffer, 0.05 molar dimedone and 1.25 mmolar hydrogen peroxide. 
Test solutions were prepared by adding the reagents to a cuvette in the 
following order: 790 .mu.l leuco dye solution, 200 .mu.l of peroxidase (10 
purpurogallin units/ml) and p, p'-biphenol (various amounts shown in Table 
III). 
Absorbances were determined at 670 nm in a standard spectrophotometer at 
30.degree. C. The resulting data, shown in Table III as the change in 
absorbance (.DELTA.A) after 1 minute, is the average of 4 readings and 
indicates an increased rate of leuco dye oxidation as catalyzed by 
peroxidase in the presence of p, p'-biphenol. 
TABLE III 
______________________________________ 
-p, -p'-Biphenol 
Concentration (Molar) 
.DELTA.A 
______________________________________ 
0 0.004 
10.sup.-5 0.382 
2 .times. 10.sup.-5 
0.670 
3 .times. 10.sup.-5 
0.886 
5 .times. 10.sup.-5 
1.145 
10.sup.-4 1.406 
______________________________________ 
EXAMPLE 4 
Determination of Digoxin Using an Analytical Element 
This example is taken from copending and commonly assigned U.S. Ser. No. 
884,249 of Eikenberry, noted above. This example illustrates the practice 
of this invention for the determination of digoxin. 
An element of the present invention was prepared having the following 
format and components: 
______________________________________ 
Spread- 
Poly(vinyltoluene- --co- -p- .sub.-t- 
50-300 g/m.sup.2 
ing butylstyrene- --co-meth- 
Layer acrylic acid) beads 
Poly(methacrylate- --co-2- 
1-10 g/m.sup.2 
acrylamido-2-methylpro- 
pane sulfonic acid-co-2- 
acetoacetoxyethyl meth- 
acrylate) adhesive 
Staphylococcus aureus 
0.2-5 g/m.sup.2 
coated with digoxin 
antibodies 
2-(3,5-Dimethoxy-4-hydroxy 
0.01-1 g/m.sup.2 
phenyl)-4,5-bis(4-di- 
methylaminophenyl)imid- 
azole leuco dye 
SURFACTANT 10G surfactant 
0.1-10 g/m.sup.2 
Water- 
Poly(vinyl alcohol) 0.1-10 g/m.sup.2 
Sol- ZONYL FSN surfactant 
0.01-1 g/m.sup.2 
uble Potassium phosphate 0.01-1 g/m.sup.2 
Layer buffer (pH 7) 
Digoxin peroxidase 10.sup.-6 -10.sup.-4 
g/m.sup.2 
conjugate 
Rea- Gelatin (hardened) 1-100 g/m.sup.2 
gent SURFACTANT 10G 0.02-2 g/m.sup.2 
Layer surfactant 
Potassium phosphate 0.05-5 g/m.sup.2 
buffer 
.alpha.-Glycerol phosphate 
200-20,000 
I.U./m.sup.2 
oxidase 
4'-Hydroxyacetanilide 
0.01-1 g/m.sup.2 
/ Poly(ethylene terephthalate) / 
/ Support / 
______________________________________ 
Digoxin was determined using this element in the following manner. A series 
of test samples containing various amounts of the ligand, digoxin, were 
prepared in a buffered solution (pH 7). A 10 .mu.l sample of each test 
sample was applied to the element prior to incubation for about 5 minutes 
at 37.degree. C. At this time, a 10 .mu.l sample of a wash fluid 
containing 100 mmole of .alpha.-glycerol phosphate was applied to the 
element over the area of the spreading layer contacted with the test 
sample to wash uncomplexed ligand analog horizontally away from complexed 
ligand analog, and to initiate the enzymatic reactions which produce a 
detectable dye. Complexed ligand analog was then determined by monitoring 
reflection densities at 670 nm in the center of the spotted area using a 
standard reflectometer. The rate of change in dye density was calculated 
from measurements taken between 60 and 120 seconds into the incubation. 
The Williams-Clapper transform (J. Optical Soc. Am., 43, 595, 1953) was 
used to determine transmission density values from reflectance density 
values. The concentration of digoxin in the test fluid was observed to be 
inversely related to the rate of dye formation. 
EXAMPLE 5 
Determination of Phenytoin Using an Analytical Element 
This example is taken from copending and commonly assigned U.S. Ser. No. 
818,303, filed Jan. 13, 1986 by Danielson et al and entitled LABELED 
HYDANTOIN CONJUGATE AND ITS USE IN ANALYTICAL ELEMENT AND IMMUNOASSAY. 
This example illustrates the use of a phenol in an immunoassay for 
phenytoin. 
An analytical element for the determination of phenytoin was prepared 
having the format and components illustrated below. Phenytoin is also 
known as diphenylhydantoin. 
______________________________________ 
Spreading 
Polystyrene Beads (5-20 .mu.m) 
25-180 g/m.sup.2 
Layer coated with normal rabbit 
serum 
Poly( -n-butyl acrylate- --co- 
1-18 g/m.sup.2 
styrene- --co-2-acrylamido 
2-methylpropane sulfonic 
acid, sodium salt) 
[75:20:5 weight ratio] 
adhesive 
ZONYL FSN surfactant 
0.1-2.5 g/m.sup.2 
S. aureus coated with 
2-20 g/m.sup.2 
phenytoin anti-serum 
Interlayer 
Gelatin (hardened) 
1-20 g/m.sup.2 
ZONYL FSN surfactant 
0.1-2.5 g/m.sup.2 
Reagent Gelatin (hardened) 
2-20 g/m.sup.2 
Layer Leuco Dye* 0.025-0.6 g/m.sup.2 
5,5-dimethyl-1,3-cyclo- 
0.01-0.5 g/m.sup.2 
hexanedione 
Glucose 0.9-6 g/m.sup.2 
4'-Hydroxyacetanilide 
0.01-0.2 g/m.sup.2 
Sodium dodecyl sulfate 
0.5-10 g/m.sup.2 
Peroxidase 1,000-50,000 
I.U./m.sup.2 
/ Poly(ethylene terephthalate) 
/ 
/ Support / 
______________________________________ 
*4,5-bis(4-dimethylaminophenyl)-2-(4-hydroxy-3,5-dimethoxyphenyl)imidazol 
 
A series of test samples containing various amounts of phenytoin were 
prepared in a buffered solution comprising 0.01 molar 3-(N-morpholino) 
propanesulfonic acid buffer (pH 7), 0.15 molar sodium chloride and 0.05% 
rabbit gamma globulin. The concentrations of phenytoin in the test samples 
are listed in Table IV below. 
The following labeled conjugate was used in the tests: 5-ethyl, 
5-phenylhydantoin-valerate-glucose oxidase. 
The label was mixed with each test sample and tested by spotting an 8 .mu.l 
sample of the resulting mixture on the element and incubating the element 
for 7 minutes at 37.degree. C. During the incubation, reflectance 
densities were monitored at 670 nm using a reflectometer. The rate of 
change in dye density was calculated from measurements taken between 60 
and 120 seconds into the incubation. The Williams-Clapper transform (J. 
Optical Soc. Am., 43, 595, 1953) was used to determine transmission 
density values from reflectance density values. 
The results are shown in Table IV below. These data show that the observed 
rate, shown as the change in transmission density (D.sub.T) with time, is 
inversely proportional to the concentration of phenytoin in the test 
sample. From dose response curves plotted using these data, a dynamic 
range of 0.155 was obtained. 
TABLE IV 
______________________________________ 
Phenytoin 
Concentration D.sub.T /min. 
(molar) (Invention) 
______________________________________ 
0 0.307 
10.sup.-8 0.289 
10.sup.-7 0.267 
10.sup.-6 0.225 
10.sup.-5 0.188 
10.sup.-4 0.154 
2 .times. 10.sup.-4 
0.152 
______________________________________ 
EXAMPLE 6 
Determination of Cholesterol 
This example illustrates the practice of this invention for the 
determination of cholesterol using an analytical element of the invention. 
This element was prepared having the following components and format: 
______________________________________ 
Spread- 
Barium sulfate 70-140 g/m.sup.2 
ing/ Cellulose acetate 6-12 g/m.sup.2 
Rea- Polyurethane resin 
0.5-1.5 g/m.sup.2 
gent Potassium phosphate 
1-2 g/m.sup.2 
Layer buffer (pH 5.5-6.5) 
l-2 g/m.sup.2 
TRITON X-100 surfactant 
5-11 g/m.sup.2 
Peroxidase 5,000-160,000 
I.U./m.sup.2 
4'-Hydroxyacetanilide 
0.01-1 g/m.sup.2 
Cholesterol oxidase 
2,000-4,000 
I.U./m.sup.2 
Cholesterol ester 1,500-12,000 
I.U./m.sup.2 
hydrolase 
2-(3,5-Dimethoxy-4- 
0.8-3 g/m.sup.2 
hydroxyphenyl)-4,5- 
bis(4-dimethylamino- 
phenyl)imidazole 
5,5-Dimethyl-1,3-cyclo- 
0.2-0.8 g/m.sup.2 
hexanedione 
Sub- Poly(N--isopropylacryl- 
0.2-0.8 g/m.sup.2 
bing amide) 
Layer 
Binder Gelatin (hardened) 
10-25 g/m.sup.2 
Layer Potassium phosphate buffer 
0.1-2 g/m.sup.2 
TRITON X-200E surfactant 
0.005-0.02 g/m.sup.2 
SURFACTANT 10G 0.005-0.01 g/m.sup.2 
surfactant 
/ Poly(ethylene terephthalate) / 
/ Support / 
______________________________________ 
To evaluate this element, a series of cholesterol standards were prepared 
from bovine and human serum, varying in cholesterol concentration from 41 
mg/dl to 876 mg/dl. 
The element was spotted with 10 .mu.l drops of these standards and the 
reflection densities were read at 540 nm and 37.degree. C. using an 
EKTACHEM Clinical Chemistry Analyzer (Eastman Kodak Co., Rochester, N.Y.) 
using standard procedures. The reflection densities (D.sub.R) obtained at 
the various concentrations are shown in Table V below. 
TABLE V 
______________________________________ 
Cholesterol 
Concentration (mg/dl) 
D.sub.R, 540 nm, 37.degree. C. 
______________________________________ 
41.0 0.522 
93.0 0.756 
144.0 0.926 
198.0 1.060 
268.0 1.205 
308.3 1.288 
392.0 1.387 
438.0 1.456 
441.8 1.526 
533.0 1.589 
649.0 1.691 
876.0 1.770 
______________________________________ 
EXAMPLE 7 
Determination of Theophylline 
An analytical element for the determination of theophylline was prepared 
having the following format and composition: 
______________________________________ 
Spreading 
Polystyrene beads 20-140 g/m.sup.2 
Layer Poly( -n-butyl acrylatre --co- 
0.5-12 g/m.sup.2 
styrene- --co-2-acrylamido- 
2-methylpropane sulfonic 
acid, sodium salt) (75:20:5 
weight ratio) adhesive 
ZONYL FSN surfactant 
0.1-0.3 g/m.sup.2 
Interlayer 
Hardened gelatin 1-10 g/m.sup.2 
ZONYL FSN surfactant 
0.05-0.2 g/m.sup.2 
Reagent Hardened gelatin 2-20 g/m.sup.2 
Layer Glucose 0.5-5 g/m.sup.2 
4'-Hydroxyacetanilide 
0.01-1 g/m.sup.2 
Peroxidase 1,000-50,000 
I.U./m.sup.2 
Sodium dodecyl sulfate 
1-8 g/m.sup.2 
4,5-Bis(4-dimethyl amino- 
0.04-0.5 g/m.sup.2 
phenyl)-2-(4-hydroxy- 
3,5-dimethoxyphenyl) 
imidazole 
Dimedone 0.01-0.5 g/m.sup.2 
/ Poly(ethylene terephthalate) 
/ 
/ Support / 
______________________________________ 
A series of solutions containing a theophylline-glucose oxidase conjugate, 
varying in concentration from 0.5.times.10.sup.-8 molar to 
8.times.10.sup.-8 molar, were prepared with theophylline (10.sup.-3 molar) 
and without theophylline. Samples (10 .mu.l) of these solutions were then 
spotted onto a finite area of the spreading layer of the element described 
above. 
After incubation at 37.degree. C. of 2-3 minutes, the reflection density 
was measured in the center of the finite area of each element at 670 nm 
using a modified standard reflectometer. The Williams-Clapper transform 
(J. Optical Soc. Am. 43, 595, 1953) was used to determine transmission 
density values (D.sub.T). 
The resulting data in Table VI below show a difference in rate (D.sub.T 
/min.) between the solutions with and without theophylline. The data 
indicate that this element is useful for the determination of theophylline 
(Theo). 
TABLE VI 
______________________________________ 
Conjugate 
Concentration 
Rate (D.sub.T /min.) 
(.times. 10.sup.-8 Molar) 
No Theo 10.sup.-3 molar Theo 
.DELTA. Rate 
______________________________________ 
0.5 0.025 0.012 0.013 
1.0 0.045 0.020 0.025 
2.0 0.082 0.045 0.037 
4.0 0.162 0.080 0.082 
8.0 0.220 0.155 0.065 
______________________________________ 
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.