Method for measurement of liquid samples

In a method for assaying an analyte comprising applying a liquid sample containing an analyte to a spreading layer of a multilayer analysis element comprising a support having laminated thereon at least one reagent layer containing a substrate capable of converting a detectable chemical species upon reaction with the analyte and a liquid sample-spreading layer and then detecting the chemical species, a wetting liquid is applied onto the spreading layer, prior to applying the liquid sample to the spreading layer. A liquid sample having a high analyte content can be measured with high accuracy.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to an improved method for quantitative 
measurement of liquid samples using a dry type procedure (multilayer 
analysis film technology), more particularly it is concerned with a method 
for assaying liquid samples especially effective for the quantitative 
determination of viscous liquid samples such as whole blood that spread 
poorly when the samples are spotted on the multilayer film. 
2. Development of the Invention 
Multilayer analysis films are already known which can be used to determine 
chemical components (especially components that increase the viscosity of 
the system, e.g., glucose, total protein, triglycerides, etc.) contained 
in a liquid sample with ease and with high speed via a dry type procedure. 
For instance, specific examples of such analysis sheets are described in 
Japanese Patent Application (OPI) No. 53888/74 (corresponding to U.S. Pat. 
No. 3,992,158), No. 137192/75 (corresponding to U.S. Pat. No. 3,983,005), 
No. 40191/76 (corresponding to U.S. Pat. No. 4,042,335), No. 3488/77 
(corresponding to U.S. Pat. No. 4,006,403), No. 131786/77 (corresponding 
to U.S. Pat. No. 4,056,898), No. 131089/78 (corresponding to U.S. Pat. No. 
4,144,306), No. 29700/79 (corresponding to U.S. Pat. No. 4,166,093) and 
No. 34298/79 (corresponding to British Pat. No. 2,000,869A) (the term 
"OPI" as used herein refers to a published unexamined Japanese patent 
application), U.S. Pat. Nos. 4,110,079 and 4,132,528, Clinical Chemistry, 
vol. 24, pages 1335 to 1350 (1978), and so on. 
Such multilayer analysis elements have a common format wherein a spreading 
layer capable of spreading a liquid sample, layers containing reagents 
essential to the analysis elements and so on are multi-coated in advance 
on, a support and, upon the actual chemical analyses using these sheets, 
quantitative analysis can be conducted through two basic processes: 
spotting of the sample liquid into the sheet and evaluation of the extent 
of dye formation by a specific reaction using a densitometer. These 
processes are referred to as dry chemical analyses as they do not require 
processes which are indispensable for conventional methods, such as: 
arrangement of test tubes; preparation, volume measurement and addition of 
reagent solutions; accurate weighing-out of samples; and so on. 
The basic structure of the multilayer chemical analysis sheet of the type 
described above comprises a support, a reagent layer and a sample 
spreading layer, which are arranged in this order. 
A liquid drop of a liquid sample dropped onto a spreading layer of the 
analysis sheet is uniformly diffused over the spreading layer and, at the 
same time, permeates into a reagent layer, where an analyte contained in 
the liquid drop is converted into a detectable chemical species (normally 
color formation or a color change occurs). By observation and quantitative 
determination of the degree of color formed or the color change, an 
analyte contained in the liquid sample can be quantitatively assayed. 
A major reason for such dry type analysis procedures being operable with 
good precision is because a liquid sample-spreading layer is arranged to 
supply a liquid sample to a reagent layer at an approximately constant 
volume per unit area. Depending upon the volume of the liquid sample 
applied, this layer acts as a spreader for the liquid sample. In order to 
obtain a spreading layer having excellent capability for spreading a 
liquid sample, a variety of materials have recently been studied and 
developed. In the case of using a liquid sample having high viscosity, 
especially whole blood or preserved blood having a markedly high 
hematocrit value as a liquid sample, however, a satisfactory spreading 
action can be obtained only with difficulty with conventional materials 
for spreading layers, and regions in which measurement was substantially 
impossible were present. Taking into account the fact that detection of a 
disease factor of an abnormally high concentration (also highly viscous) 
is principally practical in the field of clinical examination, it has been 
desired that substantially undetectable regions should be minimized as 
much as possible and the detection range should be broadened as much as 
possible. 
In the prior art, a method in which a highly viscous liquid sample is 
previously diluted to a suitable region prior to analytical operations 
(hereafter simply often referred to as "simple dilution") has been 
adopted. According to this simple dilution method, the degree of dilution 
can be controlled depending upon the viscosity of a liquid sample so that 
it must be theoretically possible to measure an analyte utilizing a 
spreading action of a multilayer analysis film, even though a liquid 
sample having a high vicosity is employed. However, the level 
(concentration of color formed or changed) of a detectable signal which is 
to be formed in a reagent layer is reduced in inverse proportion to the 
degree of dilution as the degree of dilution of a liquid sample becomes 
high, and, as a result, there is the danger that optical reading might be 
inaccurate. 
Furthermore, it is difficult to accurately weigh small quantities of a 
blood sample, particularly having a high hematocrit value and a high 
viscosity; accordingly, error due to dilution tends to be serious. An 
error in volume in collecting a sample is particularly serious when small 
quantities of less than 100 .mu.l are weighed using a micropipet, etc. 
Particularly when an amount of a liquid sample is less than several ten 
times (twenty to fourty) .mu.l, it is no exaggeration to say that 
quantitative weighing would be impossible unless a particular device is 
used and the severest possible attention is paid. Further, in actual 
dilution, a considerable amount of water must be added to a liquid sample 
(for example, dilution of a 5 .mu.l liquid sample using 5 .mu.l of water 
is substantially impossible). 
In addition, it is required that dilution be carried out outside the 
reaction system and, as a result, additional processes are added to the 
measurement procedure. Furthermore, test tubes are required for dilution 
procedures. Moreover, quantitative determination of an analyte is 
impossible unless dilution is so accurate that one can determine the 
degree of dilution. This is because color formed or changed which is 
actually detected is of the thus diluted liquid sample and the absolute 
amount cannot be determined unless the degree of dilution is considered. 
These procedures thus reduce the most advantageous features involved in 
multilayer analysis films which do not require precise measurement of a 
definite volume of a liquid sample and permit simple measurement. It has 
thus been desired to develop a quantitative assay method without impairing 
these advantages of multilayer analysis films. 
One method for improving spreading comprises treating the surface of a 
liquid sample-spreading layer with a surface active agent, thereby 
assisting spreading of a liquid sample when the liquid sample is dropped 
on the spreading layer; such is described in Japanese Patent Application 
(OPI) No. 131786/77 (U.S. Pat. No. 4,050,898) and No. 164356/80. Further, 
a method which comprises physically activating a material for a spreading 
layer by a glow discharge treatment, etc., to thereby assist spreading of 
a liquid sample is described in Japanese Patent Application No. 140532/80 
(OPI No. 66359/82). 
In these methods, however, a liquid sample is applied to a multilayer 
analysis film which is almost completely dried and, as a natural 
consequence, spreading is not rapidly facilitated since the multilayer 
analysis film is firstly wetted and then spreading follows. It is known 
that when surface active agents or highly hygroscopic components such as 
glycerin or the like are incorporated in a spreading layer, the spreading 
property is improved; however, the high water content of multilayer 
analysis materials generally results in the disadvantages of poor reagent 
stability during storage, undesired color formation (fogging during 
storage), etc. Thus, in practice there is a limit for imparting 
hydrophilicity and even in the case that multilayer analysis films having 
provided the thus improved spreading layer are employed, highly viscous 
liquid samples that involve disadvantages such as poor spreading, a 
prolonged period of time for spreading, etc., still exist, and it has been 
desired to develop multilayer analysis films which can cope with any 
liquid sample. 
SUMMARY OF THE INVENTION 
The present invention provides an improved method for measurement which 
eliminates the foregoing disadvantages involved in the prior art described 
above. 
An object of the present invention is thus to provide an improved method 
for measurement of an analyte which can broaden the latitude for 
measurement without impairing the advantages of multilayer analysis films. 
A further object of the present invention is to provide a method for 
measurement in a simple manner in which poor spreading is not encountered 
even when a highly viscous liquid sample is employed.

DETAILED DESCRIPTION OF THE INVENTION 
In a method for measurement of an analyte which comprises applying a liquid 
sample containing the analyte to the spreading layer of a multilayer 
analysis element comprising a support having provided thereon, in 
sequence, at least one reagent layer containing a substance capable of 
converting the analyte to a detectable chemical species, upon reaction 
with the analyte, through measurements using not only visible rays but 
also electron rays, UV rays, X-rays, etc., and a liquid sample-spreading 
layer, and then detecting the detectable chemical species described above, 
the present invention is characterized in that a wetting liquid is applied 
to the spreading layer of the multilayer analysis element prior to 
applying the liquid sample to the multilayer analysis element described 
above. 
The term "detectable chemical species" refers to a detectable signal or 
change that is directly or indirectly indicative of the presence and/or 
concentration of a desired analyte, or a reaction or decomposition product 
of the analyte, e.g., the optical density of a color formed, fluorometric 
density, electromagnetic (including radiation) intensity or a change in 
these densities or intensities, etc. For further details, reference may be 
made to U.S. Pat. No. 3,992,158 and EPC Publication No. 0002963. 
Preferably, the detectable change that is produced is optically 
detectable. 
The term "liquid sample-spreading layer" refers to a constituent layer 
having the capability of spreading a liquid sample which meters and 
distributes a definite volume of an applied liquid sample to a separate 
reagent layer. Hereafter the liquid sample-spreading layer is often simply 
referred to as a spreading layer. Details of the function of a spreading 
layer are described in, e.g., U.S. Pat. No. 4,292,272. 
The term "reagent layer" is used to refer to a layer containing at least 
one component of an interactive composition which, upon interaction with 
an analyte of the sample liquid, produces or releases a detectable 
chemical species. 
The term "wetting liquid" is used herein to refer to a liquid which is 
applied onto a spreading layer of a multilayer analysis film and renders 
the surface and/or interior of the spreading layer wet and to a liquid 
which is miscible at least with the sample liquid. Accordingly, in the 
case that the sample liquid is a body fluid such as blood, urine, 
cerebrospinal fluid, etc., the wetting liquid is a liquid miscible with 
water. That is, the wetting liquid refers to water or a liquid which can 
uniformly wet over the spreading layer of a multilayer analysis element 
and has a low viscosity generally from about 1 to about 50 cps, preferably 
1 to 30 cps, measured at 25.degree. C. (hereafter the same). 
Representative examples of such wetting liquids include (1) water; (2) 
glycerin; (3) alcohols (e.g., methanol, ethanol, propanol, etc.); (4) 
polar solvents (e.g., acetone, tetrahydrofuran, etc.); (5) mixtures of 
(2), (3) or (4) and water; etc. The mixing ratio of (2), (3) or (4) with 
water is at least 0.1 vol%, preferably ranges from 1 to 50 vol% and, more 
preferably ranges from 10 to 50 vol%, based on 100 vol% of water. 
Of these wetting liquids, water, glycerin and a mixture thereof (glycerin 
content, about 10 to about 50 vol%) are particularly preferred. 
The term "wetting" is used herein to refer to a state of a spreading layer 
being uniformly wetted by the wetting liquid described above and in which 
0.1 .mu.l or more preferably 0.1 to 20 .mu.l, more preferably 0.5 to 20 
.mu.l, of the wetting liquid is contained per 1 cm.sup.2 of the spreading 
layer. 
For purposes of improving or accelerating detection reactions (in addition 
to performing a wetting function), the wetting liquid may contain, in 
addition to glycerin, a surface active agent, a water-soluble high 
molecular weight substance such as albumin, etc., water-soluble salts such 
as sodium chloride, calcium chloride, etc., agents for eliminating 
interfering substances such as a buffering agent, a bilirubin dissociation 
agent, a hydrolase, ascorbic acid oxidase, etc. 
In the measurement in accordance with the present invention, an analyte is 
quantitatively determined using a multilayer analysis element (film) 
comprising a support having multi-coated and/or laminated, in sequence, a 
reagent layer and a liquid sample-spreading layer in discrete form, where 
a wetting liquid is firstly applied onto the spreading layer followed by 
applying a liquid sample thereto. Supply of a wetting liquid onto the 
spreading layer prior to the application of the liquid sample renders the 
surface of the spreading layer more wettable and at the same time renders 
the whole of the spreading layer wettable though a spreading action of the 
spreading layer. When a liquid sample is dropped onto the spreading layer 
which has thus been rendered wet, a uniform spreading can be realized in a 
short period of time even in the case of using a highly viscous liquid 
sample that undergoes poor spreading per the prior art. The spreading 
layer per se has the action of uniformly spreading a liquid; however, once 
uniform spreading of the wetting liquid is effected, the spreading layer 
is in a state in which the wetting agent is retained. In this case, the 
physical properties of the spreading layer are not considered to be quite 
the same as when it is in the dry state. Accordingly, it was assumed that 
a liquid sample subsequently applied thereto would likewise be uniformly 
spread. Against this assumption, however, the volume of the liquid sample 
supplied to the reagent layer is approximately constant per unit area, 
irrespective of the amount of the liquid sample supplied. Thus, it was 
found that quite the same function as in the dry state was realized so far 
as the spreading action of the spreading layer was concerned. It was also 
found that not only this spreading action was likewise realized even in 
the case of employing a relatively highly viscous liquid sample but also 
spreading was effected at an extremely rapid speed as compared to the 
spreading action of a multilayer analysis film in the dry state to which 
no wetting liquid is applied. As described above, in the case of 
previously applying the wetting liquid to the spreading layer prior to the 
application of a sample liquid, the poor spreading observed in the prior 
art does not occur. 
According to the method of the present invention, a multilayer analysis 
film is rendered wet at the time of use thereof. The multilayer analysis 
film is stored in a completely dry state until it is actually employed so 
that there is no fear of fogging during storage which is considered to be 
caused by rendering the multilayer analysis film wet. 
As described above, it is assumed that the effects of the present invention 
would be based not only on the fact that the wet state of the spreading 
layer results from supplying a wetting liquid thereto, but also on the 
fact that the spreading rate of a liquid sample would be accelerated and, 
at the same time, a dilution phenomenon as observed in simple dilution 
would occur microscopically at portions of the spreading layer. This is 
because when a liquid sample is applied or dropped onto the thus wetted 
spreading layer, an approximately constant volume of the liquid sample per 
unit area is supplied to a reagent layer provided beneath the spreading 
layer in a uniformly diluted state which corresponds to a lower 
concentration resulting from supplying the wetting liquid. However, such a 
dilution phenomenon accompanied by the supply of a liquid in a constant 
volume which occurs in the method of the present invention is quite 
different from a mere simple dilution. This is supported by the fact that 
the disadvantage with the simple dilution of the prior art that color 
density is reduced as a sample liquid is diluted is not observed, rather, 
a peak in the color density obtained is observed at a certain dilution, 
depending upon the kind of sample liquid, as will later be described in 
detail. 
The present invention relates to an improved method for assaying a liquid 
sample using a multilayer analysis element. If conditions are 
appropriately chosen, it is possible to effect the dilution operation on 
the spreading layer. Thus, in the case of simple dilution, agitation of a 
dilution liquid and a sample liquid in any manner is essential to good 
analytical results, whereas in the present invention, intentional 
agitation is unnecessary, which is a further characteristic feature of the 
present invention. 
In the present specification, the term "wetting liquid ratio" is used to 
refer to the ratio of the amount of the wetting liquid to the amount of 
the sample liquid applied to a multilayer analysis element. Unlike 
ordinary dilution in which a sample liquid is diluted from 2 times to 
several ten (20 or 30) times, the wetting liquid used in accordance with 
the present invention aims at wetting a spreading layer and it is thus 
preferred that the wetting liquid be employed in the same amount as or 
less than that of a sample liquid, that is, the wetting liquid ratio be 
equal to or less than 1. While it is of course within the scope of the 
invention to use a wetting liquid ratio of 1 or more, the wetting liquid 
would fill up an area of the multilayer analysis element which is 
substantially concerned with measurement if the wetting liquid is 
excessively employed, and, as a result, smooth reaction with an analyte 
contained in the sample liquid would be prevented. 
The wetting liquid employed in accordance with the present invention is not 
particularly limited as long as it has a viscosity lower than that of a 
liquid sample applied, does not contain any interfering substance that 
could lead to an error in measurement of an analyte, and has good 
compatibility with the liquid sample. 
Most typical examples of wetting liquids include water; glycerin, an 
alcohol, a polar solvent and a mixture thereof with water. In addition, a 
physiological saline can also be employed. The wetting liquid may further 
contain salts (e.g., sodium chloride, phosphates, calcium chloride), 
buffers (combinations of buffers as described in HANDBOOK OF CHEMISTRY, 
Element II, pages 1312 to 1320 (1966), published by Maruzen Publishing 
Co., Ltd., Tokyo, and Biochemistry, 5, 467 (1966), e.g., Na.sub.2 
HPO.sub.4 -KH.sub.2 PO.sub.4, Na.sub.2 HPO.sub.4 -citric acid, 
tris(hydroxymethyl)aminomethanehydrochloride, etc.), surface active agents 
(e.g., anionic, cationic and nonionic surface active agents); natural or 
synthetic high molecular weight substances such as proteins (e.g., 
albumin); organic or inorganic acids (e.g., citric acid, acetic acid, 
phosphoric acid, hydrochloric acid, tartaric acid, etc); organic or 
inorganic alkalis (e.g., sodium hydroxide, sodium carbonate, sodium 
bicarbonate, ammonia, organic amines such as triethylamine, etc.); 
oxidation-reduction substances (e.g., ascorbic acid, etc.); or the like. 
It is preferred that these additions be soluble in water. 
The wetting liquid primarily functions to wet a developing layer but, if 
necessary or desired, it can also serve additional functions. For example, 
for purposes of further improving analytical accuracy by eliminating 
and/or inhibiting endogenous competing substances or interfering 
substances, a variety of additives can also be added to the wetting 
liquid. The kind of such additives and the actions or functions thereof 
are widely known in conventional analysis of the wet procedure type but 
representative examples thereof are given below: 
TABLE 1 
______________________________________ 
Analyte Additive Object, Function, etc. 
______________________________________ 
Urine pH Buffer A sample is neutralized 
Neutralizing to optimal pH since 
Agent pH widely changes 
depending on the sample. 
Sugar Ascorbic Removal of interfering 
Oxidase material (ascorbic acid) 
Sodium Azide Removal of interfering 
material (catalase) 
Neutral Fats 
Esterase Diffusion of triglyce- 
rides 
Bilirubin Cafein Dissociation of protein- 
Benzoic Acid binding bilirubin and 
improvement in spread- 
ing bilirubin 
Cholesterol 
Cholesterol Improvement in diffusion 
esterase of cholesterol 
Lipase 
Components Protein- Removal of proteins 
in Whole Blood 
removing agent 
Plasma or Serum 
Components Anticoagulant 
Prevention of blood 
in Whole Blood coagulation 
or Plasma 
______________________________________ 
In addition, the following additives may also be incorporated into the 
wetting liquid to improve or assist the function of the wetting liquid as 
given below: 
(1) Surface active agents: Affinity (or wettability) to the spreading layer 
is increased. 
(2) Salts: Permeation of a sample liquid into the spreading layer is 
improved. The addition of salts is particularly effective for blood used 
as a sample liquid since blood is maintained under a physiological 
condition, especially where hemolysis is accompanied by the use of pure 
water. 
(3) Acids or alkalis: Where enzyme activity depends upon the pH of the 
system, the addition of acids or alkalis accelerates or discontinues an 
enzyme reaction(s) by controlling pH of the system. For example, in 
assaying urease for determining the urea content, the sensitivity is 
reduced when acids are previously added to the system; when alkalis are 
previously added, ammonia is released to thereby accelerate an enzyme 
reaction(s). 
(4) Oxidation-reduction substances: Side reactions are prevented. 
The present invention is concerned with an improvement in multilayer 
analysis elements which can broaden the latitude of measurement in a 
simple manner without impairing the advantages of multilayer analysis 
elements, such as high accuracy. In the present invention intentional 
agitation of a wetting liquid and a sample liquid is not required. A major 
object of using a wetting agent is to effect uniform spreading over the 
surface of a multilayer analysis element. By the uniform spreading action 
of a spreading layer of a multilayer analysis element, a wetting agent is 
also uniformly spread. Accordingly, even a wetting liquid need not be 
accurately weighed if conditions are chosen appropriately. Conditions for 
wetting a spreading layer with good reproducibility vary depending upon 
material, structure, layer thickness, surface treatment, etc., for a 
spreading layer as well as properties of a wetting liquid. 
The wetting liquid can be supplied to a spreading layer directly or 
indirectly, e.g., by directly spotting or dropping onto a spreading layer 
(e.g., using a micropipet), by means of indirectly supplying a wetting 
liquid by impregnating a liquid-retaining carrier (e.g., a cotton 
applicator, a filter-paper stick) with a wetting liquid and then pushing 
it onto a spreading layer, by means of immersing a paper strip in a 
wetting liquid to thereby get the stick wet, putting the paper stick on a 
spreading layer and then pressing, or by means of spraying a wetting 
liquid onto a spreading layer, etc. 
The wetting liquid is applied prior to application of a sample liquid to a 
spreading layer. It is preferred that the wetting liquid be applied to a 
spreading layer as close as possible to application of a sample liquid; 
that is, a sample liquid is applied to a spreading layer immediately after 
spreading of the wetting liquid. In the case that the amount of the 
wetting liquid is large, e.g., more than 20 .mu.l, the timing of the 
subsequent application of a sample liquid is particularly important. In 
the case where the wetting liquid is employed in an amount of 5 
.mu.l/cm.sup.2, poor results were obtained when 10 seconds or more passed 
after wetting until a sample liquid was applied, as compared to 
application immediately after wetting. It is generally preferred that the 
time period from wetting to subsequent application of a sample liquid be 
within 30 seconds immediately after the wetting. In the case that the 
wetting liquid is employed in a small amount e.g., less than 1 
.mu.l/cm.sup.2, the time period from wetting to subsequent application of 
a sample liquid is greatly prolonged. Particularly when an aqueous 
component which has low volatility, such as glycerin, etc., is contained 
in a wetting liquid, the time period from wetting to subsequent 
application of a sample liquid is not overyly important. 
The amount of the wetting liquid applied is sufficient as long as the 
wetting liquid renders at least the surface of a spreading layer of a 
multilayer analysis film wet. Accordingly, the amount of the wetting 
liquid is determined depending upon the size and material of a spreading 
layer but it is generally preferred that the wetting liquid be employed in 
an amount ranging from about 0.1 .mu.l/cm.sup.2 to about 10 
.mu.l/cm.sup.2. In case that the amount of sample liquid applied ranges 
from 2 to 10 .mu.l, a particularly preferred amount of the wetting liquid 
is between 0.5 and 15 .mu.l and the volume of the ratio of the wetting 
liquid to the sample liquid (wetting liquid ratio) is between 0.1 and 2, 
preferably between 0.5 and 1, inclusive. In order to improve poor 
spreading and at the same time realize dilution and metering effects, it 
is desired that the whole of a spreading layer be rendered wet. 
In the case that a sample liquid contains an analyte at a markedly high 
concentration or a relatively high viscosity (approximately 20 to 50 cps), 
a wetting liquid is previously supplied in a predetermined amount onto a 
spreading layer so as to provide a 1:1 to 0.1:1, preferably 1:1 to 0.5:1, 
wetting liquid ratio; subsequently a sample liquid is supplied in a 
predetermined amount, for example, 10 .mu.l. Then, the sample liquid thus 
diluted to a desired degree is supplied to a reagent layer beneath the 
spreading layer in an approximately constant amount per a unit area. 
In the case that glucose or cholesterol at a high concentration is detected 
using an oxidase enzyme, there are optimal conditions for the relationship 
between wetting liquid ratio and a detectable chemical species produced in 
a reagent layer, e.g., the color density of a colored substance. According 
to simple dilution conventionally employed, color density becomes small in 
inverse proportion to the degree of dilution as the degree of dilution 
becomes large. To the contrary, such an inversely proportional relation is 
not observed in the present invention; rather, a peak of color density at 
a certain wetting liquid ratio is observed. 
The degree of dilution showing the maximum color density varies depending 
upon the kind of detectable species, i.e., kind of an analyte. For 
example, in measurement of glucose in whole blood, it is observed that a 
system using 1 volume of whole blood and 1 volume of a wetting liquid 
(wetting liquid ratio of 1) provides higher color density than a system 
using 1 volume of whole blood and 0.2 volume of sa wetting liquid (wetting 
liquid ratio of 0.2). An example will be given hereafter. As described 
above, if the amount of wetting liquid is small, uniform spreading of a 
whole blood sample is prevented but dilution alone is effected and poor 
spreading results so that good color formation is obtained only with 
difficulty. If the amount of wetting liquid is excessively large, color 
density is also decreased. This is believed to be because the supply of 
oxygen required for oxidation of an analyte with an oxidase enzyme is 
inhibited. 
The multilayer analysis element (or film) employed in the method of the 
present invention is conventional and essentially composed of three layers 
comprising a support, at least one reagent layer and a spreading layer; a 
typical construction of the multilayer analysis element (or film) is shown 
in, e.g., U.S. Pat. No. 4,292,272, which is hereby incorporated by 
reference. 
A thickness of each of those layers is conventional and appropriately 
chosen by common knowledge in the art. A general guideline is as follows: 
______________________________________ 
reagent layer: 1 to 50 .mu.m 
spreading layer: 50 to 500 .mu.m 
support: 50 .mu.m to 2 mm 
______________________________________ 
Turning firstly to the spreading layer, as long as the spreading layer is 
constructed of materials having the property of spreading a sample liquid, 
there is no particular limit thereon. As paper for the spreading layer, a 
wide variety of paper is employed as long as it is water-permeable. For 
example, filtering paper can be employed and thin, fine filtering paper is 
particularly preferred. Indian paper; Japanese paper such as Broussonetia 
kazinoki, Edgeworthia papyrifera, etc., can also be advantageously 
employed. Not only paper made of natural cellulose but also paper obtained 
by subjecting synthetic high molecular weight substances to paper-making 
to thereby obtain a paper-form and impart water-permeability to the same, 
paper obtained by subjecting a mixture of synthetic high molecular weight 
substance fibers (pulps) and natural pulps, asbestos and glass 
fiber-filtering paper can also be employed as the spreading layer. 
In more detail, as materials that can be used in the spreading layer, 
non-fibrous materials such as polymer filtering membranes--which are known 
membranes for filtering polymers--having a variety of pore sizes, 
cephadex, agarose, dextran, etc.; natural fibers such as pulp, cotton, 
silk, wool, etc.; semi-synthetic fibers such as cellulose esters, viscose 
rayons, etc.; synthetic fibers such as polyamides, polyesters, 
polyolefins, etc.; fibrous inorganic materials, e.g., glass fibers, 
colored glass fibers, asbestos formed into a woven cloth, felt or 
non-woven cloth shape can be employed. 
Typical examples of membrane filters include Microfilter (made by Fuji 
Photo Film Co., Ltd.), Millipore (made by Millipore Corporation), etc. 
These membrane filters generally possess a pore diameter of about 0.2 to 
about 20 .mu.m, preferably 0.3 to 5.0 .mu.m, more preferably 0.5 to 1.2 
.mu.m. 
A wide variety of fabrics can be employed as the reaction layer, and of 
various fabric tissues, plain weave, which is formed by weaving warp and 
weft yarns alternately, is preferably used. As for the warp and weft of 
the plain weave, a desirable count ranges from 20 to 120. Of fabrics 
having the tissue form called a plain weave, cotton fabrics of the types 
named close cloth, canequim, broadcloth and poplin are preferably 
employed. In addition to other natural fibers woven in the same manner as 
the above described cotton fabrics (e.g., kapok, flax, hemp, ramie, silk 
and so on), fabrics obtained by weaving mixed yarns of chemical fiber 
(e.g., viscose rayon, cupro-ammonium rayon, cellulose acetate, vinylon, 
polyethylene terephthalate or so on) and cotton fiber in the same manner 
as in the above described cotton fabrics, and fabrics obtained by weaving 
chemical fiber yarns in the same manner as in the above described cotton 
fabrics can also be employed. 
It is preferred that the porous material possess voids of about 20 to about 
90%, preferably 50 to 90%, while void percentage varies depending upon the 
kind of the porous material, pore size, etc. The term "void" refers to a 
ratio of a space per a unit volume 1 m.sup.3 and is expressed by 
percentage. 
Porous material having various pore sizes of about 0.2 to about 20 .mu.m 
can be appropriately chosen depending upon the kind of an analyte. For 
example, in the case that the analytes are low molecular weight substances 
such as insulin, drugs, etc. (these have a molecular weight of from about 
400 to about 10,000), porous materials having a relatively small pore size 
are preferably used; if analytes have a relatively high molecular weight 
such as immunoglobulins (the molecular weight of which is about 160,000) 
or albumin (the molecular weight of which is about 75,000), porous 
materials having a relatively large pore size are used. 
It is preferred that these fabrics be rendered hydrophilic. 
As examples of processes for rendering fabrics hydrophilic, mention may be 
made of a process in which commercially produced fabrics are washed and 
rinsed thoroughly with water to remove starch and other processing 
materials therefrom and optionally they are further dipped with a 1 to 5% 
aqueous solution of a surface active agent(s); a process in which such a 
surface active agent(s) is incorporated into a fabric in a proportion of 
0.1 to 10% per unit weight of fabric by spraying an aqueous solution of a 
surface active agent(s) onto the fabric to wet the same and then drying; 
etc. 
In another type of process for rendering fabrics hydrophilic, fabrics are 
wet with hydrophilic polymer solutions, which may contain a fine powder(s) 
such as titanium oxide, barium sulfate and the like, and a wetting 
agent(s) such as glycerin, polyethylene glycol and the like, in addition 
to hydrophilic polymers such as gelatin, polyvinyl alcohol and the like, 
and then dried. Hydrophilic polymers are incorporated in fabrics in 
proportion of from about 0.05 to 10% by weight and preferably from about 
0.1 to 5 wt%, per unit weight of fabric. Further details thereon are given 
in Japanese Patent Application (OPI) No. 164356/80 and U.S. Pat. No. 
4,292,272. 
Further when finely divided granules such as dextran, agarose, acrylamides, 
celluloses, etc., are intermixed with these porous materials, especially 
with fibrous porous materials the, water-retaining property of a sample 
liquid is improved. 
The construction of the spreading layer and materials used therefor are 
described in, e.g., Japanese Patent Application OPI Nos. 164356/80 and 
53888/74 and Japanese Patent Application No. 34370/81. 
The spreading layer described about is located as an outermost layer of the 
multilayer analysis film and has a thickness of from about 50 to about 500 
.mu.m, preferably 100 to 200 .mu.m. 
The reagent layer used for an analysis element in accordance with the 
present invention is a layer 1 .mu.m to 100 .mu.m thick of a composition 
prepared by dispersing a reagent for determining the specific component in 
a liquid sample in a hydrophilic binder such as gelatin, polyvinyl 
alcohol, polyvinyl pyrrolidone, agarose, sodium polyvinylbenzenesulfonate, 
etc. For example, a representative composition for the reagent layer for 
assaying glucose in a sample liquid comprises glucose oxidase, peroxidase, 
4-aminoantipyrine and 1,7-dihydroxynaphthalene. Such a reagent layer for 
glucose assay is prepared by forming a layer having a thickness of 10 to 
20 .mu.m from the foregoing 4 components using gelatin as a binder. 
The reagent used for determining an analyte in a liquid sample will be 
self-evident to one skilled in the art and hence is not explained in 
detail in this specification. 
The spreading layer described above is provided by laminating the foregoing 
spreading layer on the reagent layer and then adhering these layers to 
each other or by coating a dispersion of a composition constituting a 
spreading layer followed by drying. 
The reagent layer can be divided into two or more layers. This is because 
stepwise reactions are sometimes more effective in the case of producing a 
detectable species utilizing conjugated reactions. 
As the support, conventional water-impermeable transparent supports 
preferably having a thickness of about 50 .mu.m to about 2 mm, such as 
polyethylene terephthalate films, cellulose esters (cellulose diacetate, 
cellulose triacetate, cellulose acetate propionate, etc.,) films, 
polycarbonate films, polymethyl methacrylate films, etc., and glass sheets 
are conveniently used. Further, as the support, opaque supports such as 
parting papers, etc., prepared by dispersing a pigment such as carbon 
black, titanium oxide, phthalocyanine copper, etc., in the aforesaid 
transparent supports can be used. In this case, after finishing the 
analysis reaction, the support is peeled off prior to performing 
measurement. 
In addition to the foregoing basic layers, various layers can also be 
optionally provided for purposes of assisting the structure and/or 
function of the multilayer analysis element. For example, such additional 
layers include an adhesion layer for improving adhesion between the basic 
layers, a color shielding layer or a light reflection layer for assisting 
measurement, etc. 
As materials for an adhesion layer, hydrophilic polymers used for binders 
of layers for assisting analytical functions such as a color shielding 
layer, a light reflection layer, etc. can be used. Examples of binders 
which can be employed in these layers include natural hydrophilic high 
molecular weight substances such as gelatin, agarose, sodium alginate, 
carboxymethyl cellulose, methyl cellulose, etc.; hydrophilic synthetic 
high molecular weight substances such as polyacrylamide, polyvinyl 
alcohol, polyvinylpyrrolidone, sodium polyacrylate, polyhydroxyethyl 
methacrylate, copolymers containing acrylic acid (e.g., styrene-acrylic 
acid copolymer), copolymers containing maleic acid (e.g., maleic 
anhydride-methyl vinyl ether copolymer), etc. In this case, a liquid 
sample-spreading layer is put on an adhesion layer in a state where a 
hydrophilic polymer of the adhesion layer is half-dried or after the 
hydrophilic polymer is wet with water or with water containing a surface 
active agent; thereafter these layers are pressed under an appropriate 
pressure. The thickness of the adhesion layer ranges from 0.5 to 15 .mu.m, 
preferably 0.5 to 5 .mu.m. 
When the method of the present invention is applied to the thus constructed 
multilayer analysis film, quantitative assay can be performed in a simple 
manner even with a sample liquid containing an analyte having a high 
concentration which was impossible to measure per the prior art, or with a 
sample liquid having high viscosity. According to the method of the 
present invention, results can be obtained with high accuracy. 
Liquid samples which are advantageously applied to the method of the 
present invention are those having high viscosity (more than 20-23 cps) 
for some reason, e.g., whole blood, especially preserved blood, blood 
having a high hematocrit value, blood where coagulation was initiated, 
blood having high protein content, blood where substances for increasing 
viscosity are present (for example, blood having a high content of 
glucose, total protein, triglycerides, etc. ), etc. 
Advantages of the present invention are summarized below: 
(1) Spreading of a liquid sample is improved or spreading of a liquid 
sample that was said to be impossible is made possible. 
(2) The effects of uniform dilution and metering a liquid sample to a 
reagent layer are achieved. 
(3) A container for dilution is unnecessary. 
(4) The method of the present invention can be applied to a sample liquid 
collected from a patient having exceptionally high concentration of an 
analyte, without requiring any special equipment and the latitude of 
measurement is broadened. 
(5) A simple operation in a short period of time is sufficient. 
(6) Dilution can be effected over a wide range. 
Hereinafter, the method of the present invention will be described in 
detail with reference to the examples below. In the examples, the 
multilayer analysis slide employed was prepared in a manner similar to 
Example 2 of Japanese Patent Application OPI No. 164356/80 except that one 
surface of a mixed spun broadcloth of cotton and polyethylene 
terephthalate was subjected to a glow discharge treatment and the thus 
obtained multilayer analysis film was mounted in a slide frame as 
disclosed in Japanese Utility Model Application OPI No. 142454/81. 
EXAMPLE 1 
Glucose was added to blood collected from normal human to prepare a sample 
liquid containing a glucose content of about 650 mg/dl. This sample liquid 
(6 .mu.l) was dropped on the above described slide for assaying whole 
blood. After incubation for 6 minutes, measurement was performed at an 
optical reflection density of 500 nm. The glucose content found was 0.87 
g; a color formed was unstable and reproducibility was poor. 
Next, prior to dropping of the same sample liquid of whole blood, 10 .mu.l 
of a saline solution containing 10% glycerine and 20 mM phosphate buffer 
(pH 7.0) was dropped on the slide and the whole blood sample was dropped 
thereon 5 seconds after. Subsequent measurement operations were repeated 
as described above. Optical reflection density at 500 nm was 0.981; 
uniform color formation was obtained with good reproducibility. 
EXAMPLE 2 
In place of the glycerin aqueous solution used in Example 1, 10 .mu.l of a 
7% aqueous albumin solution was dropped on the spreading layer of the 
above described multilayer analysis slide. Optical reflection density at 
500 nm was 0.968. After repeating the described procedure, good 
reproducibility was obtained. 
EXAMPLE 3 
On a spreading layer of the multilayer analysis slide, 10 .mu.l of a 
wetting liquid consisting of a 10% glycerin aqueous solution containing 
0.9% sodium chloride was dropped. Then, the same procedures of dropping 
and incubation as in Example 1 were performed using preserved whole blood 
containing various concentrations of glucose, i.e., 100, 200, 300, 400, 
500 and 600 mg/dl. Optical reflection densities were measured at 500 nm. 
The results are shown in the FIG. 1 by the line connected with x marks. 
After diluting the thus obtained samples in dilution cells using a wetting 
liquid having the same composition as described above, the respective 
samples thus diluted were dropped onto the spreading layer of the 
multilayer analysis slide. After incubating in the same manner as 
described above, optical reflection densities were measured, respectively. 
The results obtained are shown in FIG. 1 by the line connected with dots. 
As is seen from FIG. 1, the glucose content becomes high in the case of 
performing dilution outside the system; when the glucose content exceeds 
400 mg/dl, the proportional relation between glucose content and optical 
reflection density is lost. That is, according to conventional dilution, 
the results indicate that quantitative assay is impossible with whole 
blood having a glucose content of 400 mg/dl or more. On the contrary, 
according to the method of the present invention a good linear 
relationship was obtained between glucose content and optical reflection 
density even with whole blood showing a glucose content of 400 mg/dl or 
more. From this result, it is understood that the range for measurement is 
broadend in the method of the present invention. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent from one skilled in the 
art that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.