Quantitative analysis apparatus and method

A method and apparatus for the quantitative determination of an analyte in a liquid employs a liquid-permeable solid medium defining a liquid flow path. The medium includes a number of reaction-containing reaction zones spaced apart along the flow path and in which reaction occurs with the analyte or an analyte derivative (e.g., a labeled analyte) to result in the formation of a predetermined product. Detector means are employed to detect analyte, analyte derivatives, reactant or predetermined product in the reaction zones, the number of such zones in which such detection occurs indicating the amount of analyte in the liquid.

FIELD OF THE INVENTION 
The invention is in the field of quantitative chemical analysis, and is 
particularly useful in the detection and analysis of small amounts of 
chemical substance in such biological fluids as milk, blood, urine, etc. 
DESCRIPTION OF THE PRIOR ART 
Procedures for quantitatively determining the concentration of chemical 
substances in solutions are legion. Many of these procedures are long and 
tedious, and are highly susceptible to human error. Many procedures 
involve the reaction of the chemical moiety--the analyte--to be detected 
with a reactant to form a product, the procedures including a step of 
determining the amount of reactant that is consumed (e.g., as in 
titrations), or the amount of product that is produced (e.g., as by 
measuring the absorption of light by the product of a chromogenic 
reaction), or as by measuring the amount of the chemical moiety or 
reaction product that can be separated from the solution (e.g., by 
distillation), etc. Some quantitative analysis procedures, such as are 
used in radioimmunoassays, involve competitive reactions between labeled 
analytes (e.g., labeled with radioisotopes of iodine, enzymes, or 
fluorescent, chromogenic or fluorogenic molecules) in known quantities and 
unknown amounts of unlabeled analytes, the amount of analyte in the 
unknown solution being related to the measured radioactivity or other 
property of a specimen resulting from the test after suitably separating 
the reacted or bound analyte from the unreacted or unbound analyte, or 
through properties of the bound and unbound labeled analyte that permit 
them to be distinguished. Many of such procedures involve changes in color 
(as when chemical indicators are employed that respond by color changes to 
differences in hydrogen ion concentration), or in turbidity (as when the 
procedure involves the formation of a solid reaction product). 
Certain analyses involve the passage of a fluid, such as air, through a 
column containing a reactant which may change color upon contact with an 
ingredient of the air. For example, U.S. Pat. No. 3,286,506 describes a 
gas analyzing technique in which a measured amount of gas is passed 
through a glass cartridge containing an indicator, the amount of gas to be 
detected being proportional to the amount of indicator within the column 
that changes color. Similar devices are shown in U.S. Pat. Nos. 3,312,527 
and 3,545,930. 
There is a recurring trend in the field to provide analytical procedures 
which are characterized by speed, simplicity, and by a reduction in the 
vulnerability of such procedures to human error. Simple, rapid tests, for 
example, have been marketed for determining the approximate level of blood 
sugar for diabetics. Such tests, however, often are relatively imprecise. 
It would be highly desirable to provide a quantitative test for chemical 
moieties that on the one hand would be characterized by high sensitivity 
and that yet on the other hand would be characterized by simplicity, 
rapidity and relative freedom from human error. 
SUMMARY OF THE INVENTION 
In one embodiment, the invention provides an apparatus for the quantitative 
analysis of a chemically reactive substance (hereafter referred to as an 
"analyte"), in a carrier fluid such as a liquid. The apparatus includes a 
fluid-permeable solid medium that has a predetermined number of 
successive, spaced reaction zones and which defines a path for fluid flow 
sequentially through such zones. "Fluid" herein is typified as a liquid. 
Predetermined quantities of a reactant are bound to the solid medium in 
such zones and are capable of reaction with the analyte or with an analyte 
derivative, to result in the formation of a predetermined product. The 
apparatus may further include detector means for detecting, in the spaced 
zones, the presence of the analyte or its derivative, the reactant, or the 
predetermined product resulting from the reaction between the analyte or 
its derivative and the reactant. In addition, the apparatus may include 
means for suppressing the detectability of trace amounts of the analyte or 
its derivative, the reactant, or the predetermined product resulting from 
the reaction between the analyte or its derivative and the reactant. 
As used herein, the terms "reactant", "reactive" and the like when used in 
connection with the reaction between the analyte or its derivative and the 
reactant refers to the ability of the reactant to react, by covalent or 
hydrogen bonding or by any other means, with the analyte or its derivative 
to form or result in the formation of a predetermined product. That is, 
such terms are used in their broadest sense as referring to the ability of 
the reactant to in any way act upon, be acted upon, or interact with the 
analyte or analyte derivative in a manner that detectably alters the 
analyte or its derivative, the reactant or both to thereby result in the 
formation of a reaction product. Similarly, "reaction product" means any 
product resulting from the reaction of the analyte or its derivative and 
the reactant and that is detectably different from both. "Analyte 
derivative" means a chemical moiety derived from an analyte, and desirably 
is a tagged or labeled form of the analyte as may be employed in 
analytical procedures involving competing reactions between an analyte and 
its tagged or labeled derivative. 
In the apparatus of the invention, the reactant is bound to the permeable 
solid medium in the successive, spaced zones through which the analyte 
passes. A procedure employing the apparatus may take the form in which the 
analyte or its derivative, as it passes through the reaction zones, 
becomes bound to the reactant and the presence of the analyte or its 
derivative within the reaction zones is detected, as by color change or 
the like. Similarly, in a slightly modified embodiment, the analyte or its 
derivative may react with the reactant to result in the formation of a 
product that itself remains bound in the reaction zones, and the product 
itself is then detected. In these embodiments, one may determine with 
considerable precision the concentration of the analyte by detecting how 
many of the successive reaction zones, beginning with the upstream zone, 
show the presence of the analyte or its derivative, or of the product 
resulting from the reaction between the reactant and the analyte or 
analyte derivative. In another embodiment, the reactant that is bound to 
the permeable solid medium may itself be capable of detection by suitable 
detection means and may be disabled from such detection when reacted with 
an analyte or analyte derivative. In this manner, as the analyte or 
analyte-analyte derivative composition passes through successive reaction 
zones, the reactant in the successive zones is disabled from such 
detection until substantially all of the analyte or analyte-analyte 
derivative composition has been exhausted, while remaining downstream 
reaction zones still contain reactant that can be detected. In a modified 
form, the reaction between the analyte or analyte derivative and the 
reactant may cause the latter to become unbound from the solid medium to 
which it was attached and hence be washed from the successive zones. When 
the analyte or analyte derivative or both has thus been exhausted, 
subsequent or downstream reaction will display reactant that is yet bound 
to the permeable medium and which can be detected. In such embodiments, 
one may count the number of zones in which the reactant has been disabled 
beginning with the upstream zone. 
As used herein, "analyte" refers not only to the particular chemical moiety 
for which analysis is desired, but also to chemical moieties that are 
reaction products of the moiety to be determined with another chemical 
moiety. For example, a biological fluid containing an unknown amount of a 
chemical moiety may be reacted in solution or otherwise with another 
chemical moiety to provide a product, the concentration of which is 
related to the initial concentration of the chemical moiety to be 
measured. The resulting product, then, may become the "analyte" for use in 
the apparatus and method of the invention. Accordingly, "analyte" refers 
to any chemical moiety which is to be measured quantitatively. 
In a preferred embodiment, the invention employs immunochemical reactions 
in which the analyte and the reactant represent different parts of a 
specific ligand-antibody (antiligand) binding pair.

DETAILED DESCRIPTION 
With reference to FIG. 1, a transparent, hollow column of glass or the like 
is designated 12 and has open top and bottom ends (12.1, 12.2). The top 
end (12.1) preferably is flared outwardly as shown at (12.3). A standard 
(14) is provided at the bottom end of the column, and may have a hollow, 
upstanding portion (14.1) into which the bottom portion (12.4) of the 
column may be snugly fitted as by a press fit. The standard includes a 
relatively wide bottom portion (14.2) having a flat, horizontal surface 
(14.3) such as a table. The interior (14.4) of the standard preferably is 
hollow, and an upper wall (14.5) of the standard preferably is provided 
with a breathing aperture (14.6) so as to permit air to escape from the 
column when liquid is poured into the upper end (12.1) of the column. The 
aperture (14.6) may, if desired, be fitted with a loose, porous plug, such 
as a cotton plug, to retard leakage from the device when it has been 
disposed of in a trash container or the like. It may also, if desired, be 
fitted with a flexible tubing which may connect it with a pump (e.g., 
peristaltic, syringe drive withdrawal, etc.) typifying flow control means 
to control the flow rate of the liquid through the assay column. 
Within the column are positioned successive, spaced reaction zones (16, 
16.1, 16.2, 16.3, 16.4 etc.), occupied by a permeable solid medium such as 
beaded agarose, beaded polyacrylamide, porous glass, cellulose or other 
materials permeable to liquid and compatible with the analyte, analyte 
derivative, reactant and detector means. To the medium in the reaction 
zones is bound a reactant, as will be described more fully below. The 
interior of the column, as will now be understood, describes a generally 
vertical liquid flow path, and the permeable solid medium positioned in 
the reaction zones desirably occupies the entire cross-section of the flow 
path. Between the spaced reaction zones are positioned preferably 
nonreactive spacer layers (18, 18.1, 18.2, 18.3 and so on) of a 
liquid-permeable solid medium through which liquid may flow, the spacer 
layers preferably being in intimate contact with the reaction zones. The 
spacer layers desirably are of the same permeable solid medium as the 
reaction zones, and, preferably, spacer layers 18.4, 18.5 are provided at 
the top and bottom of the columns as well so that each reaction zone is 
sandwiched between spacer layers. At its upper end, the column (12) may be 
provided with an aperture (12.5) spaced a given distance above the spacer 
layer (18.4) so as to provide a predetermined volume between the aperture 
and top surface of the spacer layer. In a known manner, as a liquid 
(represented as (19) in the drawing) is poured into the open upper end 
(12.1) of the column, it will occupy the open volume at the top of the 
column and any amount of the liquid in excess of that desired may escape 
outwardly through the aperture (12.5), thereby insuring that no more than 
a given, predetermined amount of the liquid passes downwardly in the 
column. The space (19) may, if desired, be filled or partially filled with 
a porous, nonreactive material such as glass wool or similar material to 
avoid splashing of the liquid within the upper end of the column. 
To the liquid-permeable solid medium within the spaced reaction zones (16, 
16.1, etc.) is bound a reactant that is reactive with a given analyte or 
analyte derivative to form a product, all in accordance with the above 
definitions and as exemplified herein. As a typical example, the reactant 
and analyte may be so chosen that the analyte or its derivative becomes 
chemically attached to the reactant as the solution of analyte or analyte 
and analyte derivative solution (the "test solution") passes downwardly 
through the column, care being taken that the total amount of reactant 
within the column is in excess of that required to so react with the 
expected quantity of analyte and analyte derivative in the solution. After 
the test solution has begun its passage downwardly through the column, a 
wash solution, typically distilled and deionized water, optionally may be 
poured into the open end of the column to further aid the downward passage 
of the test solution through the column. Finally, an indicator or detector 
material that detects the presence of analyte or analyte derivative, 
reaction product or reactant, as by causing a color change within the 
zones (16, 16.1 and so on) may be poured into the upper end of the column. 
As the test solution flows downwardly through the column, predetermined 
amounts of the analyte or its derivative are reacted with or bound to the 
reactant in each such layer until the analyte or its derivative have been 
exhausted from the solution. The concentration of analyte in the solution 
can be determined by merely counting the number of successive zones, 
beginning at the top of the column, that have changed color. In another 
embodiment, the reactant that is bound to the medium in the zones (16, 
16.1 and so on) may be deactivated or disenabled by reaction with the 
analyte or analyte derivative or both, and the detector which is employed 
may determine, as by a color change, the presence of nondisabled reactant. 
In this embodiment, the reactant in successive zones contacted by the test 
solution will be disabled until analyte and analyte derivative have been 
exhausted from the solution. Upon detecting which of the reaction zones 
contain reactant that has not been disabled, one may determine the 
concentration of analyte in the solution by counting the number of zones 
beginning at the top of the column in which reactant is not detected. Of 
course, in this embodiment as in the embodiment set out above, one may 
also count the number of zones beginning with the bottom of the column as 
well. 
Another physical embodiment of the apparatus of the invention is shown in 
FIGS. 2 and 3 in which "wicking", or upward capillary flow of a liquid 
through a strip of filter paper or similar material is employed, the strip 
having successive, spaced reaction zones. In this embodiment, the 
permeable solid medium may take the form of a strip of filter paper, which 
is designated generally as (20) in FIGS. 2 and 3. Within spaced zones 
(20.1) of the filter paper strip is bound a reactant, as above-described, 
the spaced zones being separated by spacer layers or sections (20.2). One 
method of preparing the strip (20) involves binding a reactant to small, 
individual rectangular paper filter pieces, and then alternating these 
pieces, which form the reaction zones, with similar pieces of filter paper 
that do not contain the reactant, the alternating pieces of filter paper 
being held together, for example, by a thin strip of adhesive tape. Other, 
more sophisticated methods of manufacture will be apparent to those 
skilled in the art. 
As shown in FIGS. 2 and 3, the strip (20) of filter paper may be positioned 
in an elongated plastic holder (22) having a generally C-shaped 
cross-section. The bottom of the holder is adapted to receive the end of a 
wick (24) which may consist of twisted strands of cotton or other fibrous 
material. A similar wick (24.1) is received in the upper end of the 
plastic holder. The ends of the wicks (24, 24.1) contact the ends of the 
filter strip (20). As shown in FIG. 2, the upper and lower sections of the 
filter paper strip which come into contact with the wicks (24, 24.1), are 
spacer layers (20.2) so that each reaction zone (20.1) is sandwiched 
between spacer layers (20.2). The filter paper strip and holder are 
adapted for insertion into a test tube (26) or other container so that the 
lower wick (24) contacts the bottom of the test tube and the upper wick 
(24.1) extends out of the test tube and then downwardly toward its bottom, 
all as shown in FIG. 2. A test solution (28) placed in the bottom of the 
test tube (26) is thus caused to flow by capillary action upwardly through 
the length of the filter paper strip, successively contacting the reaction 
zones (20.1) in a manner analogous to the flow of test solution through 
the column depicted in FIG. 1. As will be described more fully below, the 
filter paper strip and holder can be moved from one test tube to another 
so that different solutions can be caused to sequentially flow through its 
length. 
Referring now to FIG. 4, an apparatus of the invention is embodied in a 
disc of permeable, solid medium such as filter paper, porous glass, etc. 
(30). The disc (30) may be placed horizontally in a suitable container 
such as a petri dish. At its center, the disc (30) is provided with a well 
(30.1) to receive a test solution or other solution. Reaction zones, 
spaced radially from the well (30.1), are shown as annular rings (30.2), 
and are separated from one another by spacer layers also in the form of 
annular rings (30.3). Spacer layers preferably form the innermost and 
outermost rings of the disc. The reaction zones (30.2) and the spacer 
layers (30.3) are concentric. Test solution that is admitted to the 
central well (30.1) is thus carried radially outwardly of the well by 
capillary action or by diffusion, aided by centrifugal force if desired, 
the test solution successively passing through the spaced reaction zones 
(30.2). 
FIGS. 5 and 6 show another embodiment of a device of the invention. The 
device includes a filter paper strip (40) similar to that of FIGS. 2 and 3 
and containing spaced reaction zones (40.1) separated by spacer layers 
(40.2). A holder, preferably of plastic, is designated (42) and has a flat 
base (42.1) with upwardly extending legs (42.2, 42.3) carried at its ends. 
The leg (42.2) is provided with an upwardly open well (42.4) into which 
may be inserted the upper end of the filter paper strip (40), care being 
taken that the end (42.5) of the filter paper extends downwardly to the 
floor of the well. The strip of filter paper extends obliquely downwardly 
from the well, and its lower end is captured in a slot (42.6) formed in 
the leg (42.3). In use, the test solution or other solution is placed in 
the well (42.4), and is carried downwardly of the strip by both gravity 
and capillary action, the solution sequentially encountering the spaced 
reaction zones (40.1). 
FIG. 7 shows yet another embodiment of a device of the invention which can 
be used for multiple concurrent tests. The device, designated (50), 
includes a pair of spaced plates (50.1, 50.2). Referring to the right-hand 
portion of FIG. 7, the space between the plates is divided into generally 
vertical channels by means of elongated spacers (52, 52.1). As shown in 
the drawing, the channel (52.2) formed by the spacers has a wide upper 
section and a narrow lower section. The lower section is provided with a 
series of vertically spaced reaction zones (54) comprising a 
liquid-permeable solid medium to which is bound a reactant, the medium 
being any of those described above. Between the reaction zones, are placed 
spacer layers (56), the spacer layers sandwiching between them the 
reaction zones (54). Between the spacers (52, 52.1), at the upper end of 
the channel is placed an elongated vertical divider (52.3) which divides 
the upper portion of the channel into two sections (52.4) and (52.5). A 
plug (58), which may be made of the same material as the spacers, has an 
upper, finger-gripping portion (58.1) and a lower, tapering plug portion 
(58.2) adapted to be inserted in the channel (52.5). The flat surfaces of 
each of the spacers and plug, of course, contact the facing surfaces of 
both glass plates to prevent leakage of material from the channels. 
In use, a solution such as a test solution is poured into the upper end of 
the channels formed by the spacers (52, 52.1), and the plug (58) is then 
inserted to provide an air-tight upper seal in the one channel (52.5). As 
a result, liquid in the other channel (52.4) preferentially flows 
downwardly through the reaction zones and spacer layers. When the liquid 
level in the channel (52.4) falls below the lower end of the spacer 
(52.3), air can bubble upwardly through the channel (52.5), permitting the 
contents of that channel to empty downwardly through the reaction zones as 
well. In this manner, the sequential flow of liquid, first from channel 
(52.4) and then from channel (52.5), is rendered automatic. Preferably, 
one of the plates (e.g., plate 50.1) is transparent so that the results of 
any color change in the reaction zones may readily be observed. The other 
plate (50.2) may be transparent or may be of an opaque white or other 
light color to serve as a background against which color changes can 
readily be seen. 
ANALYTES-REACTANTS 
Analytes that can be detected in accordance with the present invention 
include substantially all chemical substances that are reactive with a 
reactant to form a product, as above discussed. It will be understood that 
the invention is not limited to any particular analyte or reactant, but is 
useful for substantially any analyte-reactant combination. 
Many analytes may be analyzed simply by adapting known chemical reactions 
to the invention. 
For example, carbon dioxide may be analyzed with phenolphthalein at a 
slightly alkaline pH. Calcium ion may be analyzed utilizing a reactant 
comprising calmodulin and mammalian phosphodiesterase or another 
calmodulin-sensitive enzyme (Maekawa and Abe, Biochemical and Biophysical 
Research Communications 97:621 (1980)). Ferrous ion may be analyzed 
utilizing, as a reactant, a ferrocene derivative (Katz, et al, J. Am. 
Chem. Soc. 104:346 (1982)). A large number of additional examples may be 
selected from the list of organic analytical reagents compiled by John H. 
Yoe in Handbook of Chemistry and Physics, p.D 126-129, 57th Edition, 
Robert C. Weast, Ed., CRC Press, Cleveland, 1976, and in other references 
cited therein. 
Typical analyte-reactant pairs selected from the field of organic chemistry 
similarly may be chosen by adapting known chemical reactions to the 
invention. For example, almost any phenol may be analyzed with Gibbs 
Reagent (2,6-dichloro-p-benzoquinone-4-chlorimine) (Dacre, J., Analytical 
Chemistry 43:589 (1971)). A reagent for Indoles is 
p-dimethylaminobenzaldehyde (Fieser and Fieser, Reagents For Organic 
Synthesis, Volume 1, p.273, John Wiley & Sons, Inc., New York, (1967). The 
last-mentioned reference also shows the use of phenylhydrazine as a 
reactant for cortisone and similar steroids, and the use of sulfoacetic 
acid as a reactant in the Liebermann--Burchard test for unsaturated 
sterols. Amino acids and ammonium salts may be analyzed using the reagent 
ninhydrin (indane-1,2,3-trione hydrate) (Pasto, et al Organic Structure 
Determination, p.429, Prentice-Hall, Inc., Englewood Cliffs, N.J. 1969). 
Reducing sugar may be measured with Red Tetrazolium (2,3,5-triphenyl 
-2H-tetrazolium Chloride) (Fieser, Organic Experiments, p.135, Raytheon 
Education Co., Lexington, Mass., 1968). 
Various other analyte reagent pairs may be selected from the field of 
chemistry for adaptation to the invention from such reference works as: 
Schuurs, et al, U.S. Pat. No. 3,654,090 (Method For The Determination Of 
Antigens And Antibodies); Kay, U.S. Pat. No. 3,789,116 (Fluorescent 
Labeled Antibody Reagent); Rubenstein, et al, U.S. Pat. No. 3,817,837 
(Enzyme Amplification Assay); Ling, U.S. Pat. No. 3,867,517 (Direct 
Radioimmunoassay For Antigens And Their Antibodies); Giaever, U.S. Pat. 
No. 3,960,490 (Method And Apparatus For Detecting Immunologic Reactions By 
Diffusion In Gel); Ullman, U.S. Pat. No. 3,996,345 (Fluorescence Quenching 
With Immunological Pairs In Immunoassays); Maggio, U.S. Pat. No. 4,233,402 
(Reagents And Method Employing Channeling); Boguslaski, et al, Canadian 
Patent No. 1,082,577 (Hapten-Cofactor Homogeneous Enzyme Immunoassay); 
Schonfeld, H., Ed., "New Developments in Immunoassays", Antibiotics and 
Chemotherapy, Volume 26, 1979; O'Sullivan, et al, "Enzyme Immunoassays: A 
Review", Annals of Clinical Biochemistry 16:221 (1979); Schuurs, et al, 
Enzyme Immunoassay, Clin. Chim. Acta. 81:1 (1977); Feldmann, et al, Eds., 
First International Symposium On Immunoenzymatic Techniques, INSERM Symp. 
No. 2, North Holland Publishing Co., Amsterdam, 1976; Williams, et al, 
Methods in Immunology and Immunochemistry, Volume 3, Academic Press, New 
York, 1971; and Yalow, et al, J. Clin. Invest. 39:1157 (1960). 
Yet other analyte-reactant pairs may be found in: reference works such as 
Feigl, F., Spot Tests in Inorganic Analysis, 6th edition, Elsevier 
Publishing Co., New York, 1972; Feigl, Fritz, Spot Tests in Organic 
Analysis, 7th edition, Elsevier Publishing Co., New York, 1966; Snell, F. 
and Snell, C., Colorimetric Methods of Analysis, Vols. 1-4AAA, Van 
Nostrand Reinhold Co., New York, 1967-74; and Braibanti, A., Ed. 
Bioenergetics and Thermodynamics: Model Systems--Synthetic and Natural 
Chelates and Macrocycles as Models for Biological and Pharmaceutical 
Studies, D. Reidel Publishing Co., Boston, 1980. 
The references identified above are incorporated herein by reference. 
Of particular importance to the instant invention are those 
analyte-reactant combinations that form specific binding pairs of which 
one is an antibody and in which the other is a ligand to which the 
antibody is specific. 
Such immunochemical reactant pairs are well-known in the art, and a wide 
variety of tests have been devised to detect the presence or quantity or 
both of an analyte, particularly when the analyte is present only in 
exceedingly small concentrations. Reference is made to the 
above-identified patents and publications. 
DETECTORS 
The detectors useful in the invention are capable of detecting the presence 
in the successive reaction zones, of analyte, analyte derivatives, 
reactants, or the predetermined reaction product, all as described above. 
The means of detection may take various forms. In the preferred 
embodiment, detection is signaled by a change of color, or a lack of a 
change of color, in the respective reaction zones of the apparatus. 
However, detection may be signaled by other means as well, such as by 
luminescence or fluorescence of the zones, radioactivity of the zones, 
etc. For many reactions, detection is signaled by a change in pH, and the 
detector may hence take the form of a pH color indicator such as 
phenolphthalein, Nile Blue A, Thymol Blue, and Methyl Violet. In other 
tests, one may detect the presence or absence of the appropriate chemical 
moiety in a reaction zone by observing whether a solid reaction product 
has settled upon the succeeding spacer layer. Various detector mechanisms 
are known to the art, and need not be described in detail. In the 
preferred embodiment, however, which makes use of immunochemical reaction 
between the analyte or the analyte and its derivative and the reactant, 
often very small concentrations of analyte are to be measured and 
accordingly a magnifying or amplifying mechanism may suitably be employed. 
One such mechanism makes use of enzymes to promote the reaction of a 
reaction product with a detector moiety to in turn provide a visual color 
indication. For example, the analyte to be tested may be provided in 
admixture with a known quantity of an analyte-glucose oxidase conjugate as 
an analyte derivative, the spaced, sequential reaction zones of the 
apparatus containing an antibody specific to the analyte. A signal 
generating system, such as horseradish peroxidase bound to the antibody in 
the permeable solid medium in such zones and a chromogenic material such 
as o-dianisidine (added, with glucose, to the test solution) can be 
employed. The addition of the test solution, containing the analyte, the 
analyte-glucose oxidase conjugate, glucose, catalase and o-dianisidine, is 
then flowed through the apparatus, such as the column depicted in FIG. 1. 
The analyte and the analyte-glucose oxidase conjugate compete for binding 
sites on the bound antibody, thereby resulting in a color formation due to 
the reaction of the o-dianisidine with hydrogen peroxide-produced by the 
glucose oxidase-catalyzed reaction of oxygen with glucose. Unreacted 
analyte and analyte-glucose oxidase conjugate flow to successive zones 
until the admixture has been exhausted of analyte and analyte-glucose 
oxidase conjugate. A variety of modifications of this procedure, of 
course, are known to the art. 
Example I 
The chromogen 
5,5'[3-(2-Pyridyl)-1,2,4-triazine-5,6-diyl]bis-2-furansulfonic acid, 
disodium salt, ("Ferene", a trademarked product of Chemical Dynamics 
Corp.) is used for the determination of serum iron in soluble assays 
through measurement of the absorbance at 593 nm, at which wavelength any 
interference from other pigments in the serum should be minimized. It may 
be covalently coupled to useful carrier derivatives through nitration, 
reduction, diazotization and diazonium coupling to proteins (such as 
albumin) immobilized on agarose beads, paper strips or other suitable 
permeable solid medium. This immobilized signal-generating reagent 
(chromogenic chelating agent) is physically arranged in sequential spaced 
layers or bands (reaction zones) through which the test fluid will 
migrate. 
Small columns are prepared from silanized Pasteur pipettes by breaking off 
both ends, attaching a short piece of tubing to the bottom (constricted) 
end and inserting glass wool plugs in the tube bottoms. The columns are 
packed by sequential insertion of layers of agarose-Ferene separated by 
layers of unmodified agarose. Typically, 0.4 ml of a 1:1 agarose 
suspension is applied directly above the support, followed by alternating 
layers of 50 microliters of a 1:1 suspension of agarose-Ferene and 0.2 ml 
of agarose suspension. After each addition to the column, the walls of the 
columns are rinsed with phosphate-buffered saline (PBS) and the solution 
above the gel is allowed to flow into the gel before addition of the next 
layer. 
For use in the assay, the tubing at the bottom of the prepared column is 
connected to a peristaltic pump to control the flow rate of the assay. An 
appropriate dilution of test sample for iron analysis is applied to the 
column. The iron solution ("test" solution) is passed through the assay 
column at controlled flow rates, typically between 10 and 15 minutes for 
complete entrance. When all the solution is into the gel bed, the columns 
are rinsed with water. As the test solution flows through, color develops 
in some of the Ferene-containing reaction zones. The number of colored 
zones resulting is a function of the concentration of iron in the test 
solution. 
Example II 
A. The enzyme cholinesterase reacts with and is inhibited by toxic 
organophosphate and carbamate agents. Cholinesterase and the chromogenic 
sulfhydryl reagent 5,5'-Dithiobis-(2-nitrobenzoic acid) (Ellman's Reagent) 
are immobilized upon agarose beads, which are then assembled into columns 
according to Example I. A test solution (diluted blood serum) is added to 
the column and migrates through the reaction zones following which a 
solution of butyryl-thiocholine iodide is added. The zones retaining 
catalytically active cholinesterase will generate a yellow color through 
reaction of the thiocholine produced by the hydrolytic activity of the 
cholinesterase, with the immobilized Ellman's Reagent. The presence of 
reactive choline-sterase-inhibiting toxin in the test sample will result 
in fewer colored bands, which will be in the downstream end region of the 
column. 
B. Amino acids and other nucleophilic amine compounds are measured by use 
of the chromogenic reagent 2,4-dinitrofluorobenzene (FDNB), which produces 
a yellow product upon reaction. A 0.1 ml aqueous sample, containing about 
0.1-1.0 micromoles of amino analyte, is transferred to a siliconed glass 
vessel. The pH is adjusted to 7.0, if necessary, and two milligrams (25 
micromoles) of NaHCO.sub.3 is added and dissolved. Next is added 0.12 ml 
of 0.15% FDNB in absolute alcohol (1.5 micromoles). This solution is 
prepared fresh shortly before use. After the reaction has neared 
completion, it is analyzed for remaining FDNB (reactant) content by flow 
exposure to the assay system prepared according to Example I. In this 
case, a similar amine-containing analyte is immobilized in the zones in a 
permeable solid medium at a known content (e.g., 0.1-0.25 micromoles per 
reaction zone). After rinsing with 50% ethanol in aqueous solution, the 
number of yellow reaction zones produced and remaining after rinse will be 
inversely related to the amount of analyte in the test sample. 
Example III 
The IgG fraction from rabbit anti-penicilloylbovine gamma globulin was 
partially purified by precipitation with 33% saturated ammonium sulfate. 
The precipitate was redissolved and dialyzed against phosphate buffered 
saline (PBS). This IgG preparation was used for immobilizing antibodies 
onto beaded agarose. The agarose was suspended in dioxane, ten reacted 
with carbonyldiimidazole. After being washed with dioxane, it was 
suspended in water, and then in aqueous borate buffer, pH 9.0. The IgG was 
then added to the activated agarose and the gel suspension stirred by 
rocking at 4.degree. C. for 2 days. After extensive washing with PBS, the 
gel containing immobilized antibody was ready for use in the assay. 
Small columns were prepared from silanized Pasteur pipettes by breaking off 
both ends, attaching short pieces of tubing to the bottom (constricted) 
ends and inserting glass wool plugs in the column bottoms. The columns 
were packed by sequentially inserting alternating layers of agarose-IgG 
separated by layers of unmodified agarose. Typically 0.4 ml of 1:1 agarose 
suspension was applied directly above the plugs, followed by alternating 
layers of 50 microliters of a 1:1 suspension of agarose-IgG (to form the 
reaction zones) and 0.2 ml of agarose suspension (to form spacer layers). 
After each addition, the walls of the columns were rinsed with PBS and the 
solution above the gel was allowed to flow into the gel before addition of 
the next layer. 
For use in an assay, tubing at the bottom of the prepared column was 
connected to a peristaltic pump to control the flow rate of the assay. An 
appropriate dilution of penicilloyl-glucose oxidase ("Pen-GO") (typically 
0.1 microgram Pen-GO in 1 ml PBS) with or without known amounts of the 
analyte (penicilloyl-epsilon amino caproate) (Pen-EAC), was applied to the 
column. The Pen-GO was prepared by reacting penicillin G with glucose 
oxidase in borate buffer, pH 9.0, for 2-3 days at 4.degree. C. The Pen-GO 
solution was passed through the assay column at controlled flow rates, 
typically between 10 and 15 minutes for complete entrance into the gel. 
When all the solution was into the gel bed, a detector solution was added 
to the column. The detector solution was prepared as follows: 0.20 ml of 
horseradish peroxidase (HRP) solution (2 mg/ml), 2 ml of 18% glucose 
solution, 1 ml of 0.2 M phosphate buffer (pH 6.0) and 0.100 ml of 1% 
o-dianisidine was diluted 1:10 in PBS and 1 ml or less was applied to the 
columns at the same flow rate as the previous solutions. Brown color 
developed in some of the reaction zones. Presence of the penicilloyl 
moiety in the Pen-GO solution results in the upper reactive zone or zones 
being lighter in color, with color being generated in zones further down 
the column. 
This Example may be repeated for the analysis of serum albumin (a large 
protein molecule) by replacement of the penicillin-glucose oxidase 
conjugate with an albumin-glucose oxidase conjugate. 
Example IIIA 
Peroxidase-labeled IgG prepared from rabbit antiserum against penicillin 
was immobilized in small strips of filter paper by the method reported in 
Example III. Catalase was bound to other, similar strips of filter paper. 
The first and second mentioned strips were then cut into rectangular 
shapes to provide, respectively, reaction zones and spacer layers. The 
small rectangular pieces of filter paper were then layed onto a strip of 
adhesive tape, alternating the reaction zones and spacer layers with edges 
of the sequential pieces of paper overlapping or at least touching one 
another to provide a continuous capillary flow path. 
Penicilloyl-glucose oxidase ("Pen-GO") in a solution of Human Serum Albumin 
("HSA") was freeze-dried inside a test tube. Within another test tube, 
made of brown glass for protecting the contents from light, was 
freeze-dried a solution of o-dianisidine and glucose in phosphate-buffered 
saline at pH 6.0. 
A short wick was attached to the bottom of the prepared filter paper strip 
described above, and a longer wick was placed in contact with the upper 
end of the strip. The strips themselves can be stored under refrigeration, 
and preferably are retained in a wet condition resulting from the 
preparation described above. 
In one example of use, a test solution consisting of a measured volume of 
milk containing a known concentration of penicillin G is added to the test 
tube containing the freeze-dried Pen-GO, and the tube is shaken gently to 
mix the contents. The filter paper strip is then inserted into the tube 
with its upper, longer wick extending over the lip of the tube and then 
downwardly as shown in FIG. 2. When the entire solution has been taken up 
by the strip (or, alternatively, when the solution reaches an arbitrary 
flow line marked on the upper wick and designated "F" in FIG. 2) the 
filter paper strip is removed from the test tube and is placed in the 
brown glass tube to which previously has been added water to dissolve the 
freeze-dried contents thereof. The latter solution similarly wicks 
upwardly through the filter paper strip, causing color development to 
occur in certain of the reaction zones as determined by the quantity of 
penicillin G in the initial test solution. 
In this example, the penicillin G in the milk and the penicillin of the 
Pen-GO compete for binding sites on the antibodies immobilized in the 
reaction zones of the filter paper strip. Of course, larger concentrations 
of penicillin G in the milk sample cause the penicillin G and the Pen-GO 
to migrate further through the filter paper strip. The presence of Pen-GO 
in any of the zones is indicated through the development of color from the 
reaction of H.sub.2 O.sub.2 with o-dianisidine, the H.sub.2 O.sub.2 being 
formed from the glucose oxidase in the presence of glucose and oxygen, and 
as catalyzed by the peroxidase. The catalase in the spacer layers 
catalyzes the conversion of H.sub.2 O.sub.2 into O.sub.2 and H.sub.2 O, 
and thus prevents migration of H.sub.2 O.sub.2 from one reactive zone to 
another. 
As with each of the apparatuses described herein, the device of this 
example may be calibrated by determining how many of the reaction zones 
become colored as a result of the test procedure. For example, one of the 
reaction zones may change color only when the test solution (e.g., milk) 
contains at least 9 nanograms of analyte (e.g., penicillin G) per ml. For 
a sample of milk containing an unknown concentration of penicillin G, one 
merely counts the number of reaction zones that have changed color to find 
the narrow, defined concentration range within which lies the penicillin G 
concentration. 
Example IV 
Antibody against a polyvalent antigen (e.g., serum albumin) analyte is 
labeled with peroxidase and bound to permeable solid medium according to 
Example III to form reaction zones in a column. Another batch of the same 
or similar antibody is labeled with an enzyme such as glucose oxidase. 
Into the column is poured a test sample containing an unknown amount of 
analyte antigen. Through the column is then flowed the soluble glucose 
oxidase-antibody in the presence of glucose plus catalase plus 
o-dianisidine. The number of colored bands resulting is directly related 
to the amount of analyte antigen in the test sample relative to the 
antigen binding capacity of the antibody zones. In this example, the 
antigen first reacts with the bound antibody and binds to the antibody, 
forming a predetermined product. The latter, in turn, is detected by the 
coupling of the glucose oxidase antibody conjugate to available antigenic 
sites on the antigen followed by the color forming reaction. 
Example V 
An analyte or a derivative thereof (e.g., penicillin-peroxidase) is 
covalently bound to a permeable solid medium according to Example III. An 
enzyme-labeled receptor (e.g., glucose oxidase-antibody against 
penicillin) is prepared and exposed to the immobilized analyte to form the 
specific binding complex (e.g., immune complex). The assay unit is 
assembled according to Example III. Subsequent exposure to a test sample 
containing an unknown amount of analyte is done at elevated temperature 
(e.g., 60.degree. C.) to hasten the attainment of equilibrium through 
competitive binding of the immobilized analyte and analyte in the test 
sample with the enzyme-labeled antibody. Analyte in the test sample under 
such conditions will competitively displace the labeled antibody from the 
immobilized analyte. The number of colored reaction zones resulting from 
the procedure is inversely related to the amount of analyte in the test 
sample. These bands will appear in the terminal or downstream portion of 
the column. 
Example VI 
Three assay columns with 4 reaction zones each were prepared according to 
Example III, except that the top reaction zone was prepared with 75 
microliters of IgG-agarose suspension (1:1) and the lower 3 zones with 50 
microliters. Test samples containing 0, 50 and 200 nanograms Pen-EAC, were 
placed in different columns, with each test sample containing 200 ng 
Pen-GO per ml. Flow time for sample application was 20 min. Application of 
the solution of signal generating reagents produced 2 colored zones with 
the 0 ng Pen-EAC sample, 3 in the 50 and 4 with the 200 ng sample. 
A wider and more precise range of analyte content, of course, may be 
measured by using a larger number of assay zones. 
In a preferred embodiment, only a single pass through the apparatus of a 
single liquid material is required. An analyte may be mixed with an 
analyte derivative, chromogen or other material and flowed through the 
apparatus to yield an appropriate test result. In a further preferred 
embodiment, the apparatus is chemically complete in that it includes all 
reactants and other chemicals necessary or desirable for the quantitative 
analysis of an analyte; that is, all that is required is that the analyte 
in a liquid carrier be flowed through the apparatus. Elements of the 
apparatus that, if combined, would undergo reaction in the absence of the 
analyte may be maintained in different zones. For example, the bottom-most 
layer (20.2) of the strip of FIG. 2 may contain a reactant physically 
separated from reactants in the adjacent reaction zone. When the analyte 
in a carrier liquid is flowed through the layer (20.2), the reactant in 
this layer together with the analyte and carrier liquid is flowed into the 
first reaction zone. If desired, a reactant may be provided in the form of 
a solid and may merely be placed upon the upper layer (18.4) of the column 
of FIG. 1, the reactant being dissolved by and carried with the liquid 
carrier and analyte into the column. 
The above-described embodiments are typified by the following Examples 
VII-IX which also describe and exemplify a preferred format of the 
invention. 
This format requires at least two enzymes, one of which is coupled to an 
analyte to form an analyte derivative and catalyzes a color-forming 
reaction, and another enzyme that is immobilized in reaction zones which 
also contain antibody to the analyte, the latter enzyme providing 
substrate for the color-generating enzyme. In this format, therefore, only 
a single solution which consists of or contains the analyte test sample is 
flowed into or through the solid medium after which color develops in the 
reaction zones, the number of colored zones being directly related to the 
concentration of analyte in the test sample. 
Example VII 
The IgG fraction from rabbit anti-penicilloylbovine serum albumin was 
partially purified by precipitation with 33% saturated ammonium sulfate. 
This protein was coupled to microcrystalline cellulose by reaction of the 
cellulose with carbonyldiimidazole in dioxane, followed by washing and 
then by reaction with the IgG preparation in borate buffer at pH 9.0 at 4 
degrees C. for two days. The cellulose was then washed extensively with 
PBS and used for preparation of banded strips. Glucose oxidase was also 
coupled to microcrystalline cellulose in the same manner. A 
penicilloyl-peroxidase was prepared by first coupling a polyacrylamide 
amine to HRP, then reacting penicillin G with that preparation. It is 
believed that the use of a linear polymer as a spacer for attaching the 
hapten to the enzyme allows more hapten molecules to be coupled to each 
enzyme molecule and renders the hapten molecules more accessible for 
binding to anti-body, thus speeding the binding rate. Polyacrylamide was 
synthesized by dissolving 0.5 gm. of acrylamide in 200 ml. of deionized 
water, degassing, then adding 0.2 ml. of 
N,N,N',N'-tetramethylethylenediamine and 0.15 gm. of ammonium persulfate. 
This solution was mixed, then allowed to sit at room temperature for 30 
min. then passed through an ultrafiltration membrane, dialyzed vs. 
deionized water and lyophilized. The polyacrylamide was then dissolved in 
1.0 ml. of 0.2 M phosphate buffer at pH 7.7 and 0.3 ml. of 25% 
glutaraldehyde was added. This solution was incubated at 37 degrees C. for 
19 hours after which it was passed through a Sephadex G-25 column to 
remove the excess glutaraldehyde. The void volume fractions which absorbed 
strongly at 230 nm were pooled and added to a solution of 
diaminodipropylamine (0.5 ml. in 2.0 ml. of water) at pH 9.0. This 
solution was allowed to react at 4 degrees C. over night. The reaction 
mixture was then passed through a Sephadex G-150 column and the fractions 
that absorbed significantly at 230 nm. were divided into four pools of 
equal volume, the second of which was coupled to peroxidase (HRP). HRP was 
reacted with 1.25% glutaraldehyde at pH 7.0 for 15 hours at room 
temperature. After passing the reaction mixture through a Sephadex G-25 
column, the HRP-containing fractions were pooled and added to the 
polyacrylamide-diamine preparation, the pH was adjusted to 9.0, and this 
solution was allowed to react at 4 degrees C. overnight. The 
peroxidase-polyacrylamide-diamine was then passed through a Biogel P-100 
column and the void volume fractions were poled and concentrated, then 
reacted with penicillin. Fifty mg. of penicillin G was added to the 
peroxidase-polyacry-lamide-diamine, the pH adjusted to 9.0 and stirred at 
4 degrees C. over night. This preparation was then dialyzed extensively, 
then used for the assay. 
Banded strips were prepared by cutting 0.5.times.8.0 cm strips of a 
polyester film having a hydrophilic surface onto which were glued strips 
of Whatman 3 MM chromatography paper. At one end was glued a 0.5.times.4.0 
cm. long paper strip followed by a 3.5 mm. space. Then three one cm. long 
paper strips were glued onto the Mylar strip with 2.0 mm. spaces between 
them. The paper on the Mylar was wetted with a solution of 0.02% 
o-dianisidine in water. The spaces were then filled in with a suspension 
of microcrystalline cellulose prepared by mixing 50% suspensions of the 
IgG-cellulose and the glucose oxidase-cellulose in a 20:1 ratio. The first 
space was filled with 20 ul. of this suspension and the other three spaces 
each contained 10 ul. These strips were air dried, then stored dry until 
used. 
The strips were developed by placing the end with the longer paper spacer 
into a small vial containing the developing solution. This solution 
contained peroxidase-polyacrylamide-diamine-penicillin (25 ul. of a 0.25 
microgram/ml. solution), glucose (0.3 ml of a 1.125% glucose solution in 
0.2 M phosphate buffer at pH 6.0) and 10 ul. of dilutions of 
penicilloyl-aminocaproic acid (EAC) in water. Under these conditions, pink 
bands could readily be observed after 20-30 min., such that, with no 
penicilloyl-EAC in the developing solution, one band was colored; with 0.4 
micromolar hapten (penicilloyl-EAC), two bands were colored; and with 1.0 
uM penicilloyl-EAC, all three bands were colored. 
If needed or desired, antibody to peroxidase, an HRP-binding lectin or some 
other binder or inactivator of peroxidase can be included in the spacer 
layers for the purpose of improving the sharpness or decisiveness of zone 
color determinations. Furthermore, catalase immobilized in the spacer 
layers may permit more rapid color development in the reaction zones 
without generation of color in the spacer layers. 
Example VIII 
Banded strips are prepared according to Example VII, except that all of the 
components of the assay except the sample to be tested are incorporated 
into the strip. The peroxidase-polyacrylamide-diamine-penicillin is 
dissolved in a solution of between 0.5 and 1.0% gelatin containing 2.5% 
glucose and 0.2 M phosphate buffer at pH 6.0, 0.1 ml. of which is applied 
to the bottom paper strip and dried. In this example, therefore, the user 
has only to dip the strip into a solution suspected of containing the 
analyte, wait for a prescribed time, then read the results by counting the 
number of colored bands on the strip. 
EXAMPLE IX 
Assay columns are prepared according to Example III, except that the 
reaction zones are composed of a mixture of IgG-agarose and glucose 
oxidase-agarose (20:1). Peroxidase-penicillin (as prepared in Example 
VII), glucose, o-dianisidine, and phosphate buffer, stored in dry form, 
are dissolved in 1.0 ml of the test sample which is then added to the 
column and allowed to flow through. The results are read after the 
prescribed time by counting the number of colored bands on the column. The 
reagents added to the analyte test sample can be in the form of a small 
pellet or can be dried onto the under surface of the cap for a small 
vessel used to measure the volume of sample, etc. In the latter case, the 
vessel is filled, the cap placed on top, the vessel inverted a few times 
and the sample is poured into the column. The reagents to be mixed with 
the sample can even be dried onto a small plug that is stored in the top 
of the column, in which case they dissolve when the sample is added to the 
column. 
Various other enzyme pairs can be used for generating color in the reaction 
zones. For example, alkaline phosphatase can be immobilized in the 
reaction zones with beta-galactosidase coupled to the analyte. The use of 
naphthol-beta-D-galactopyranoside-6-phosphate as substrate for the 
alkaline phosphatase results in the generation of 
naphthol-beta-D-galactopyranoside, which is hydroloyzed by 
beta-galactosidase to produce naphthol which in the presence of a 
diazonium salt results in a colored product in the reaction zones. 
The accuracy and reliability of the apparatus of the invention depends to 
some extent upon how readily or easily the generation of color or other 
detectable change in the different reaction zones may be ascertained. A 
reaction zone in the direction of analyte flow desirably should show 
detectable changes only when a significant, minimum quantity of analyte or 
other material being detected has passed through the preceeding reaction 
zone; since the physical nature of the apparatus often does not permit 
reaction to go fully to completion in each such zone, a small "tail" e.g., 
trace, amount of material may flow into successive zones and may be 
marginally detected in such zones to yield readings that are difficult to 
interpret. One may largely avoid this problem, however by several means. 
Detectors may be employed that are sensitive only to minimum 
concentrations of a chemical moiety to be detected. For example, one may 
utilize o-phenylene diamine in place of o-dianisidine as a chromophore in 
the above examples, the former being less sensitive. Another method 
involves the placement in spacer layers or, less desirably, in reaction 
zones, of small quantities of "scavenger" reactants capable of 
immobilizing or deactivating trace amounts of materials", as exemplified 
in Example VII. This enables the sensitivity and operation of the 
apparatus to be tailored as desired to particular analyses. Control of 
sensitivity and reliability also may depend upon the concentration of the 
reactant in the solid reaction zones, and the solubility of materials such 
as the colored product in some analyses. 
While a preferred embodiment of the present invention has been described, 
it should be understood that various changes, adaptations and 
modifications may be made therein without departing from the spirit of the 
invention and the scope of the appended claims.