CA1206878A - Quantitative analysis apparatus and method - Google Patents
Quantitative analysis apparatus and methodInfo
- Publication number
- CA1206878A CA1206878A CA000423140A CA423140A CA1206878A CA 1206878 A CA1206878 A CA 1206878A CA 000423140 A CA000423140 A CA 000423140A CA 423140 A CA423140 A CA 423140A CA 1206878 A CA1206878 A CA 1206878A
- Authority
- CA
- Canada
- Prior art keywords
- analyte
- reactant
- reaction zones
- liquid
- derivative
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/805—Test papers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/97—Test strip or test slide
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/805—Optical property
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/81—Tube, bottle, or dipstick
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/815—Test for named compound or class of compounds
Abstract
ABSTRACT
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 reactant-con-taining 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 derivative, 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.
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 reactant-con-taining 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 derivative, 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.
Description
I
QUANTITATIVE ANALYSIS APPARATUS AND METHOD
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 o legion. Many of these procedures are long and lo-pious, and are highly susceptible to human error.
Many procedures involve the reaction Of the chemical moiety - the analyze - 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 (ego as by measuring thy 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 soul-lion (e.g., by distillation), etc. Some quantitative analysis procedures, such as are used in radiomen-assays, involve competitive reactions between labeled analyzes (erg., labeled with radioisotopes of iodine, enzymes, or fluorescent, chromogenic or fluorogenic molecules) in known quantities and unknown amounts of - -:
,;~
12~ 8
QUANTITATIVE ANALYSIS APPARATUS AND METHOD
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 o legion. Many of these procedures are long and lo-pious, and are highly susceptible to human error.
Many procedures involve the reaction Of the chemical moiety - the analyze - 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 (ego as by measuring thy 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 soul-lion (e.g., by distillation), etc. Some quantitative analysis procedures, such as are used in radiomen-assays, involve competitive reactions between labeled analyzes (erg., labeled with radioisotopes of iodine, enzymes, or fluorescent, chromogenic or fluorogenic molecules) in known quantities and unknown amounts of - -:
,;~
12~ 8
- 2 -1 unlabeled analyzes, the amount of analyze in the us-known solution being related to the measured radioac-tivity or other property of a specimen resulting from the test after suitably separating the reacted or bound analyze from the unworked or unbound analyze, or through properties of the bound and unbound labeled analyze that permit them to be distinguished. Many of such procedures involve changes in color (as when chemical indicators are employed that respond by color o 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 no-act ant which may change color upon contact with an ingredient of the air. For example, US. Patent
Certain analyses involve the passage of a fluid, such as air, through a column containing a no-act ant which may change color upon contact with an ingredient of the air. For example, US. Patent
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. Sims liar devices are shown in US. Patents 3,312,527 and 3,545,930~
There is a recurring trend in the field to pro-vise analytical procedures which are characterized by speed, simplicity, and by a reduction in the vulner-ability of such procedures to human error. Simple, rapid tests, for example, have been marketed for de-~ermining 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.
~Z~68~8 n one embodiment, the invention provides an apparatus for the quantitative analysis ox a chemical-lye reactive substance (hereafter referred to as an "annihilate), in a carrier fluid such as a liquid. The apparatus includes a fluid-permeable solid medium that has a predetermined number of successive, spaced react lion zones and which defines a path for fluid flow sequentially through such zones. "Fluid" herein is lo 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 analyze or with an analyze derivative, to result in the formation of a predetermined product. The apparatus may further in-elude detector means for detecting, in the spaced zones, the presence of the analyze or its derivative, the reactant, or the predetermined product resulting from the reaction between the analyze or its derive-live and the reactant. In addition, the apparatus may include means for suppressing the detectability of trace amounts of the analyze or its derivative, the reactant, or the predetermined product resulting from the reaction between the analyze or its derivative and the reactant.
As used herein, the terms "reactants "rear-live" and the like when used in connection with the reaction between the analyze or its derivative and the reactant refers to the ability of the reactant to no-act, by covalent or hydrogen bonding or by any other means, with the analyze 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 anal lyre or analyze derivative in a manner that delectably alters the analyze or its derivative, the reactant or .
~26~;8~8 1 both to thereby result in the formation of a reaction product. Similarly, "reaction product" means any product resulting from the reaction of the analyze or its derivative and the reactant and that is delectably different from both. "Analyze derivative" means a chemical moiety derived from an analyze, and desirably is a tagged or labeled form of the analyze as may be employed in analytical procedures involving competing reactions between an analyze and its tagged or labeled lo derivative.
In the apparatus of the invention, the reactant is bound to the permeable solid medium in the success size, spaced zones through which the analyze passes.
A procedure employing the apparatus may take the form in which the analyze or its derivative, as it passes through the reaction zones, becomes bound to the no-act ant and the presence of the analyze or its derive-live within the reaction zones is detected, as by color change or the like. Similarly, in a slightly modified embodiment, the analyze 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 analyze by detect-in how many of the successive reaction zones, begin-nine with the upstream zone, show the presence of the analyze or its derivative, or of the product resulting from the reaction between the reactant and the analyze or analyze derivative In another embodiment, the reactant that is bound to the permeable solid medium may itself be capable of detection by suitable detect lion means and may be disabled from such detection when reacted with an analyze or analyze derivative.
In this manner, as the analyze or analyte-analyte -` ~Z~;878 1 derivative composition passes through successive react lion zones, the reactant in the successive zones is disabled from such detection until substantially all of the analyze or analyte-analyte derivative compost-lion has been exhausted, while remaining downstream reaction zones still contain reactant that can be de-tooted. In a modified form, the reaction between the analyze or analyze derivative and the reactant may cause the latter to become unbound from the solid lo medium to which it was attached and hence be washed from the successive zones. When the analyze or anal lyre derivative or both has thus been exhausted, sub-sequent 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.
s used herein "analyze" refers not only to the particular chemical moiety for which analysis is desired, but also to chemical moieties that are react lion 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 con-cent ration of the chemical moiety to be measured. Thy resulting product, then, may become the annihilate for use in the apparatus and method of the invention.
Accordingly annihilate refers to any chemical moiety which is to be measured quantitatively.
In a preferred embodiment, the invention em-ploys immunochemical reactions in which the analyze and the reactant represent different parts of a spew cilia ligand-antibody (antiligand) binding pair.
6~78 Figure 1 is a broken-away view, in partial cross-section, showing an apparatus of the invention;
inure 2 is a broken-away view, in partial cross-section, showing another apparatus of the invent lion:
Figure 3 is a cross-sec~ional view taken along line 3-3 of Figure 2 Figure 4 is a plan view of another embodiment lo of an apparatus of the invention;
Figure 5 is a perspective view of yet another test apparatus of the invention;
Figure is a broken-away cross sectional view taken along line 6-6 of Figure 5 and Figure 7 is a perspective, broken-away view of another test apparatus of }he invention.
DETAILED DESCRIPTION
With reference to Figure 1, a transparent, hot-low 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 port lion (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 flay, horizontal surface 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.~) 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 ox ) 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 -` ~Z~687~
1 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 slow rate of the liquid through the assay column.
Within the column are positioned successive, spaced reaction zones (16, 16.1, 16.2, 16.3, eke.), occupied by a permeable solid medium such as beaded agrees, beaded polyacrylamide, porous glass, cell-lo lose or other materials permeable to liquid and come partible with the analyze, analyze 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 Jones 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 sand-wicked 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 ll9) in the drawing) is poured into the open upper end ~12.1~ of the column, it will occupy the open volume at the top 87~
of the column and any amount of the liquid in ox-cuss 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 down-warmly in the column. The space (~) may, if desired, be filled or partially filled with a porous, none-active material such as glass wool or similar material to avoid splashing of the liquid within the upper end of the column.
lo To the liquid permeable solid medium within the spaced reaction zones (16, 16.1, etc.) is bound a no-act ant that is reactive with a given analyze or anal lyre derivative to form a product, all in accordance with the above definitions and as exemplified herein.
As a typical example, the reactant and analyze may be so chosen that the analyze or its derivative becomes chemically attached to the reactant as the solution I of analyze or analyze and analyze 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 analyze and analyze derivative in the solution.
After the test solution has begun its passage down-warmly 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 analyze or analyze derive alive, 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 analyze or its derivative are reacted with or bound to the reactant in each such ~61!378 g 1 layer until the analyze or its derivative have been exhausted from the solution. The concentration of analyze 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 ebbed ~rneht~
another eM~b~me~*, the reactant that is bound to the medium in the zones (16, 16.1 and so on) may be de-activated or disenabled by reaction with the analyze or analyze derivative or both, and the detector which lo is employed may determine, as by a color change, the presence ox nolldisabled reactant. In this embodiment, the reactant in successive zones contacted by the test solution will by disabled until analyze and analyze 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 analyze in the solution by count-in 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 Figures 2 and 3 in which "waking, 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 figures 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) in-voles binding a reactant to small, individual fee-angular paper filter pieces, and then alternating :- ~Z~6~37~3 1 these pieces, which form the reaction zones, with similar pieces of filter paper that do no contain the reactant, the alternating pieces of filter paper being held together, for example, by a thin strip of ad-hesive tape. Other, more sophisticated methods of manufacture will be apparent to those skilled in the art.
As shown in Figures 2 and 3, the strip (20) of filter paper may be positioned in an elongated plastic lo 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 t20). As shown in Figure 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) 20 is sandwiched between spacer layers (aye) The lit-ton 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 a shown in Figure 2. A test solution to placed in the bottom of the test tube (26) is thus caused to flow by cavil-lazy 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 yin Figure 1. As will be described more sully 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 ~L2(~6~78 1 Referring now to Figure 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 putter 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.]), are shown as an-nailer rings ~30.2), and are separated from one another lo 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 centric-gal force if desired, the test solution successively passing through the spaced reaction zones ~30.2).
Figures 5 and 6 show another embodiment of a device of the invention. The device includes a filter paper strip (40) similar to that of Figures 2 and 3 and containing spaced reaction zones (40.1) separated by spacer layers (40.2). A holder, preferably of plastic, is designated t42) 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 down-warmly 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 ~L2~6~7~
1 action, the solution sequentially encountering the spaced reaction zones (40.1).
Figure 7 shows yet another embodiment of a de-vice of the invention which can be used for multiple concurrent tests. The device, designated (50), in eludes a pair of spaced plates (50.1, 50.2). Refer-ring to the right-hand portion of Figure 7, the space between the plates is divided into generally vertical channels by means of elongated spacers ~52, 52.1). As lo 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 t58.2) adapted to be inserted in the channel (5205). 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 in-sorted 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 1 - 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 fig-rid, first from channel (52.4) and then from channel 152.5), 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 lo 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
, Analyzes 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 thaw the invention is no limited Jo any particular analyze or reactant, but is useful for sub-staunchly any analyte-reactant combination.
Many analyzes may be analyzed simply by adapt-in known chemical reactions to the invention.
For example, carbon dioxide may be analyzed with phenolphthalein at a slightly alkaline phi Cal-alum ion may be analyzed utilizing a reactant comprise in calmodulin and mammalian phosphodiesterase or another calmodulin-sensitive enzyme (Mohawk and Abe, Biochemical and Biophys~cal Research Communications 97:621 ~1980)). Ferrous ion may be analyzed utilize in, as a reactant, a foreseen derivative (Katz, et at, J. Am. Chum. Sock 104:346 l1982)). A large number of additional examples may be selected from the list of organic analytical reagents compiled by John H. You in Handbook of Chemistry and Physics, pod 126-129, ....
~6~7~3 1 Thea Edition, Robert C. West, Ed., CRC Press, Clove-land, 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 invent Sheehan Pro example, almost any phenol may be analyzed with Gibbs Reagent (2,6-dichloro-p-benzoquinone-4-chlorimine) (Deere, J., Analytical Chemistry 43:589 (1971)). A reagent for Insoles is p-dimethylamino-lo benzaldehyde (Fuzzier and Fuzzier, Reagents For Organic ; Synthesis, Volume 1, p.273, John Wiley & Sons, Inc., New York, (lg67). The last-mentioned reference also shows the use of phenylhydrazine as a reactant for cortisone and similar steroids, and the use of sulfa-acetic acid as a reactant in the ~iebermann - Burchard test for unsaturated strolls. Amino acids and ammonia us salts may be analyzed using the reagent ninhydrin (indane-1,2,3-trione hydrate) post, et at Structure Determination, p.42g, Prentice-Hall, Inc., 20 Englewood Cliffs, New Jersey, 1969). Reducing sugar may be measured with Red Tetrazolium (2,3,5-triphenyl -2H-tetrazolium Chloride) (Fuzzier, Organic Export-mints, ply, Raytheon Education Co., Lexington, Massachusetts, 1968).
various other analyze reagent pairs may be so-looted from the field of chemistry for adaptation to the invention from such reference works as: Cheerios, et at, US. Patent 3,654,090 ~nzyme-Linked Immune-sorbent Assay); Kay, US. Patent 3,789,116 (Flyers-30 cent Labeled Antibody Reagents); Ruben stein, et Allis. Patent 3,817,837 (Homogeneous Enzyme Immune-assay), Lingo US. Patent 3,867,517 (Radiomen-assay); Giver, US. Patent 3,906,490 (Radial Mooney-diffusion); Pullman, US. Patent 3,996,345 (Flours pence Quenching Homogeneous Immunoassay); Maggie, US.
Patent 4,233,402 (Enzyme Channeling Homogeneous Enzyme - ~206878 Immunoassay); Boguslaski~ et at, Canadian Patent 1,082,577 (Hapten-Cofactor Homogeneous Enzyme Immune-assay); Schonfeld, H., Ed., "New Developments in Immunoassay", Antibiotics and Chemotherapy Volume 26, 1979; O'Sullivan, et at, "Enzyme Immunoassay: A
Review", Annals of Clinical Biochemistry 16:221 (197g);
Cheerios, et at, Enzyme Immunoassay, Olin. Chimp Act.
81:1 (1977); Feldman, et at, Ens., First International Symposium On Immunoenzymatic Techniques, INSERT Swamp.
No. 2, North Holland Publishing Co., Amsterdam, 1976;
Williams, et at, Methods in Immunology and Immune-chemistry, Volume 3, Academic Press, New York, 1971;
and Yule, et at, J. Olin. Invest. 39 1157 (1960).
Yet other analyte-reactant pairs may be found in. reference works such as Feign, I ox Tests in Inorganic Analysis Thea edition, levier Publishing Co., New York 1972; Feign, Fritz, Spot Tests in Organic Analysis, Thea edition, Elsevier Publishing Co., New York, 1966; Swell, F. and Swell, C., Calorimetric Methods of Analysis, Vows. AYE, Van Nostrand Reinhold Co., New York, 1967-74; and Braibanti, A., Ed. Bioener~etics and Thermodynamics: Model Systems - Synthetic and Natural Chelates and Macro cycles as yodels for Biological and Pharmaceutical Studies, D. Rudely Publishing Co., Boston, 1980.
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 of quantity or 7~1 1 both of an analyze, particularly when the analyze is present only in exceedingly small concentrations.
Reference is made to the above-identi~ied patents and publications.
DETECTORS
The detectors useful in the invention are gape-bye of detecting the presence in the successive react lion zones, of analyze, annihilate derivatives, react-ants, or the predetermined reaction product, all as lo 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 flu-rosins of the zones, radioactivity of the zones, etc. For many reactions, detection is signaled by a change in phi and the detector may hence take the form of a pi color indicator such as phenolphthalein, Nile Blue A, Thymol Blue r and Methyl Violet. In other tests, one may detect the presence or absence of the appropriate chemical moiety in a reaction zone by ox-serving whether a solid reaction product has settled upon the succeeding spacer layer. Various detector mechanisms are known to the art, and need not be de-scribed in detail. In the preferred embodiment, how-ever, which makes use of immunochemical reaction be-tweet the analyze or the analyze and its derivative and the reactant, often very small concentrations of analyze are to be measured and accordingly a magnify-in 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 analyze to be tested may be provided in admixture with a known quantity of an analyte-glucose 6!378 1 oxidize conjugate as an analyze derivative the spaced, sequential reaction zones of the apparatus containing an antibody specific to the analyze. A
signal generating system, such as horseradish porks-dBase 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 analyze, the analyte-glucose lo oxidize conjugate, glucose, kettles and o-dianisidine, is then flowed through the apparatus, such as the column depicted in Figure 1. The analyze and the anal lyte-glucose oxidize conjugate compete for binding sites on the bound antibody, thereby resulting in a color formation due to the reaction of the oddness-dine with hydrogen peroxide produced by the glucose oxidize - catalyzed reaction of oxygen with glucose.
Unrequited analyze and analyte-glucose oxidize coinage-gate flow to successive zones until the admixture has been exhausted of analyze and analyte-glucose oxidize conjugate A variety of modifications of this prove-Doria of counsel 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 ("Foreign, a trademarked product of Chemical Dynamics Corp.) is used for the determination of serum iron in soluble assays through measurement of the absorbency at 593nm, at which wavelength any inter furriness from other pigments in the serum should be minimized. It may be covalently coupled to useful carrier derivatives through nitration reduction, diazoti2ation and diazonium coupling to proteins such as albumin) immobilized on agrees beads, paper strips or other suitable permeable solid medium. This imp mobilized signal-generating reagent (chromogenic ~6~78 1 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 silenced 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 both toys. The columns are packed by sequential insertion of layers of agarose-Ferene separated by layers of lo unmodified agrees. Typically, 0.4 ml of a 1:1 age-rose suspension is applied directly above the support, followed by alternating layers of 50 micro liters of a 1:1 suspension of agarose-Ferene and 0.2 ml of agrees suspension. After each addition to the column, the walls of the columns are rinsed with phosphate-buf-freed 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 appear-private dilution of test sample for iron analysis is applied to the column. The iron solution Taoist solution is passed through the assay column at con-trolled flow rates, typically between 10 and 15 mint vies for complete entrance. When all the solution is into the gel bed the columns are rinsed with water.
us 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.
Exhume II
A. The enzyme cholinesterase reacts with and is inhibited by toxic organophosphate and carbamate agents. Cholinesterase and the chromogenic sulfhydryl 6~378 1 reagent 5,5'-Dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) are immobilized upon agrees 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 follow-o b r Al - -to owe ~-~ in which a solution ~-~uty-r-~-lS~ic~K~ e iodide is added. The zones retaining catalytically active cholinesterase will generate a yellow color through reaction of the thiocholine produced by the hydrolytic lo activity of the cholinesterase, with the immobilized Ellman's Reagent. The presence of reactive choline-sterase-inhibiting toxin in tube test sample will no-suit 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 no-agent 2,4-dinitrofluorobenzene (FDNB), which produces a yellow product upon reaction. A 0.1 ml aqueous sample, containing about 0.1-1.0 micro moles of amino analyze, is transferred to a silicone glass Bessel.
The pi is adjusted to 7.0, if necessary, and two milligrams ~25 micro moles) of Nikko is added and dissolved. Next is added 0.12 ml of 0.15~ FDNB in absolute alcohol tl.5 micro moles). 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 analyze is immobilized in the zones in a permeable solid medium at a known con-tent (e.g., 0.1-0.25 micro moles per reaction zone).
After rinsing with 50~ ethanol in aqueous solution, the number of yellow reaction zones produced and no-mining after rinse will be inversely related to the amount of analyze in the test sample.
... .
2~687 Example III
The Gig fraction from rabbit anti-penicilloyl-bovine gamma globulin was partially purified by pro-cipitation with 33~ saturated ammonium sulfate. The precipitate was redissolved and dialyzed against pros-plate buffered saline (PBS). This Gig preparation was used for immobilizing antibodies onto beaded agrees.
The agrees was suspended in Dixon, then reacted with carbonyldiimidazole. After being washed with lo Dixon, it was suspended in water, and then in aqueous borate buffer, pi 9Ø The Gig was then added to the activated agrees and the gel suspension stirred by rocking at 4 C for 2 days. After ox-tensile washing with PBS, the gel containing imp mobilized antibody was ready for use in the assay.
Small columns were prepared from silenced 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 both 20 toys. The columns were packed by sequentially insert-in alternating layers of agarose-IgG separated by layers of unmodified agrees. Typically 0.4 ml of 1:1 agrees suspension was applied directly above the plugs, followed by alternating layers of 50 micro-liters of a 1:1 suspension of agarose-IgG (to form the reaction zones) and 0.2 ml of agrees suspension to form spacer lyres 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 30 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 oxidize ~nPen-GOn) typically 0.1 microgram Pen-GO in 1 ml PBS ? with or without known amounts of the analyze aye 7~3 1 (penicilloyl-epsilon amino copyright) (Punk), was applied to the column. The Pen-GO was prepared by reacting penicillin G with glucose oxidize in borate buffer, pi 9.0, for 2-3 days at 4 C. The Pen-GO
solution was passed through the assay column at con-trolled flow rates, typically between 10 and 15 mint vies 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 pro-lo pared as follows: 0.20 ml of horseradish peroxides (HOP) solution (2 mg/ml), 2 ml of 18% glucose soul-lion, 1 ml of 0.2 phosphate buffer pi 6.0) and 0.100 ml of I 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 20 down the column.
This Example may be repeated for the analysis of serum albumin (a large protein molecule) by no-placement of the penicillin-glucose oxidize conjugate with an albumin-glucose oxidize conjugate.
Example IIIA
Peroxidase-labeled Gig prepared from rabbit antiserum against penicillin was immobilized in small strips of jilter paper by the method reported in En-ample III. Kettles was bound to other, similar strips of filter paper. The first and second men toned strips wore then cut into rectangular shapes to provide, respectively, reaction zones and spacer lay ens. The small rectangular pieces of filter paper were then lazed onto a strip of adhesive tape, alter-noting the reaction zones and spacer layers with edges of the sequential pieces of paper overlapping or at 1 least touching one another to provide a continuous capillary flow path.
Penicilloyl-glucose oxidize ("Ping) in a solution of Human Serum Albumin ("HA") 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 pi 6Ø
A short wick was attached to the bottom of the lo 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 consist tying of a measured volume of milk containing a known concentration of penicillin G is added to the test tube containing the freeze-dried Ping, 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 Figure I. 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 Figure 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 free~e-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 ox penicillin G in the initial test solution.
In this example, the penicillin G in the milk and the penicillin of the Ping compete for binding 6~8 1 sites on the antibodies immobilized in the reaction zones of the jilter paper strip. Of course, larger concentrations of penicillin G in the milk sample cause the penicillin G and the Pen-GO to migrate fur-then through the filter paper strip. 'rho presence ox Pen-GO in any of the zones is indicated through the development of color from the reaction of H202 with o-dianisidine, the H202 being formed from the glucose oxidize in the presence of glucose and oxygen, o and as catalyzed by the peroxides. The kettles in the spacer layers catalyzes the conversion of H202 into 2 and H20, and thus prevents migration of H202 from one reactive zone to another.
As with each of the apparatuses described here-in, 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 exam-pie, one of the reaction zones may change color only when the test solution (e.g., milk) contains at least 9 nanogram of annihilate ego., penicillin G) per moo For a sample of milk containing an unknown concentra-lion of penicillin Go 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.
Antibody against a polyvalent antigen (e.g., serum albumin) analyze is labeled with peroxides 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 oxidize. Into the column is poured a test sample containing an unknown amount of analyze antigen. Through the column is then flowed the soluble glucose oxidase-antibody in the presence .
~6~37~
1 of glucose plus kettles plus o-dianisidine. The numb bier of colored bands resulting is directly related to the amount of analyze antigen in the test sample rota-live 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 de-tooted by the coupling of the glucose oxidize antibody conjugate to available antigenic sites on the antigen lo followed by the color forming reaction.
Example V
An analyze or a derivative thereof (e.g., pent-cillin-peroxidase) is covalently bound to a permeable solid medium according to example III. An enzyme-la-bleed receptor (e.g., glucose oxidase-antibody against penicillin) is prepared and exposed to the immobilized analyze to form the specific binding complex (e.g., immune complex). The assay unit is assembled accord ding to Example III. Subsequent exposure to a test sample containing an unknown amount of analyze is done at elevated temperature (e.g., 60 C) to hasten the attainment of equilibrium through competitive binding of the immobilized analyze and analyze in the test sample with the enzyme-labeled antibody. Analyze in the test sample under such conditions will compete-lively displace the labeled antibody from the iamb-lived analyze. The number of colored reaction zones resulting from the procedure is inversely related to the amount of analyze 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 Jo Example III, except that the top reaction zone was prepared with 75 micro liters of IgG-agarose suspension and the lower 3 zones 1 with 50 micro liters. Test samples containing 0, 50 and 200 nanogram Punk, were placed in different columns, with each test sample containing 200 no Pen GO
per ml. Flow time for sample application was 20 min.
Application of the solution of signal generating no-agents produced 2 colored zones with the O no Punk sample, 3 in the 50 and 4 with the 200 no sample.
A wider and more precise range of analyze con-tent, of course, may be measured by using a larger o number of assay zones.
In a preferred embodiment, only a single pass through the apparatus of a single liquid material is required. An analyze may be mixed with an analyze derivative, chromogen or other material and flowed through the apparatus to yield an appropriate test result. In a further preferred embodiment, the apt pyrites is chemically complete in that it includes all reactants and other chemicals necessary or desirable for the quantitative analysis of an analyze, that is, all that is required is that the analyze in a liquid carrier be flowed through the apparatus. Elements of the apparatus that, if combined, would undergo react lion in the absence of the analyze may be maintained in different zones. For example, the bottom-most layer (20.2) of the strip of Figure 2 may contain a reactant physically separated from reactants in the adjacent reaction zone. When the analyze in a carrier liquid is flowed through the layer (20.2), the react lent in this layer together with the analyze and car-nor liquid is flowed into the first reaction zone.
If desired, a reknit may be provided in the form of a solid and may merely be placed upon the upper layer (18.4) of the column of Figure 1, the reactant being dissolved by and carried with the liquid carrier and analyze into the column.
~68~
1 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 analyze to form an analyze derivative and catalyzes a color-forming reaction, and another enzyme that is immobilized in reaction zones which also contain antibody to the analyze, the latter enzyme providing substrate for the color-generating lo enzyme. In this format, therefore, only a single solution which consists of or contains the analyze 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 analyze in the test sample.
Example VII
The Gig fraction from rabbit anti-penicilloyl-bovine serum albumin was partially purified by precip-ita~ion with 33% saturated ammonium sulfate. This pro-20 loin was coupled to microcrystalline cellulose by no-action of the cellulose with carbonyldiimidazole in dioxanet followed by washing and then by reaction with the Gig preparation in borate buffer at pi 9.0 at 4 degrees C for two days. The cellulose was then washed extensively with PBS and used for preparation of banded strips r Glucose oxidize was also coupled to microcrystalline cellulose in the same manner. A
penicilloyl-peroxidase was prepared by first coupling a polyacrylamide amine to HOP, then reacting pencil-fin G with that preparation. It is believed that these of a linear polymer as a spacer for attaching the hasten to the enzyme allows more hasten molecules to be coupled to each enzyme molecule and renders the hasten molecules more accessible for binding to anti-body, thus speeding the binding rate. Polyacrylamide was synthesized by dissolving 0.5 gym. of acrylamide in I I
200 ml. of deionized water, degas sing, then adding 0.2 ml. of N,N,N',N'-tetramethylethylenediamine and 0.15 gym. of ammonium per sulfate. This solution was mixed, then allowed to it at room temperature for 30 min.
then passed through an ultrafiltration membrane, dialyzed us deionized water and lyophilized. The polyacrylamide was then dissolved in lo ml. of 0.2 M
phosphate buffer at pi 7.7 and 0.3 ml. of 25% gluier-alluded was added. This solution was incubated at 37 degrees C for 19 hours after which it was passed through a Sephadex7'G-25 column to remove the excess glutaraldehyde. The void volume fractions which Abe sorbed strongly at 230 no were pooled and added to a solution of diaminodipropylamine (0.5 ml. in 2.0 ml.
of water) at pi 9Ø 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 a 230 no.
were divided into four pools of equal volume, the second of which was coupled to peroxides (HOP). HOP
was reacted with 1.25% glutaraldehyde at pi on for 15 hours at room temperature. Aster passing the reaction mixture through a Sephadex -25 column, the ~RP-containing fractions were pooled and added to the polyacrylamide-diamine preparation, the pi was ad-jutted to 9.0, and this solution was allowed to react at 4 degrees C overnight. The peroxidase-polyacryla-mide-diamine was then passed through a ~iogel~P-100 comma and the void volume fractions were pooled and concentrated, then reacted with penicillin. Fifty my.
of penicillin G was added to the peroxidase-polyacry-lamide-diamine, the pi adjusted to 9.0 and stirred at
There is a recurring trend in the field to pro-vise analytical procedures which are characterized by speed, simplicity, and by a reduction in the vulner-ability of such procedures to human error. Simple, rapid tests, for example, have been marketed for de-~ermining 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.
~Z~68~8 n one embodiment, the invention provides an apparatus for the quantitative analysis ox a chemical-lye reactive substance (hereafter referred to as an "annihilate), in a carrier fluid such as a liquid. The apparatus includes a fluid-permeable solid medium that has a predetermined number of successive, spaced react lion zones and which defines a path for fluid flow sequentially through such zones. "Fluid" herein is lo 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 analyze or with an analyze derivative, to result in the formation of a predetermined product. The apparatus may further in-elude detector means for detecting, in the spaced zones, the presence of the analyze or its derivative, the reactant, or the predetermined product resulting from the reaction between the analyze or its derive-live and the reactant. In addition, the apparatus may include means for suppressing the detectability of trace amounts of the analyze or its derivative, the reactant, or the predetermined product resulting from the reaction between the analyze or its derivative and the reactant.
As used herein, the terms "reactants "rear-live" and the like when used in connection with the reaction between the analyze or its derivative and the reactant refers to the ability of the reactant to no-act, by covalent or hydrogen bonding or by any other means, with the analyze 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 anal lyre or analyze derivative in a manner that delectably alters the analyze or its derivative, the reactant or .
~26~;8~8 1 both to thereby result in the formation of a reaction product. Similarly, "reaction product" means any product resulting from the reaction of the analyze or its derivative and the reactant and that is delectably different from both. "Analyze derivative" means a chemical moiety derived from an analyze, and desirably is a tagged or labeled form of the analyze as may be employed in analytical procedures involving competing reactions between an analyze and its tagged or labeled lo derivative.
In the apparatus of the invention, the reactant is bound to the permeable solid medium in the success size, spaced zones through which the analyze passes.
A procedure employing the apparatus may take the form in which the analyze or its derivative, as it passes through the reaction zones, becomes bound to the no-act ant and the presence of the analyze or its derive-live within the reaction zones is detected, as by color change or the like. Similarly, in a slightly modified embodiment, the analyze 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 analyze by detect-in how many of the successive reaction zones, begin-nine with the upstream zone, show the presence of the analyze or its derivative, or of the product resulting from the reaction between the reactant and the analyze or analyze derivative In another embodiment, the reactant that is bound to the permeable solid medium may itself be capable of detection by suitable detect lion means and may be disabled from such detection when reacted with an analyze or analyze derivative.
In this manner, as the analyze or analyte-analyte -` ~Z~;878 1 derivative composition passes through successive react lion zones, the reactant in the successive zones is disabled from such detection until substantially all of the analyze or analyte-analyte derivative compost-lion has been exhausted, while remaining downstream reaction zones still contain reactant that can be de-tooted. In a modified form, the reaction between the analyze or analyze derivative and the reactant may cause the latter to become unbound from the solid lo medium to which it was attached and hence be washed from the successive zones. When the analyze or anal lyre derivative or both has thus been exhausted, sub-sequent 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.
s used herein "analyze" refers not only to the particular chemical moiety for which analysis is desired, but also to chemical moieties that are react lion 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 con-cent ration of the chemical moiety to be measured. Thy resulting product, then, may become the annihilate for use in the apparatus and method of the invention.
Accordingly annihilate refers to any chemical moiety which is to be measured quantitatively.
In a preferred embodiment, the invention em-ploys immunochemical reactions in which the analyze and the reactant represent different parts of a spew cilia ligand-antibody (antiligand) binding pair.
6~78 Figure 1 is a broken-away view, in partial cross-section, showing an apparatus of the invention;
inure 2 is a broken-away view, in partial cross-section, showing another apparatus of the invent lion:
Figure 3 is a cross-sec~ional view taken along line 3-3 of Figure 2 Figure 4 is a plan view of another embodiment lo of an apparatus of the invention;
Figure 5 is a perspective view of yet another test apparatus of the invention;
Figure is a broken-away cross sectional view taken along line 6-6 of Figure 5 and Figure 7 is a perspective, broken-away view of another test apparatus of }he invention.
DETAILED DESCRIPTION
With reference to Figure 1, a transparent, hot-low 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 port lion (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 flay, horizontal surface 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.~) 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 ox ) 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 -` ~Z~687~
1 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 slow rate of the liquid through the assay column.
Within the column are positioned successive, spaced reaction zones (16, 16.1, 16.2, 16.3, eke.), occupied by a permeable solid medium such as beaded agrees, beaded polyacrylamide, porous glass, cell-lo lose or other materials permeable to liquid and come partible with the analyze, analyze 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 Jones 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 sand-wicked 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 ll9) in the drawing) is poured into the open upper end ~12.1~ of the column, it will occupy the open volume at the top 87~
of the column and any amount of the liquid in ox-cuss 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 down-warmly in the column. The space (~) may, if desired, be filled or partially filled with a porous, none-active material such as glass wool or similar material to avoid splashing of the liquid within the upper end of the column.
lo To the liquid permeable solid medium within the spaced reaction zones (16, 16.1, etc.) is bound a no-act ant that is reactive with a given analyze or anal lyre derivative to form a product, all in accordance with the above definitions and as exemplified herein.
As a typical example, the reactant and analyze may be so chosen that the analyze or its derivative becomes chemically attached to the reactant as the solution I of analyze or analyze and analyze 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 analyze and analyze derivative in the solution.
After the test solution has begun its passage down-warmly 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 analyze or analyze derive alive, 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 analyze or its derivative are reacted with or bound to the reactant in each such ~61!378 g 1 layer until the analyze or its derivative have been exhausted from the solution. The concentration of analyze 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 ebbed ~rneht~
another eM~b~me~*, the reactant that is bound to the medium in the zones (16, 16.1 and so on) may be de-activated or disenabled by reaction with the analyze or analyze derivative or both, and the detector which lo is employed may determine, as by a color change, the presence ox nolldisabled reactant. In this embodiment, the reactant in successive zones contacted by the test solution will by disabled until analyze and analyze 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 analyze in the solution by count-in 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 Figures 2 and 3 in which "waking, 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 figures 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) in-voles binding a reactant to small, individual fee-angular paper filter pieces, and then alternating :- ~Z~6~37~3 1 these pieces, which form the reaction zones, with similar pieces of filter paper that do no contain the reactant, the alternating pieces of filter paper being held together, for example, by a thin strip of ad-hesive tape. Other, more sophisticated methods of manufacture will be apparent to those skilled in the art.
As shown in Figures 2 and 3, the strip (20) of filter paper may be positioned in an elongated plastic lo 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 t20). As shown in Figure 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) 20 is sandwiched between spacer layers (aye) The lit-ton 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 a shown in Figure 2. A test solution to placed in the bottom of the test tube (26) is thus caused to flow by cavil-lazy 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 yin Figure 1. As will be described more sully 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 ~L2(~6~78 1 Referring now to Figure 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 putter 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.]), are shown as an-nailer rings ~30.2), and are separated from one another lo 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 centric-gal force if desired, the test solution successively passing through the spaced reaction zones ~30.2).
Figures 5 and 6 show another embodiment of a device of the invention. The device includes a filter paper strip (40) similar to that of Figures 2 and 3 and containing spaced reaction zones (40.1) separated by spacer layers (40.2). A holder, preferably of plastic, is designated t42) 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 down-warmly 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 ~L2~6~7~
1 action, the solution sequentially encountering the spaced reaction zones (40.1).
Figure 7 shows yet another embodiment of a de-vice of the invention which can be used for multiple concurrent tests. The device, designated (50), in eludes a pair of spaced plates (50.1, 50.2). Refer-ring to the right-hand portion of Figure 7, the space between the plates is divided into generally vertical channels by means of elongated spacers ~52, 52.1). As lo 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 t58.2) adapted to be inserted in the channel (5205). 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 in-sorted 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 1 - 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 fig-rid, first from channel (52.4) and then from channel 152.5), 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 lo 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
, Analyzes 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 thaw the invention is no limited Jo any particular analyze or reactant, but is useful for sub-staunchly any analyte-reactant combination.
Many analyzes may be analyzed simply by adapt-in known chemical reactions to the invention.
For example, carbon dioxide may be analyzed with phenolphthalein at a slightly alkaline phi Cal-alum ion may be analyzed utilizing a reactant comprise in calmodulin and mammalian phosphodiesterase or another calmodulin-sensitive enzyme (Mohawk and Abe, Biochemical and Biophys~cal Research Communications 97:621 ~1980)). Ferrous ion may be analyzed utilize in, as a reactant, a foreseen derivative (Katz, et at, J. Am. Chum. Sock 104:346 l1982)). A large number of additional examples may be selected from the list of organic analytical reagents compiled by John H. You in Handbook of Chemistry and Physics, pod 126-129, ....
~6~7~3 1 Thea Edition, Robert C. West, Ed., CRC Press, Clove-land, 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 invent Sheehan Pro example, almost any phenol may be analyzed with Gibbs Reagent (2,6-dichloro-p-benzoquinone-4-chlorimine) (Deere, J., Analytical Chemistry 43:589 (1971)). A reagent for Insoles is p-dimethylamino-lo benzaldehyde (Fuzzier and Fuzzier, Reagents For Organic ; Synthesis, Volume 1, p.273, John Wiley & Sons, Inc., New York, (lg67). The last-mentioned reference also shows the use of phenylhydrazine as a reactant for cortisone and similar steroids, and the use of sulfa-acetic acid as a reactant in the ~iebermann - Burchard test for unsaturated strolls. Amino acids and ammonia us salts may be analyzed using the reagent ninhydrin (indane-1,2,3-trione hydrate) post, et at Structure Determination, p.42g, Prentice-Hall, Inc., 20 Englewood Cliffs, New Jersey, 1969). Reducing sugar may be measured with Red Tetrazolium (2,3,5-triphenyl -2H-tetrazolium Chloride) (Fuzzier, Organic Export-mints, ply, Raytheon Education Co., Lexington, Massachusetts, 1968).
various other analyze reagent pairs may be so-looted from the field of chemistry for adaptation to the invention from such reference works as: Cheerios, et at, US. Patent 3,654,090 ~nzyme-Linked Immune-sorbent Assay); Kay, US. Patent 3,789,116 (Flyers-30 cent Labeled Antibody Reagents); Ruben stein, et Allis. Patent 3,817,837 (Homogeneous Enzyme Immune-assay), Lingo US. Patent 3,867,517 (Radiomen-assay); Giver, US. Patent 3,906,490 (Radial Mooney-diffusion); Pullman, US. Patent 3,996,345 (Flours pence Quenching Homogeneous Immunoassay); Maggie, US.
Patent 4,233,402 (Enzyme Channeling Homogeneous Enzyme - ~206878 Immunoassay); Boguslaski~ et at, Canadian Patent 1,082,577 (Hapten-Cofactor Homogeneous Enzyme Immune-assay); Schonfeld, H., Ed., "New Developments in Immunoassay", Antibiotics and Chemotherapy Volume 26, 1979; O'Sullivan, et at, "Enzyme Immunoassay: A
Review", Annals of Clinical Biochemistry 16:221 (197g);
Cheerios, et at, Enzyme Immunoassay, Olin. Chimp Act.
81:1 (1977); Feldman, et at, Ens., First International Symposium On Immunoenzymatic Techniques, INSERT Swamp.
No. 2, North Holland Publishing Co., Amsterdam, 1976;
Williams, et at, Methods in Immunology and Immune-chemistry, Volume 3, Academic Press, New York, 1971;
and Yule, et at, J. Olin. Invest. 39 1157 (1960).
Yet other analyte-reactant pairs may be found in. reference works such as Feign, I ox Tests in Inorganic Analysis Thea edition, levier Publishing Co., New York 1972; Feign, Fritz, Spot Tests in Organic Analysis, Thea edition, Elsevier Publishing Co., New York, 1966; Swell, F. and Swell, C., Calorimetric Methods of Analysis, Vows. AYE, Van Nostrand Reinhold Co., New York, 1967-74; and Braibanti, A., Ed. Bioener~etics and Thermodynamics: Model Systems - Synthetic and Natural Chelates and Macro cycles as yodels for Biological and Pharmaceutical Studies, D. Rudely Publishing Co., Boston, 1980.
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 of quantity or 7~1 1 both of an analyze, particularly when the analyze is present only in exceedingly small concentrations.
Reference is made to the above-identi~ied patents and publications.
DETECTORS
The detectors useful in the invention are gape-bye of detecting the presence in the successive react lion zones, of analyze, annihilate derivatives, react-ants, or the predetermined reaction product, all as lo 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 flu-rosins of the zones, radioactivity of the zones, etc. For many reactions, detection is signaled by a change in phi and the detector may hence take the form of a pi color indicator such as phenolphthalein, Nile Blue A, Thymol Blue r and Methyl Violet. In other tests, one may detect the presence or absence of the appropriate chemical moiety in a reaction zone by ox-serving whether a solid reaction product has settled upon the succeeding spacer layer. Various detector mechanisms are known to the art, and need not be de-scribed in detail. In the preferred embodiment, how-ever, which makes use of immunochemical reaction be-tweet the analyze or the analyze and its derivative and the reactant, often very small concentrations of analyze are to be measured and accordingly a magnify-in 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 analyze to be tested may be provided in admixture with a known quantity of an analyte-glucose 6!378 1 oxidize conjugate as an analyze derivative the spaced, sequential reaction zones of the apparatus containing an antibody specific to the analyze. A
signal generating system, such as horseradish porks-dBase 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 analyze, the analyte-glucose lo oxidize conjugate, glucose, kettles and o-dianisidine, is then flowed through the apparatus, such as the column depicted in Figure 1. The analyze and the anal lyte-glucose oxidize conjugate compete for binding sites on the bound antibody, thereby resulting in a color formation due to the reaction of the oddness-dine with hydrogen peroxide produced by the glucose oxidize - catalyzed reaction of oxygen with glucose.
Unrequited analyze and analyte-glucose oxidize coinage-gate flow to successive zones until the admixture has been exhausted of analyze and analyte-glucose oxidize conjugate A variety of modifications of this prove-Doria of counsel 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 ("Foreign, a trademarked product of Chemical Dynamics Corp.) is used for the determination of serum iron in soluble assays through measurement of the absorbency at 593nm, at which wavelength any inter furriness from other pigments in the serum should be minimized. It may be covalently coupled to useful carrier derivatives through nitration reduction, diazoti2ation and diazonium coupling to proteins such as albumin) immobilized on agrees beads, paper strips or other suitable permeable solid medium. This imp mobilized signal-generating reagent (chromogenic ~6~78 1 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 silenced 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 both toys. The columns are packed by sequential insertion of layers of agarose-Ferene separated by layers of lo unmodified agrees. Typically, 0.4 ml of a 1:1 age-rose suspension is applied directly above the support, followed by alternating layers of 50 micro liters of a 1:1 suspension of agarose-Ferene and 0.2 ml of agrees suspension. After each addition to the column, the walls of the columns are rinsed with phosphate-buf-freed 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 appear-private dilution of test sample for iron analysis is applied to the column. The iron solution Taoist solution is passed through the assay column at con-trolled flow rates, typically between 10 and 15 mint vies for complete entrance. When all the solution is into the gel bed the columns are rinsed with water.
us 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.
Exhume II
A. The enzyme cholinesterase reacts with and is inhibited by toxic organophosphate and carbamate agents. Cholinesterase and the chromogenic sulfhydryl 6~378 1 reagent 5,5'-Dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) are immobilized upon agrees 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 follow-o b r Al - -to owe ~-~ in which a solution ~-~uty-r-~-lS~ic~K~ e iodide is added. The zones retaining catalytically active cholinesterase will generate a yellow color through reaction of the thiocholine produced by the hydrolytic lo activity of the cholinesterase, with the immobilized Ellman's Reagent. The presence of reactive choline-sterase-inhibiting toxin in tube test sample will no-suit 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 no-agent 2,4-dinitrofluorobenzene (FDNB), which produces a yellow product upon reaction. A 0.1 ml aqueous sample, containing about 0.1-1.0 micro moles of amino analyze, is transferred to a silicone glass Bessel.
The pi is adjusted to 7.0, if necessary, and two milligrams ~25 micro moles) of Nikko is added and dissolved. Next is added 0.12 ml of 0.15~ FDNB in absolute alcohol tl.5 micro moles). 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 analyze is immobilized in the zones in a permeable solid medium at a known con-tent (e.g., 0.1-0.25 micro moles per reaction zone).
After rinsing with 50~ ethanol in aqueous solution, the number of yellow reaction zones produced and no-mining after rinse will be inversely related to the amount of analyze in the test sample.
... .
2~687 Example III
The Gig fraction from rabbit anti-penicilloyl-bovine gamma globulin was partially purified by pro-cipitation with 33~ saturated ammonium sulfate. The precipitate was redissolved and dialyzed against pros-plate buffered saline (PBS). This Gig preparation was used for immobilizing antibodies onto beaded agrees.
The agrees was suspended in Dixon, then reacted with carbonyldiimidazole. After being washed with lo Dixon, it was suspended in water, and then in aqueous borate buffer, pi 9Ø The Gig was then added to the activated agrees and the gel suspension stirred by rocking at 4 C for 2 days. After ox-tensile washing with PBS, the gel containing imp mobilized antibody was ready for use in the assay.
Small columns were prepared from silenced 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 both 20 toys. The columns were packed by sequentially insert-in alternating layers of agarose-IgG separated by layers of unmodified agrees. Typically 0.4 ml of 1:1 agrees suspension was applied directly above the plugs, followed by alternating layers of 50 micro-liters of a 1:1 suspension of agarose-IgG (to form the reaction zones) and 0.2 ml of agrees suspension to form spacer lyres 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 30 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 oxidize ~nPen-GOn) typically 0.1 microgram Pen-GO in 1 ml PBS ? with or without known amounts of the analyze aye 7~3 1 (penicilloyl-epsilon amino copyright) (Punk), was applied to the column. The Pen-GO was prepared by reacting penicillin G with glucose oxidize in borate buffer, pi 9.0, for 2-3 days at 4 C. The Pen-GO
solution was passed through the assay column at con-trolled flow rates, typically between 10 and 15 mint vies 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 pro-lo pared as follows: 0.20 ml of horseradish peroxides (HOP) solution (2 mg/ml), 2 ml of 18% glucose soul-lion, 1 ml of 0.2 phosphate buffer pi 6.0) and 0.100 ml of I 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 20 down the column.
This Example may be repeated for the analysis of serum albumin (a large protein molecule) by no-placement of the penicillin-glucose oxidize conjugate with an albumin-glucose oxidize conjugate.
Example IIIA
Peroxidase-labeled Gig prepared from rabbit antiserum against penicillin was immobilized in small strips of jilter paper by the method reported in En-ample III. Kettles was bound to other, similar strips of filter paper. The first and second men toned strips wore then cut into rectangular shapes to provide, respectively, reaction zones and spacer lay ens. The small rectangular pieces of filter paper were then lazed onto a strip of adhesive tape, alter-noting the reaction zones and spacer layers with edges of the sequential pieces of paper overlapping or at 1 least touching one another to provide a continuous capillary flow path.
Penicilloyl-glucose oxidize ("Ping) in a solution of Human Serum Albumin ("HA") 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 pi 6Ø
A short wick was attached to the bottom of the lo 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 consist tying of a measured volume of milk containing a known concentration of penicillin G is added to the test tube containing the freeze-dried Ping, 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 Figure I. 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 Figure 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 free~e-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 ox penicillin G in the initial test solution.
In this example, the penicillin G in the milk and the penicillin of the Ping compete for binding 6~8 1 sites on the antibodies immobilized in the reaction zones of the jilter paper strip. Of course, larger concentrations of penicillin G in the milk sample cause the penicillin G and the Pen-GO to migrate fur-then through the filter paper strip. 'rho presence ox Pen-GO in any of the zones is indicated through the development of color from the reaction of H202 with o-dianisidine, the H202 being formed from the glucose oxidize in the presence of glucose and oxygen, o and as catalyzed by the peroxides. The kettles in the spacer layers catalyzes the conversion of H202 into 2 and H20, and thus prevents migration of H202 from one reactive zone to another.
As with each of the apparatuses described here-in, 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 exam-pie, one of the reaction zones may change color only when the test solution (e.g., milk) contains at least 9 nanogram of annihilate ego., penicillin G) per moo For a sample of milk containing an unknown concentra-lion of penicillin Go 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.
Antibody against a polyvalent antigen (e.g., serum albumin) analyze is labeled with peroxides 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 oxidize. Into the column is poured a test sample containing an unknown amount of analyze antigen. Through the column is then flowed the soluble glucose oxidase-antibody in the presence .
~6~37~
1 of glucose plus kettles plus o-dianisidine. The numb bier of colored bands resulting is directly related to the amount of analyze antigen in the test sample rota-live 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 de-tooted by the coupling of the glucose oxidize antibody conjugate to available antigenic sites on the antigen lo followed by the color forming reaction.
Example V
An analyze or a derivative thereof (e.g., pent-cillin-peroxidase) is covalently bound to a permeable solid medium according to example III. An enzyme-la-bleed receptor (e.g., glucose oxidase-antibody against penicillin) is prepared and exposed to the immobilized analyze to form the specific binding complex (e.g., immune complex). The assay unit is assembled accord ding to Example III. Subsequent exposure to a test sample containing an unknown amount of analyze is done at elevated temperature (e.g., 60 C) to hasten the attainment of equilibrium through competitive binding of the immobilized analyze and analyze in the test sample with the enzyme-labeled antibody. Analyze in the test sample under such conditions will compete-lively displace the labeled antibody from the iamb-lived analyze. The number of colored reaction zones resulting from the procedure is inversely related to the amount of analyze 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 Jo Example III, except that the top reaction zone was prepared with 75 micro liters of IgG-agarose suspension and the lower 3 zones 1 with 50 micro liters. Test samples containing 0, 50 and 200 nanogram Punk, were placed in different columns, with each test sample containing 200 no Pen GO
per ml. Flow time for sample application was 20 min.
Application of the solution of signal generating no-agents produced 2 colored zones with the O no Punk sample, 3 in the 50 and 4 with the 200 no sample.
A wider and more precise range of analyze con-tent, of course, may be measured by using a larger o number of assay zones.
In a preferred embodiment, only a single pass through the apparatus of a single liquid material is required. An analyze may be mixed with an analyze derivative, chromogen or other material and flowed through the apparatus to yield an appropriate test result. In a further preferred embodiment, the apt pyrites is chemically complete in that it includes all reactants and other chemicals necessary or desirable for the quantitative analysis of an analyze, that is, all that is required is that the analyze in a liquid carrier be flowed through the apparatus. Elements of the apparatus that, if combined, would undergo react lion in the absence of the analyze may be maintained in different zones. For example, the bottom-most layer (20.2) of the strip of Figure 2 may contain a reactant physically separated from reactants in the adjacent reaction zone. When the analyze in a carrier liquid is flowed through the layer (20.2), the react lent in this layer together with the analyze and car-nor liquid is flowed into the first reaction zone.
If desired, a reknit may be provided in the form of a solid and may merely be placed upon the upper layer (18.4) of the column of Figure 1, the reactant being dissolved by and carried with the liquid carrier and analyze into the column.
~68~
1 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 analyze to form an analyze derivative and catalyzes a color-forming reaction, and another enzyme that is immobilized in reaction zones which also contain antibody to the analyze, the latter enzyme providing substrate for the color-generating lo enzyme. In this format, therefore, only a single solution which consists of or contains the analyze 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 analyze in the test sample.
Example VII
The Gig fraction from rabbit anti-penicilloyl-bovine serum albumin was partially purified by precip-ita~ion with 33% saturated ammonium sulfate. This pro-20 loin was coupled to microcrystalline cellulose by no-action of the cellulose with carbonyldiimidazole in dioxanet followed by washing and then by reaction with the Gig preparation in borate buffer at pi 9.0 at 4 degrees C for two days. The cellulose was then washed extensively with PBS and used for preparation of banded strips r Glucose oxidize was also coupled to microcrystalline cellulose in the same manner. A
penicilloyl-peroxidase was prepared by first coupling a polyacrylamide amine to HOP, then reacting pencil-fin G with that preparation. It is believed that these of a linear polymer as a spacer for attaching the hasten to the enzyme allows more hasten molecules to be coupled to each enzyme molecule and renders the hasten molecules more accessible for binding to anti-body, thus speeding the binding rate. Polyacrylamide was synthesized by dissolving 0.5 gym. of acrylamide in I I
200 ml. of deionized water, degas sing, then adding 0.2 ml. of N,N,N',N'-tetramethylethylenediamine and 0.15 gym. of ammonium per sulfate. This solution was mixed, then allowed to it at room temperature for 30 min.
then passed through an ultrafiltration membrane, dialyzed us deionized water and lyophilized. The polyacrylamide was then dissolved in lo ml. of 0.2 M
phosphate buffer at pi 7.7 and 0.3 ml. of 25% gluier-alluded was added. This solution was incubated at 37 degrees C for 19 hours after which it was passed through a Sephadex7'G-25 column to remove the excess glutaraldehyde. The void volume fractions which Abe sorbed strongly at 230 no were pooled and added to a solution of diaminodipropylamine (0.5 ml. in 2.0 ml.
of water) at pi 9Ø 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 a 230 no.
were divided into four pools of equal volume, the second of which was coupled to peroxides (HOP). HOP
was reacted with 1.25% glutaraldehyde at pi on for 15 hours at room temperature. Aster passing the reaction mixture through a Sephadex -25 column, the ~RP-containing fractions were pooled and added to the polyacrylamide-diamine preparation, the pi was ad-jutted to 9.0, and this solution was allowed to react at 4 degrees C overnight. The peroxidase-polyacryla-mide-diamine was then passed through a ~iogel~P-100 comma and the void volume fractions were pooled and concentrated, then reacted with penicillin. Fifty my.
of penicillin G was added to the peroxidase-polyacry-lamide-diamine, the pi adjusted to 9.0 and stirred at
4 degrees C over night. This preparation was then dialyzed extensively, then used for the assay ,)~ I
:~2~78 1 Banded strips were prepared by cutting 0.5 X
8.0 cm. strips of a polyester film having a hydrophilic surface onto which were glued strips ox Whatman~3MM
chromatography paper. At one end was glued a 0.5 X 4.0 cm. long paper strip followed by a 3.5 mm. space. Then three one cm. long paper strips were glued onto the strip with 2.0 mm spaces between them. The Cicero paper on the was wetted with a solution of 0.02%
o-dianisidine in water. The spaces were then filled in lo with a suspension of microcrystalline cellulose pro pared 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 us. of this suspend soon and the other three spaces each contained 10 us.
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 con-twining the developing solution. This solution I contained peroxidase-polyacrylamide-diamine-penicillin (25 us. of a 0.25 microgram/ml. solution), glucose (0.3 ml of a 1~125~ glucose solution in 0.2M phosphate buffer at pi 6.0) and 10 us. of dilutions of pencil-loyl-amino~aproic acid EKE) 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 micro molar hasten (penicilloyl-~AC), two bands were colored; and with 1.0 us penicilloyl-EAC, all three bands were colored.
If needed or desirer antibody Jo peroxides, an HRP-binding pectin or some other binder or iniquity-valor of peroxides can be included in the spacer lay-ens for the purpose of improving the sharpness or decisiveness of zone color determinations. Further-more, kettles immobilized in the spacer layers may I R
lo 7~3 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
lo phosphate buffer at pi 6.0, 0.1 ml. of which is apt plied 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 analyze, 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 Jo Example III, except that the reaction zones are composed of a mixture of Gig agrees and glucose oxidase-agarose 20 (20~ Peroxidase-penicillin (as prepared in Example VOW glucose, o-dianisidine, and phosphate buffer, stored in dry form, are dissolved in 1~0 ml of the jest sample which is then added to the column and at-lowed 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 analyze 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 ox 30 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 ISSUER
1 Various other enzyme pairs can be used for gent crating color in the reaction zones. Pro example, alkaline phosphates can be immobilized in the react lion zones with beta-galactosidase coupled to the analyze. The use of naphthol-beta-D-galactopyrano-side-6-phosphate as substrate for the alkaline pros-photos results in the generation of naphthol-beta-D-galactopyranoside, which is hydrolyzed by beta-galac-tosidase to produce naphthol which in the presence of o 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 veneration ox color or other detectable change in the different reaction zones may be ascer~ainedO A reaction zone in the direction of analyze wow desirably should show detectable changes only when a significant, minimum quantity of analyze or other material being detected has passed through 20 the proceeding reaction zone: since the physical nature of the apparatus often does not permit reaction to go fully to completion in each such zoner 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, how-ever 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 30 utilize o-phenylene Damon 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 ` ~Z~6Z!~78 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 ox the reactant in the solid reaction zones, and the volubility of mate-fiats such as the colored product in some analyses.
While a preferred embodiment of the present in-mention has been described, it should be understood Jo 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.
:~2~78 1 Banded strips were prepared by cutting 0.5 X
8.0 cm. strips of a polyester film having a hydrophilic surface onto which were glued strips ox Whatman~3MM
chromatography paper. At one end was glued a 0.5 X 4.0 cm. long paper strip followed by a 3.5 mm. space. Then three one cm. long paper strips were glued onto the strip with 2.0 mm spaces between them. The Cicero paper on the was wetted with a solution of 0.02%
o-dianisidine in water. The spaces were then filled in lo with a suspension of microcrystalline cellulose pro pared 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 us. of this suspend soon and the other three spaces each contained 10 us.
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 con-twining the developing solution. This solution I contained peroxidase-polyacrylamide-diamine-penicillin (25 us. of a 0.25 microgram/ml. solution), glucose (0.3 ml of a 1~125~ glucose solution in 0.2M phosphate buffer at pi 6.0) and 10 us. of dilutions of pencil-loyl-amino~aproic acid EKE) 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 micro molar hasten (penicilloyl-~AC), two bands were colored; and with 1.0 us penicilloyl-EAC, all three bands were colored.
If needed or desirer antibody Jo peroxides, an HRP-binding pectin or some other binder or iniquity-valor of peroxides can be included in the spacer lay-ens for the purpose of improving the sharpness or decisiveness of zone color determinations. Further-more, kettles immobilized in the spacer layers may I R
lo 7~3 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
lo phosphate buffer at pi 6.0, 0.1 ml. of which is apt plied 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 analyze, 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 Jo Example III, except that the reaction zones are composed of a mixture of Gig agrees and glucose oxidase-agarose 20 (20~ Peroxidase-penicillin (as prepared in Example VOW glucose, o-dianisidine, and phosphate buffer, stored in dry form, are dissolved in 1~0 ml of the jest sample which is then added to the column and at-lowed 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 analyze 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 ox 30 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 ISSUER
1 Various other enzyme pairs can be used for gent crating color in the reaction zones. Pro example, alkaline phosphates can be immobilized in the react lion zones with beta-galactosidase coupled to the analyze. The use of naphthol-beta-D-galactopyrano-side-6-phosphate as substrate for the alkaline pros-photos results in the generation of naphthol-beta-D-galactopyranoside, which is hydrolyzed by beta-galac-tosidase to produce naphthol which in the presence of o 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 veneration ox color or other detectable change in the different reaction zones may be ascer~ainedO A reaction zone in the direction of analyze wow desirably should show detectable changes only when a significant, minimum quantity of analyze or other material being detected has passed through 20 the proceeding reaction zone: since the physical nature of the apparatus often does not permit reaction to go fully to completion in each such zoner 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, how-ever 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 30 utilize o-phenylene Damon 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 ` ~Z~6Z!~78 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 ox the reactant in the solid reaction zones, and the volubility of mate-fiats such as the colored product in some analyses.
While a preferred embodiment of the present in-mention has been described, it should be understood Jo 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.
Claims (25)
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Apparatus for the quantitative analysis of an analyte in a fluid, comprising a fluid-permeable solid medium defining a path for fluid flow and having a predetermined number of successive, spaced reaction zones in the path of flow, the reaction zones having immobilized therein predetermined quantities of a reactant capable of competitively reacting with or competitively binding to the analyte or an analyte derivative to result in the formation of a predetermined product; the analyte or its derivative, the reactant or the predetermined product in the reaction zones being detectable and the number of reaction zones in which such detection occurs indicating quantitatively the amount of analyte in said fluid.
2. The apparatus of claim 1 wherein said fluid-permeable solid medium includes spacer sections in flow communication with but separating said reaction zones.
3. The apparatus of claim 1 wherein the fluid-permeable medium in the reaction zones has immobilized therein an antibody specific to an analyte to be quantitatively analyzed.
4. The apparatus of claim 1 including detection means for detecting, in the reaction zones, the analyte or its derivative, the reagent, or the predetermined product.
5. The apparatus of claim 2 wherein said reaction zones are vertically spaced in a column.
6. The apparatus of claim 2 wherein the reaction zones are spaced along the length of a strip of solid, fibrous material through which fluid is drawn by capillary action.
7. The apparatus of claim 2 wherein the reaction zones are in the shape of concentric rings and the spacer sections are in the shape of concentric rings sandwiching between them the reaction zones.
8. The apparatus of claim 2 including flow rate controlling means for controlling the rate of fluid flow along the fluid flow path.
9. Method for the quantitative analysis of an analyte in a carrier liquid, comprising the steps of:
providing a liquid-permeable solid medium defining a flow path and having a predetermined number of successive, spaced reaction zones in the path of flow, said reaction zones having immobilized therein a reactant competitively reactive with or competitively binding to the analyte or an analyte derivative or both to result in the formation of a predetermined product;
flowing said liquid along the flow path and sequentially through the spaced reaction zones; and detecting the presence of analyte, analyte derivative, reactant or predetermined product in the reaction zones;
the amount of analyte in the determining liquid being a function of a number of zones in which such detection occurs.
providing a liquid-permeable solid medium defining a flow path and having a predetermined number of successive, spaced reaction zones in the path of flow, said reaction zones having immobilized therein a reactant competitively reactive with or competitively binding to the analyte or an analyte derivative or both to result in the formation of a predetermined product;
flowing said liquid along the flow path and sequentially through the spaced reaction zones; and detecting the presence of analyte, analyte derivative, reactant or predetermined product in the reaction zones;
the amount of analyte in the determining liquid being a function of a number of zones in which such detection occurs.
10. The method of claim 9 wherein said analyte and said reactant are ones of a specific ligand-antiligand binding pair.
11. The method of claim 10 wherein said reactant includes an antibody specific to said reactant.
12. The method of claim 9 wherein reaction between said analyte and reactant renders the analyte, analyte derivative, reactant or predetermined reaction product nondetectable during said detection step.
13. The method of claim 9 wherein said liquid contains said analyte and a known concentration of said analyte labeled with a chemical moiety, the presence of which chemical moiety is detected in said detection step.
14. The method of claim 13 wherein the chemical moiety is an enzyme.
15. The method of claim 13 wherein the liquid is milk, the analyte is penicillin and the reactant includes an anti-penicillin antibody.
16. The method of claim 15 wherein the liquid includes a known concentration of an enzyme-labeled penicillin.
17. Method for the quantitative determination of an analyte in a fluid containing a known quantity of a labeled analyte, comprising the steps of:
providing a fluid-permeable solid medium defining a fluid flow path and having immobilized therein a reactant reactive with the labeled analyte in competition with the analyte to result in the formation of a predetermined product;
flowing said fluid along the flow path; and detecting the presence of the labeled analyte or the predetermined product along the flow path;
the length of the flow path in which such detection occurs being a function of the amount of analyte in the fluid.
providing a fluid-permeable solid medium defining a fluid flow path and having immobilized therein a reactant reactive with the labeled analyte in competition with the analyte to result in the formation of a predetermined product;
flowing said fluid along the flow path; and detecting the presence of the labeled analyte or the predetermined product along the flow path;
the length of the flow path in which such detection occurs being a function of the amount of analyte in the fluid.
18. Method for the quantitative determination of penicillin in a liquid containing a known quantity of a labeled penicillin, comprising the steps of:
providing a liquid-permeable solid medium defining a liquid flow path and having immobilized therein a reactant including an antibody to said penicillin and to said labeled penicillin and reactive competitively therewith to result in the formation of a predetermined product;
flowing said liquid along said flow path; and detecting the presence of the labeled penicillin or the predetermined product along the flow path;
the length of the flow path in which such detection occurs being a function of the amount of penicillin in the liquid.
providing a liquid-permeable solid medium defining a liquid flow path and having immobilized therein a reactant including an antibody to said penicillin and to said labeled penicillin and reactive competitively therewith to result in the formation of a predetermined product;
flowing said liquid along said flow path; and detecting the presence of the labeled penicillin or the predetermined product along the flow path;
the length of the flow path in which such detection occurs being a function of the amount of penicillin in the liquid.
19. The apparatus of claim 1 including means in the reaction zones or spacer sections for suppressing therein the detection of trace amounts of the analyte or its derivative, the reactant or the predetermined product.
20. The apparatus of claim 3 wherein said reaction zones are particularly adapted to change colour upon contact therein with an analyte derivative that is a conjugate of an analyte and an enzyme, the reaction zones having immobilized therein another enzyme, one of said enzymes being capable of generating substrate for the other said enzyme.
21. The apparatus of claim 20 wherein said analyte-enzyme conjugate is contained in a layer upstream from a reaction zone, whereby flow of an analyte in a fluid carrier through said layer carries the analyte conjugate into said reaction zone.
22. The method of claim 9 wherein only a single liquid containing an analyte is flowed in said flow path.
23. The method of claim 22 wherein said liquid consists essentially of the analyte and carrier liquid.
24. The method of claim 22 wherein said liquid includes an analyte and an analyte derivative.
25. The method of claim 9 including the step of suppressing the detection of trace amounts of analyte, analyte derivative, reactant or predetermined product.
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US35645982A | 1982-03-09 | 1982-03-09 | |
US06/467,229 US5073484A (en) | 1982-03-09 | 1983-02-23 | Quantitative analysis apparatus and method |
US467,229 | 1983-02-23 | ||
US356,459 | 1989-05-25 |
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CA1206878A true CA1206878A (en) | 1986-07-02 |
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CA000423140A Expired CA1206878A (en) | 1982-03-09 | 1983-03-08 | Quantitative analysis apparatus and method |
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EP (1) | EP0088636B1 (en) |
JP (3) | JPH0670625B2 (en) |
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BR (1) | BR8301191A (en) |
CA (1) | CA1206878A (en) |
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US5141875A (en) * | 1982-01-08 | 1992-08-25 | Environmental Diagnostics, Inc. | Rotary fluid manipulator |
US4444193A (en) * | 1982-01-11 | 1984-04-24 | Medtronic, Inc. | Fluid absorbent quantitative test device |
US5073484A (en) * | 1982-03-09 | 1991-12-17 | Bio-Metric Systems, Inc. | Quantitative analysis apparatus and method |
US4435504A (en) * | 1982-07-15 | 1984-03-06 | Syva Company | Immunochromatographic assay with support having bound "MIP" and second enzyme |
US4459358A (en) * | 1982-12-29 | 1984-07-10 | Polaroid Corporation | Multilayer element for analysis |
US4552839A (en) * | 1983-08-01 | 1985-11-12 | Syntex (U.S.A.) Inc. | Determination of analytes in particle-containing medium |
DE3484505D1 (en) * | 1983-12-19 | 1991-05-29 | Daiichi Pure Chemicals Co Ltd | IMMUNTEST. |
DE3445816C1 (en) * | 1984-12-15 | 1986-06-12 | Behringwerke Ag, 3550 Marburg | Flat diagnostic agent |
-
1983
- 1983-02-23 US US06/467,229 patent/US5073484A/en not_active Expired - Lifetime
- 1983-03-08 CA CA000423140A patent/CA1206878A/en not_active Expired
- 1983-03-08 IL IL68082A patent/IL68082A/en not_active IP Right Cessation
- 1983-03-09 EP EP83301294A patent/EP0088636B1/en not_active Expired
- 1983-03-09 AU AU12196/83A patent/AU560552B2/en not_active Expired
- 1983-03-09 DE DE8383301294T patent/DE3382384D1/en not_active Expired - Lifetime
- 1983-03-09 BR BR8301191A patent/BR8301191A/en not_active IP Right Cessation
- 1983-03-09 JP JP58038884A patent/JPH0670625B2/en not_active Expired - Lifetime
-
1992
- 1992-06-01 US US07/891,864 patent/US6020147A/en not_active Expired - Lifetime
- 1992-06-01 US US07/891,932 patent/US5654162A/en not_active Expired - Fee Related
-
1993
- 1993-06-24 JP JP5177306A patent/JPH07111431B2/en not_active Expired - Lifetime
-
1997
- 1997-03-03 JP JP9063818A patent/JP2913281B2/en not_active Expired - Lifetime
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7883899B2 (en) | 2002-10-11 | 2011-02-08 | Zbx Corporation | Diagnostic devices |
Also Published As
Publication number | Publication date |
---|---|
US5654162A (en) | 1997-08-05 |
EP0088636A2 (en) | 1983-09-14 |
JPH0670625B2 (en) | 1994-09-07 |
US6020147A (en) | 2000-02-01 |
JP2913281B2 (en) | 1999-06-28 |
BR8301191A (en) | 1983-11-22 |
JPH07111431B2 (en) | 1995-11-29 |
JPH1010126A (en) | 1998-01-16 |
EP0088636A3 (en) | 1985-12-04 |
EP0088636B1 (en) | 1991-08-28 |
DE3382384D1 (en) | 1991-10-02 |
JPH06258323A (en) | 1994-09-16 |
IL68082A (en) | 1986-12-31 |
US5073484A (en) | 1991-12-17 |
IL68082A0 (en) | 1983-06-15 |
AU1219683A (en) | 1983-09-15 |
JPS58179357A (en) | 1983-10-20 |
AU560552B2 (en) | 1987-04-09 |
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