CA1317876C - Bi-directional lateral chromatographic test device - Google Patents
Bi-directional lateral chromatographic test deviceInfo
- Publication number
- CA1317876C CA1317876C CA000585262A CA585262A CA1317876C CA 1317876 C CA1317876 C CA 1317876C CA 000585262 A CA000585262 A CA 000585262A CA 585262 A CA585262 A CA 585262A CA 1317876 C CA1317876 C CA 1317876C
- Authority
- CA
- Canada
- Prior art keywords
- zone
- sample
- application
- absorbent means
- reaction zone
- 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 - Fee Related
Links
Classifications
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
-
- 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
-
- 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
-
- 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
- Y10S436/808—Automated or kit
-
- 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
- Y10S436/809—Multifield plates or multicontainer arrays
-
- 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
- Y10S436/825—Pretreatment for removal of interfering factors from sample
Abstract
Abstract of the Disclosure:
A chromatic test device for the performance of immuno- or chemical assays wherein a unitary planar fibrous filter body incorporates a sample application zone, a separation zone and a reaction zone and said application zone is in fluid communication with first absorbent means and said reaction zone is in fluid communication with second absorbent means to establish bilateral flow of the fluid component of said sample and of analyte applied to said zones during the performance of an assay.
A chromatic test device for the performance of immuno- or chemical assays wherein a unitary planar fibrous filter body incorporates a sample application zone, a separation zone and a reaction zone and said application zone is in fluid communication with first absorbent means and said reaction zone is in fluid communication with second absorbent means to establish bilateral flow of the fluid component of said sample and of analyte applied to said zones during the performance of an assay.
Description
t317876 BI-DIRECTIONAI. L~TERAL CHRO~TOGRAPHIC TEST DEVICE
Back round of the Invention:
The invention relates to a diagnostic device for performing solid phase immunoassays to detect the presence of antigens or antibodies in biological or non-biological 5. fluids. The teachings of the invention are incorporated in a bi-directional lateral chromatographic device for use in solid phase immunoassays or for the non-immunological detection or quantitation of proteins or substances in biological or non-bioloqical fluids.
10. More particularly, the invention relates to devices and methods which utilize filter means for testing biological fluids to detect the presence of analytes such as bacterial, viral, parasitic, or funqal antigens and immunoglobulins, hormones, serum proteins, drugs and the - 15. like.
$ypical of prior art devices presently in use are the teachings of U.S. Patent 4,623,461, which discloses a filter body located in a housing having an openin~ therein for the reception of a suspension sample to permit the 20. upper face of the filter to trap colored or particulate matter contained within the specimen and prevent such matter from reaching the bottom face of the reaction zone, which has been previously treated with a suitable reactant.
The perimeter of the filter is engaged with a suitahle absorbent body and the absorbent body is intended to receive 5. the outward diffusion of liquids applied to the filter.
One of the disadvantages of the '461 construction lies in the fact that the fluid flow from ~he point of application of the suspension to the absorbent hody is uni-directional and a subtantial accumulation of solids at the 10. point of application of the sample suspension is inevitable, which will seriously i~pinge upon the resultant chroma-tological or other type of test reading imparted by the device.
U.S. Letters Patent 3,825,410 discloses a disposable 15. combined storage and reaction cell for use in the performance o chemical and biological reactions which receives reactants dispensed therein and maintains the same in stored condition so that they remain stable. Reactants will not mutually react until such time as it is required to initiate the 20. reaction.
The immobilization of the reactants is accomplished by such procedures as freeze drying and the reaction is initiated by the introduction of a sample to be analyzed, ~, ' \
whereafter separation of bound and free ligand can be performed either within the unit itself or externally.
The reaction cell of the '410 patent may include a filter so that the entire process of separation can be 5. completed within the reaction cell and the filter be removed from the reaction cell and submitted for radioactivity or other tracer counts.
~ J.S. Letters Patent 3,888,629 discloses a reaction cell for performing various types of assays which incorpo-LO. rates a matrix pad of absorbent material retaining the necessary reagents for the reaction and serving as a site in which the reaction totalIy occurs. A se~arable lower chamber incorporates absorbent material abutting the matrix pad to promote filtration through the pad after the reaction lS. has taken place~ ~
Both patents are characterized by the mere uti-lization of the filter as a pass-through device which is time-consuming and which is hi~dered by the deposition of solids out of the suspension sample.
20. Objects and Advanta~es of the Invention:
One of the objects of ~he invention is the pro-vision of a diagnostic ~evice which incorporates a planar filter body havinq sample application, separation Ind reaction zones, said filter body bein~ configured in such a manner that the bulk o the solids in the suspension sample are retained in the sample application zone and the 5. fluid ls caused to mi~rate bilaterally through the inter-stices of the filter by the 1uid communication of absorbent means with the application and reaction zones.
Conse~uently, the unilateral directional flow which causes accumulation of solids in the reaction zone 10. in the previously discussed prior art devices is eliminated in the test device of our construction because the rapid bilateral flow achieved by the construction of the device causes i~mediate deposition of solids out of the fluid component of the suspension.
15. Another object of the invention is the provision of a diagnostic test device of the aforementioned character wherein the planar filter incorporates a sample application zone which is relatively large and which is connected to the reaction zone by a separation zone, the length of the 20. separation zone being proportioned to the character of the suspension sample applied to the a~plication zone, and the separation and application zones cooperate to retain the bulk of the solids or particulate matter in the application and separating zones so that the immuno-reagent or chemical test reagent deposited in the reaction zone, when subjected to the test procedures and analytes, will have a minimum of or no particulates embodied therein which would cause the 5. emission of high background signals, thu~ creating a negative effect on the tes~ readout.
~ nother important object of the invention is the provision of a composite housing filter conjugate which is characterize~ by ease of assembly and application. The 10. test device of the invention is designed particularly for use in the field or the testing of various human and animal diseases or for various chemical tests involving the utilization of blood samples from humans and animals, and it is capable of giving test results equal to labora-15. tory results within a matter of minutes depending upon thesample solution which is applied to the specific device.
Another object of the invention is the provision of a method of performing a test by the utilization of the device of the invention which incorporates a plurality of 20. simple steps which can be carried forth by non-laboratory personnel in the field and which can provide such personnel with an almost immediate readout of the presence or absence of the sought-after infection or dru~, or the like.
According tG one aspect of the invention there is provided in a test device for per~orming solid phase immunoassays, the combination of a planar fiber filter matrix, said matrix including a test sample application zone, a separation zone and a reaction zone, said separation zone separating said application zone from said reaction zone and being sufficiently long to prevent migration of particulates in said sample into said reaction zone, first absorption means contiguous to said sample application zone, and second absorption means contiguous to said reaction zone whereby bilateral flow of the liquid componerlt of said test sample occurs.
According to another aspect of the inveDtion there is provided in a test device for performing solid phase immunoassays, the combination of a housing, a planar filter matrix located in said housing, said filter matrix iDcorporating a test sample application ~one, a separation zone integIal with said test sample application zone and a reaction zone communicating with said separation zone, a first absorbent means located in said housing in fluid communication with said application z~lne, and second absorbent means in said housing in spaced relationship with said first absorbent means and in fluid communicatlon with said Ieaction zone whereby bilateral flow of the fluid component of said test sarnple occurs.
According to a further aspect of the invention there is provided in a chromatographic test device for performing solid phase immunoassays, the combination a planar filter matrix including a body portion having a plurality of laterally extending integral arrns disposed in spaced relationship with one another, each of said anns having an expanded end portion, first absorbent means in fluid communication with said expanded end portions and second absorbent means in fluid communication with said body portion whereby bilateral flow of fluid deposited upon said expanded end portions may be achieved.
According to yet a further aspect of the invention there is provided a method of perfonning an immunoassay in coryunction with a chromatographic test device which incorporates a planar filter matrix having sample application, separation and reaction zones, and fiIst and second absorbent means in fluid comrnunication, respectively, with said application and reaetion zones, the steps of applying a suitable volume of liquid sample to said sample application zone, applying a suitable volume of wash reagent to said sample application zone, permitting capillary forces within said filter matrix to bilaterally draw the fluid portion of said sample primarily in the direction of said separation zone, but secondarily in the direction of said first absorbent means, entrapping particulates in said application and separation zones, permitting the fluid portion of said sample to flow into said reaction zone which is treated with a reagent immobilized to the fiber matrix, applying a suitable volume of wash reagent to said reaction zone to wash away ur~eacted sample components in bilateral directions away ~rom the reaction zone and toward the separation and sample application zones, applying a tracer reagent to said reaction zone, washing said reaction zone, and applying chromatic-eliciting substrate to said reaction zone to produce a c}~omatic reaction.
Brief Descri~tion of the Drawin~s:
~_.
Other objects and advantages of the invention will be apparent from the following specificat:ion and the accom-panying drawings, which are fox the purpose of illustration 5. only, and in which:
FIG. 1 is a top plan view of a ~ypical device of the invention;
FIG. 2 is a device similar to FIG. 1 with the exception that it incorporates a plurality of application 10. and reaction zones;
FIG. 3 is a top plan view showing the housing of the device of FI~. 2 with the cover removed therefrom to ` .
illustrate the location of and configuration of the filter and the relation thereof with tha absorbent means and the 15. particular design of the housing to encapsulate the filter and absorbent means;
FIG. 4 is a view similar to FIG. 3 illustrating removal of the filter to disclose the relationship of the absorbent means with the housing and the particular con-20. ~figuration thereof;
FIG. 5 i5 a vertical sectional view taken onthe broken line ~-5 of FIG. 2 and illustrates the eluid relationship~of the filter with the absorbent means and , the filter and absorbent components with the specific design of the housing and co~er therefor; anfl FIG. 6 is an exploded view illustrating the various components of the test device.
5. Description of the Preferred Embodiments of the Invention:
The chromatic assay device of the present ~n~en-tion may be used to perform sol.id phase immunoassays for the detection of antigens or antibodies, hereinafter referred to as analytes, in biological and non-biological 10. fluids. The device may be non-immunologically used to identify and/or quantitate proteins or substances in biological and non-biologi~al fluids. The device may be : utilized to perform assays such as competitive or non-: competitive enzyme-linked immunoassays, enzyme-multiplied lS. immunoassays, enzyme-inhibition assays, heterogeneous or homogeneous fluorescent immunoassays, chemiluminescent and bioluminescent assays, these assays utilizing various labelled probes, and the like.
Obviously, the particular analyte test to be 20. used will depend upon the chosen sample and the desired result to be achieved.
Referring to the drawings, and particularly to FIG. 1 thereof, we show a test device 10 constructed in accordance with the teachings of our invention which is incorporated in a housing 12, said housing consisting of 5. a lower or bottom component 14 and a cover or closure 16.
The bottom component 14 of the housing may be fabricated by injection molding from suitable synthetic plastic materials such as polyethylene and, as will appear further hereinbelow, is specifical.Ly designed to receive a flat 10. co-planar filter 20.
The closure 16 overlies the filter 20 and is : secured to the housing by pressure-sensitive adhesive or other adhering means and incorporates an application port ~ or openina 22 and a reaction port or opening 24, said ports : 15. communicating, respectively, with the application zone 26 and the Eeaction zone 28~of the filter.
The test device 10 is designed for the performance of a sinqle test, but, as best shown in FI~,. 2 of the draw-ings,~a device 30 incorporating a multiplicity of application 20. ports 22 and reaotion ports 24 can be provided in a closure or cover 32 which overlies a suitabl~ configured, as will be explained in greater detail below, lower housing portion 34 and filter 36. The ports 22 and 24 respectively overlie application and reaction zones~38fand 40 of the filter 36.
.
131~87~
The filters 20 and 36 are constituted by a planar glass fiber matrix which is sandwiched be~tween the lower component of the housing and the cover therefor.
As best shown in FIG. 3 of the drawings, the 5. filter 36 includes a plurality of application zones 38 . and reaction zones 40 which are maintained in fluid .. ..
communication by separation zones 42. The separation zones 4~ are of elongate configuration and establish fluid communication between the application zones 38 and lO. the reaction zones 40.
The application zon~s 36 are shown as being roughly trapezoidal in configuration and provide a rela-tively large area for the application of the test sample.
For a purpose which wlll be expl~ined hereinbelow, the 15. separation zones 42 are of relatively restricted width in comparison with the width of the application zones 38.
Located in fluid communication with the appli-cation zones 38 is first absorbent means 44 constituted . by an elongated strip 46 of absorbent material. Similarly, 20. a second absorbent means 48 constituted by an elongated strip 52 is disposed in fluid communication with the multiplicity of reaction zones 40 provided by the filter 36 .
:
. The lower or bottom portion 34 of the.housing of the test device 30 is configured, as best shown in FIGS. 4 and 5 of th~ drawings, to provide receptacles 55 for the application, separation and reac1:ion zones o.f 5. the filter 36 so that fluid flow is confined in the plane of the filter 36 because of the sandwich created between the closure 32 and the lower portion 34 of the ho~sing.
The receptacles are defined bv integrally molded lobes 58 in the body of the lower por~ion 34 of the housing and lO. stringently confine the relevant portions of the filter in the receptacles 56.
Juxtaposed to the receptacles 56 is a first elongated rectangular well 60 for the reception of the first absorbent means 44, and a corresponding well 62 15. is provided for the reception of the second absorbent means 48. ~
The closure or cover 32 of the device 30 can be fabricated from vinyl or other plastic sheet material and may be adhesively or otherwise secured to the bottom 20. portion 34 of the housing of the test device 30.
The application, separation and reaction zones are contiguous w;ith-in the co-planar surfaces of the glass fiber matrix. A sample or samples applied to the sample application zones 38 will mlgrate laterally by capillary and chromatographic action. As will be described herein-~elow in greater detail, the fundamental result achieved by the test devices constructed in accordance with the teachings of the invention is bilateral flow of the fluid 5. component of suspensions applied to the application zones 38.
During the bilateral migration, particulate matter present within the sample volume, i.e., cellular components of whole blood, salt crystals of urine or protein aggregates of serum or plasma, etc., are filtered from the fluid portion 10. of the applied sample by particle size exclusion dictated by the mean pore size of the glass fiber matrix. Since the mean pore size of the glass fiber matrix is not an absolute value, but, rather, represents a Poisson distribution of a range of pore sizes, the length and width of the separation 15. zone will be influenced and dictated by the mean porosity of the glass fiber matrix. Likewise, since the mean diameter o particulates within the sample will vary, a separation gradient will be realized within the body of the separation zone, with larger particulates remaining closer 20. to the application zone, while smaller particulates will mlgrate some distance from the application zone.
Therefore, the length and width of the separation zone between the reaction and sample application zone~ must be carefully established empirically in order to position the reaction zone at a proper distance from the sample application zone to prohibit an inhibitory quantum of particulates from entering the reaction zone. If the 5. separation zone length is too short, some particulates may enter the reaction one; if too long, the volume of filtered sample fluid containing the desired analyte to be detected may be insufficient for optimal detection.
Bilateral mi~ration of the fluid portion of the lO. applied sample is also channeled in a direction l80 degrees away from the separation zone and, subsequently, the reaction zone, by the tapered constriction in the lateral boundaries of the trapezoidally-shaped glass fiber matrix.
This constriction of the glass fiber matrix favors migra-15. tion of the sample through the separation zone in thedirection of the reaction zone, yet still allows for some migration Qf fluid awa~ from the separation and reaction zones~ facilitating removal of unwanted or interfering debris ~particulates, protein aggregates, unreacted test 20. reagents) from the reaction zone upon subsequent applica-tion of wash solution and/or test reagents to the reaction zone. In essence, this design functions as a safety valve and reduces or eliminates back-washing of unreacted 1317~76 components into the reaction zone which may cause high background gignals.
Sandwich relationship between the filter 36 and the cover 32 with the bottom portion 34 of the housing of 5. the filter and with the associated ahsorbent means 44 and 48 is illustrated in the cross-sectional view of FIG. 5, which, of course, is equally applicable to the test device of FIG. 1, as well as the test device of FIG. 2. It will be noted rom the showings of F~GS. 3 and 5 that a portion 10. of each application zone 38 is in fluid communication with the correspondiny first absorbent means 44, as best shown at 66 in FIGS. 3 and 5 of the drawings. Consequently, the absorbent means 44 is in fluid communication with the application zones 38 and causes a bilateral flow of fluid 15. from the sample being applied to the application zones simultaneously with flow in the opposite direction from the application zones 38 into the separation zones 42.
The bilateral flow through the separation zones 42 is facilitated by the location of the reaction zones 40 20. adjacent to a relatively large area of the filter 36, shown, in the particular embodiment of the test device, as generally rectangular in configuration and overlying the second absorbent means 48.
Consequently, bilateral flow established in this manner reduces the hydraulic pressure in ~he application zones 38 and causes rapid settling of par.ticulates or other inclusions in the sample suspension, thus causing rapid 5. settling out of the particulates or othex detritus before reaching the reaction zones 40.
It will also be noted that the secona absorbent means 48 is of much larger dimensions than the absorbent means 44, causing more rapid absorption of the excess ~luid 10. of the sample and causing the accentuation of the bilateral flow phenomenon achieved b.y the ~ilter design and its association with the first and second absorbent means.
It will be readily apparent to those skilled in . the art that the configuration of the application zones 38 15. can be readily altered to accommodate the needs of the particular samples being tested by the devices 10 and 30 and, furthermore, as specified hereinabove, the length and width of the separation zones be empirically established to con~orm to the bilateral flow patterns to be established 20. ~or the particular sample.
Moreover, the relativ~ dimensions and depth of the absorbent means 44 and 48 can be altered to establish qreater or lesser fluid communication between the application zones 38 and reaFtion zones 40, respectively.
1 3 1 7~76 A typical glass fiber matrix filter has its source in Eaton-Dikeman Division of Filtration Sciences, Mount Holly Springs, Pennsylvania. The weight is 71 gm/m2;
the depth is 0.43 mm; the mèan pore size is 0.6 micron (u~;
5. the mean fiber diameter is 0.7u ~0.25u to 1.5u); and the composition is borosilicate glass.
The dimensions of the application zone are 8 mm ; in diameter, and the separation zone 4 mm X 9mm.
These dimensions are suitable to effect se~aration 10~ of cells, protein aggregates or other debris from a 30-40 ul sample of human whole blood or serum a~plied to the sample application zone followed by a wash volume of S0-60 ul applied to the same. Modification of the preferred embodi-ment is indicated i~ the whole blood sample is from an 15. animal other than a human, such as equine or bovine whole blood, which generally has smaller red blood cell diameters (5.5u or 5.9u, respectively) than human (6.9u-8.1u). In this instance, a glass ~iber matrix of a smaller pore size would be desirable, or, alternately, a longer or shorter 20. separation zone may be re~uired.
` Although the filters 20 and 36 are described as fahricated in accordance with the previously set forth specifications, it will be obvious to those skilled in the .
`
1 3 ~ 7876 art that the filter means can be made of any porous material capable of drawing liquid through its structure by capillary action. The pores of the filter matrix should/ obviously, be sufficiently small to accomplish filter separation of the 5. insolubilized components of the test sample from solubilized components.
The filter may be composed of such materials as glass fiber filter paper, nitrocellulose,-plastic, synthetic polymer, cellulose, cellulose acetate, and various other 10. equivalent materials having the qualities and characteristics described hereinabove.
Of course, it is desirable to utilize materials which are inert and chemically non-reactive with the analytes and washing solvents with which the test device lS to be 15. utilized.
The test devices 10 and 30, of course, have their respective reaction zones 28 and 40 treated with specific analyte reactants. ~ocalized regions o~ the respective filters 20 and 36 are treated to provide the reaction zones 20. 28 and 40 to prepare the test devices 10 and 30 for use with a predetermined test specimen without any preparatory addi~ions to the test devices. For example, a binding protein could be placed in the reaction zones to which an antibody is bound, which antibody is im~unologically 25. reactivé with a specific antigen.
1 31 787~
Consequently, when a specimen is applied to the application zones 26 and 3~ throu~h the application ports 22, the fluid component of the suspension is immediately sub~ected to the bilateral action achieved by ~he specific construction 5. of the test devices alluded to hereinabove.
The absor~ent material utilized in the first and second absorbent means 44 and 48 may be of any suitable material, such as hydrophilic polymers, particulate absor-bents, glass fiber, carbon fiber, cellulose iber, wood pulp 10. or sponge material.
As previously mentioned, the size and shape of the respective absorbent means 44 and 48 is dictated by the volumetric considerations apDlicable to the specific test for which the test devices 10 and 30 are designed, and 15. corresponding diminishment or enhancement of the absorptive capacity of the first and second absorbent means 44 and 48 result from empirical calculations of the needs for the establishment of greater or lesser bilateral flow of the fluid components of the test specimen.
20. Method of the Invention In practicing the method o the invention, a suitable volume of sample is applied directly to the sample application zone of the glass fiber matrix. A suitable :
:
1317~76 volume of wash reasent is then applie~ to the same area of the sample application zone.
Capillary and chromatographic forces within the body of the glass fiber matrix draw the fluid portion of 5. the sample primarily in the direction of the separation zone but, also, secondarily in the opposite direction.
The bilateral flow is defined by the lateral boundaries of the glass fiber matrix and the fluid communication of the application and reaction zones with their respective 10. absorbent means. As the fluid migrates through the separa-tion zone, particles larger than the mean pore size of the glass fiber matrix are restricted in their lateral migration toward the reaction zone, so that only the fluid portion of the sample reaches and flows into and through the reaction 15. zone. The analyte, contained within the fluid portion o the sample reacts and binds with the specific complimentary .
immuno-reagent (antigen or antibody) or chemical test reagents, which have been immobilized to the glass fiber matrix in the area of the reaction zone.
20. Subsequently, a suitable volume of wash reagent is applied directly to th~ ~ This washes away .... ,, ~. _ unreacted sample components which may interfere with subse-quent steps, in bilateral directions, again defined by the ' ' lateral boundaries of the glass fiber matrix, the directions being 1) away from the separation and sample application zones, and 2) toward the separation and sample application zones, reversing the original direc~ion of flow.
5. Wash in the latter direction inhibits or prevents previously filtered particulates from reaching the reaction zone and actually acts as a "counter current" to back flush potential interfering particulates present in the original sample away from the reaction zone. The test analyte 10. present in the fluid portion of the sample is bound to the complimentary ~m~no-reagent or chemical test reagent immobilized to the glass fiber matrix at the reaction zone site.
Subsequently, an immuno-reagent, or chemical test 15. reagent in the case of a biochemical test, complimentary to the test analyte, conjugated with an enzyme or other suitable tracer, such as a radionuclide or fluorescent dye, is applied directly to the reaction zone. Unbound immuno-reagent conjugate or chemical test reagent is washed from 20. the reaction zone in the lateral bi-directional mode outlined above by the application of a suitable wash volume applied directly to the reaction zone~
~;;
Sequentially, a suitable substrate or chromogen is added to the reaction zone. I the analyte was pre.sent in the sample, it will be sandwiched between the immobilized and enxyme conjugate immuno-reagents within the reaction 5. zone. The enzyme conjugated to the analyte bound immuno-reagent acts upon the substrate or chromogen to produce a colored product within the reaction zone which may be viewed or measured with an instrument.
Exem lar Filter Construction and P Y__ 10. Methods of Utilizing Same Example 1. Detection of Antibody to Rubella Virus in Whole Blood Inactivated Rubella virus antigen is immobilized onto the reaction zone of the glass fiber matrix. This is 15. followed by the addition of a blocking agent such as 1.0 - bovine serum albumin or 0.5~ non-fat milk suspension to the same area and allowed to dry.
The use of a blocking agent decreases the non-specific binding of extraneous proteins present in the 20. fluid (serous) portion of whole blood to the reaction zone of the glass fiber matrix.
1 31 787h To perform an assay, approximately 30 microliters of whole blood is applied to the sample application zone of the device. This is followed by 60 microliters of a wash solution consisting of 0.53 non-fat milk in a phosphate 5. buffered saline applied to the same area. Migration of the whole blood sample through the separation zone will filter and separate the cellulax components from the sample within the area of the separation zone. Evidence of separation of the fluid portion of the whole blood sample is observed lQ. in the reaction zone by the appearance of serous fluids wetting the reaction zone area.
60 microliters of the wash solution is then applied to the wetted reaction zone. Upon absorption, the .., ,. _ . . ...
wash step is repeated. It will be noticed that the serous lS. components and pigments contained therein will be eliminated via the lateral bi-directional mode described earlier from the reaction zone by this wash procedure.
However, if the whole blood specimen contains antibodies to the Rubella virus, the antibodies in the 20. serous portion of the blood sample will bind to the Rubella virus antigens immobilized within the reaction zone of the glass fiber matrix. Thenl 60 microliters of an affinity purified rabbit anti-human IgG alkaline phosphatase con~ugate is applied to the reaction zone. This will bind to the antibody of the Rubella virus which may be present in the blood sample and will be trapped by the immobilized an~igen located in the reaction zone of the glass fiber matrix.
5. Unreacted en2yme conjugate is washed away as described above.
Finally, 60 microliters of a suitable`substrate chromogen may be applied to the reaction zone. Appearance of a colored product at the reaction zone is evidence of enzyme activity and, therefore, indicative of antibody to 10. Rubella virus present in the whole blood sample.
Example 2. Determination of Human Choriogonadotropin in Urine by _ a "Sandwich" Technique A polypeptide hormone, human choriogonadotropin 15. (HCG), is secreted into the maternal circulatory system by the trophoblasts of the developing fetus. Ultimately, this hormone is excreted in the maternal urine. Detection of ~CG in the urine is presumptive evidence of pregnancy. HCG
is collected, concentrated and purified by well known pub-20. lished methods. The purified hormone may be used to generateantibodies (polyclonal or monoclonal) in the appropriate species, i.e., rabbits or mice, respectively.
1 31 7~76 The antibody of HCG is immobilized to the reaction zone of the glass fiber matrix. A blocking protein is then applied to the reaction zone as described in the previous example.
5. To determine if a urine specimen contains HC~7, a few drops of the specimen are applied to the sampl~ appli-cation zone of the glass fiber matrix of the device. This is followed by a sufficient volume of a wash solution applied to the same area to cause the sample to migrate 10. throu~h the separation zone towar~ and through the reaction zone of the glass fiber matrix which contains the immobi-lized antibody to HCG. If HC~, is present in the sample, it will ~ind to the i~mobilized antibody located within the reaction zone.
15. Alternately, sufficient volume of urine may be applied to the sample application zone to cause the sample to chromatograph through the reaction zone without the use of a wash. In either case, migration of the urine sample through the separation zone will filter out urine particu-20. lates which may interfere in subsequent testing steps.
Then, a suitable volume of a washing solution is applied to the reaction zone. Lateral, bi directional flow of the wash solution will carry unreacted urine components away from the reaction zone, i.e., away from the separation and sample application zones as well as toward the separa tion and sample application zones, reversing the original dixection of flow. Movement toward the reaction zone of wash fluid in the latter dixection prohibîts further mo~e-5. ment of unwanted particulates by counter flow forces.
Indeed, subsequent addition of any wash or test reagent to : the area of the reaction zone will force any trapped particulates or debris located within the separation zone away from the reaction zone.
lO. Applicatian to the reaction zone of an appropriate enzyme labeled antibody to HCG (either polyclonal or mono-clonal~ will bind to the HCG of the sample which has been trapped by the immobilized antibody bound to the reaction zone of the glass fiber matrix. Again, a wash solution is 15. applied, as indicated above, to wash away, in a lateral, bi-directional mode, any unreacted enzyme conjugated anti-body.
Subsequent addition of a suitable substrate chromogen solution to the reaction zone will indicate the 20. presence of enzyme and, therefore, the presence of HCG, by : the development of a colored product at the reaction zone.
The method described above in this example is typical of a "sandwich technique", whereby the analyte, HC~. in this - .
~317876 case, is sandwiched between two antibodies, one immobilized to the glass fiber matrix of the reaction zone, the other - conjugated to an enzyme or other suitable label. The presence of the HCG analyte is indicated by the development 5. of color within the reaction zone.
Example 3. Determination of Human Choriogonadotropin in Urine by Competitive Inhibition Immunoassa Y
10. The test devices of the invention are not limited to "sandwich" methodoLogy, but ma~ be applied to aompetitive inhibition techniques as described by the following example.
Antibody immobilization, sample application and washing methods and separation/chromatographic principles are as 15. described in the prevlous example. However, instead of application of an antibody enzyme conjugate, one may apply to the reaction zone an enzyme conjugate of the analyte, i.e., HCG coupled to an appropriate enzyme.
If HCG is present in the sample~ it will bind to 20. a finite and limited number of available antibody binding sites located and lmmobilized within the reaction zone of the glass fiber matrix. If the sample contains substantial amounts of HCG, then all available antibody binding sites in the reaction zone will be saturated.
Upon subsequent application of an enzyme conjugated to HCG (instead of enzyme conjugated to an anti-HCG antibody), all available immobilized antibody binding sites are saturated with the HCG from the sample and will not bind to the enzyme-5. HCG conjugate. When a sui~able wash solution is applied,the enzyme-HCG conjugate will be washed away from the reaction zone in a lateral, bi-directional fashion.
Application of a suitable substrate chromo~en solution to the xeaction zone will not develop a color in 10. this ins~ance sinca no enzyme is available. If, howev~r, the sample contains no or insufficient quantities of HCG, to saturate all immobilized antibody bindin~ sites, then HC~
enzyme conju~ate will bind to the available immobilized HCG binding sites and will not be washed away with subse-15. quent washing steps.
Therefore, in this instance, upon subsequentapplication of a suitable substrate chromogen solution to the reaction zone of the glass fiber matrix, some color will develop, indicating the sample had little or no HC&
20. present. In a competitive inhibition assay as just des-cribed, the absence of color development in the reaction zone is indicative of the presence of the analyte (HCG) in the sample, while~he presence of color development in the reaction zone indicates little or no analyte (HC~.) in the sample. The device can also be used for competitive immuno-assays of low molecular weight analytes, such as thyroid hormones, therapeu~ic drugs, steroids and other low 5. molecular weight analytes.
Example 4. Detection of Glucose in Whole Blood __ _ _ _ _ _ The device may be used to perform assays to indicate the presence or quantitation of analytes without 10. employing immunological methods and principles. For example, one may detect the presence of glucose in whole bLood by standard enzyme analytical techniques. In this instance, a mixture of the enzymes glucose oxidase and ~ horseradish peroxidase is immobilized to the reaction 15. zone of the device. Whole blood is then applied to the sample application zone.
A suitable wash solution is then applied to the sample application zone to effect the bi-directional lateral chromatographic separation of the fluid portion of the 20. sample ~rom the ce1lular components as described previously to introduce the fluid portion containing glucose into the reaction zoneO The immobilized oxidase acts upon the giucose of the sample to produce D-glucono-3-lactone and hydrogen peroxide.
~ 3 1 7876 The horseradish peroxidase, also i~mobilized within the reaction zone, catalyses the hydrogen peroxide in situ as it is generated. Subse~uent addition to the reaction zone of a suitable chromogen test reagent will 5. react with the products of catalysis to produce a colored product, the intensity of which is proportional to the amount o glucose present in ~he original sample. The intensity of color development may be observed visually or detected by the use o~ suitable instrumentation. It 10. is evident from this example that the device of the invention accomplishes other than immunoassays with equal efféctiveness.
It will be readily apparent that the utilization of the test devices manufactured in accordance with the ~; 15. teachings of this invention provides both more effective and less time-consumin~ testing of various suspensions in the field by relativel~ inexperienced personnel. ~he bilateral mi~ration of the flui~ components of the various samples applied to the application zones attributable to 20. the unique construction of the test devices prevents the contamination of the reaction zones by the partlculates in the suspension samples and also facilitates the migra-tion of the fluid component of the sample to the reaction zones.
~`
Back round of the Invention:
The invention relates to a diagnostic device for performing solid phase immunoassays to detect the presence of antigens or antibodies in biological or non-biological 5. fluids. The teachings of the invention are incorporated in a bi-directional lateral chromatographic device for use in solid phase immunoassays or for the non-immunological detection or quantitation of proteins or substances in biological or non-bioloqical fluids.
10. More particularly, the invention relates to devices and methods which utilize filter means for testing biological fluids to detect the presence of analytes such as bacterial, viral, parasitic, or funqal antigens and immunoglobulins, hormones, serum proteins, drugs and the - 15. like.
$ypical of prior art devices presently in use are the teachings of U.S. Patent 4,623,461, which discloses a filter body located in a housing having an openin~ therein for the reception of a suspension sample to permit the 20. upper face of the filter to trap colored or particulate matter contained within the specimen and prevent such matter from reaching the bottom face of the reaction zone, which has been previously treated with a suitable reactant.
The perimeter of the filter is engaged with a suitahle absorbent body and the absorbent body is intended to receive 5. the outward diffusion of liquids applied to the filter.
One of the disadvantages of the '461 construction lies in the fact that the fluid flow from ~he point of application of the suspension to the absorbent hody is uni-directional and a subtantial accumulation of solids at the 10. point of application of the sample suspension is inevitable, which will seriously i~pinge upon the resultant chroma-tological or other type of test reading imparted by the device.
U.S. Letters Patent 3,825,410 discloses a disposable 15. combined storage and reaction cell for use in the performance o chemical and biological reactions which receives reactants dispensed therein and maintains the same in stored condition so that they remain stable. Reactants will not mutually react until such time as it is required to initiate the 20. reaction.
The immobilization of the reactants is accomplished by such procedures as freeze drying and the reaction is initiated by the introduction of a sample to be analyzed, ~, ' \
whereafter separation of bound and free ligand can be performed either within the unit itself or externally.
The reaction cell of the '410 patent may include a filter so that the entire process of separation can be 5. completed within the reaction cell and the filter be removed from the reaction cell and submitted for radioactivity or other tracer counts.
~ J.S. Letters Patent 3,888,629 discloses a reaction cell for performing various types of assays which incorpo-LO. rates a matrix pad of absorbent material retaining the necessary reagents for the reaction and serving as a site in which the reaction totalIy occurs. A se~arable lower chamber incorporates absorbent material abutting the matrix pad to promote filtration through the pad after the reaction lS. has taken place~ ~
Both patents are characterized by the mere uti-lization of the filter as a pass-through device which is time-consuming and which is hi~dered by the deposition of solids out of the suspension sample.
20. Objects and Advanta~es of the Invention:
One of the objects of ~he invention is the pro-vision of a diagnostic ~evice which incorporates a planar filter body havinq sample application, separation Ind reaction zones, said filter body bein~ configured in such a manner that the bulk o the solids in the suspension sample are retained in the sample application zone and the 5. fluid ls caused to mi~rate bilaterally through the inter-stices of the filter by the 1uid communication of absorbent means with the application and reaction zones.
Conse~uently, the unilateral directional flow which causes accumulation of solids in the reaction zone 10. in the previously discussed prior art devices is eliminated in the test device of our construction because the rapid bilateral flow achieved by the construction of the device causes i~mediate deposition of solids out of the fluid component of the suspension.
15. Another object of the invention is the provision of a diagnostic test device of the aforementioned character wherein the planar filter incorporates a sample application zone which is relatively large and which is connected to the reaction zone by a separation zone, the length of the 20. separation zone being proportioned to the character of the suspension sample applied to the a~plication zone, and the separation and application zones cooperate to retain the bulk of the solids or particulate matter in the application and separating zones so that the immuno-reagent or chemical test reagent deposited in the reaction zone, when subjected to the test procedures and analytes, will have a minimum of or no particulates embodied therein which would cause the 5. emission of high background signals, thu~ creating a negative effect on the tes~ readout.
~ nother important object of the invention is the provision of a composite housing filter conjugate which is characterize~ by ease of assembly and application. The 10. test device of the invention is designed particularly for use in the field or the testing of various human and animal diseases or for various chemical tests involving the utilization of blood samples from humans and animals, and it is capable of giving test results equal to labora-15. tory results within a matter of minutes depending upon thesample solution which is applied to the specific device.
Another object of the invention is the provision of a method of performing a test by the utilization of the device of the invention which incorporates a plurality of 20. simple steps which can be carried forth by non-laboratory personnel in the field and which can provide such personnel with an almost immediate readout of the presence or absence of the sought-after infection or dru~, or the like.
According tG one aspect of the invention there is provided in a test device for per~orming solid phase immunoassays, the combination of a planar fiber filter matrix, said matrix including a test sample application zone, a separation zone and a reaction zone, said separation zone separating said application zone from said reaction zone and being sufficiently long to prevent migration of particulates in said sample into said reaction zone, first absorption means contiguous to said sample application zone, and second absorption means contiguous to said reaction zone whereby bilateral flow of the liquid componerlt of said test sample occurs.
According to another aspect of the inveDtion there is provided in a test device for performing solid phase immunoassays, the combination of a housing, a planar filter matrix located in said housing, said filter matrix iDcorporating a test sample application ~one, a separation zone integIal with said test sample application zone and a reaction zone communicating with said separation zone, a first absorbent means located in said housing in fluid communication with said application z~lne, and second absorbent means in said housing in spaced relationship with said first absorbent means and in fluid communicatlon with said Ieaction zone whereby bilateral flow of the fluid component of said test sarnple occurs.
According to a further aspect of the invention there is provided in a chromatographic test device for performing solid phase immunoassays, the combination a planar filter matrix including a body portion having a plurality of laterally extending integral arrns disposed in spaced relationship with one another, each of said anns having an expanded end portion, first absorbent means in fluid communication with said expanded end portions and second absorbent means in fluid communication with said body portion whereby bilateral flow of fluid deposited upon said expanded end portions may be achieved.
According to yet a further aspect of the invention there is provided a method of perfonning an immunoassay in coryunction with a chromatographic test device which incorporates a planar filter matrix having sample application, separation and reaction zones, and fiIst and second absorbent means in fluid comrnunication, respectively, with said application and reaetion zones, the steps of applying a suitable volume of liquid sample to said sample application zone, applying a suitable volume of wash reagent to said sample application zone, permitting capillary forces within said filter matrix to bilaterally draw the fluid portion of said sample primarily in the direction of said separation zone, but secondarily in the direction of said first absorbent means, entrapping particulates in said application and separation zones, permitting the fluid portion of said sample to flow into said reaction zone which is treated with a reagent immobilized to the fiber matrix, applying a suitable volume of wash reagent to said reaction zone to wash away ur~eacted sample components in bilateral directions away ~rom the reaction zone and toward the separation and sample application zones, applying a tracer reagent to said reaction zone, washing said reaction zone, and applying chromatic-eliciting substrate to said reaction zone to produce a c}~omatic reaction.
Brief Descri~tion of the Drawin~s:
~_.
Other objects and advantages of the invention will be apparent from the following specificat:ion and the accom-panying drawings, which are fox the purpose of illustration 5. only, and in which:
FIG. 1 is a top plan view of a ~ypical device of the invention;
FIG. 2 is a device similar to FIG. 1 with the exception that it incorporates a plurality of application 10. and reaction zones;
FIG. 3 is a top plan view showing the housing of the device of FI~. 2 with the cover removed therefrom to ` .
illustrate the location of and configuration of the filter and the relation thereof with tha absorbent means and the 15. particular design of the housing to encapsulate the filter and absorbent means;
FIG. 4 is a view similar to FIG. 3 illustrating removal of the filter to disclose the relationship of the absorbent means with the housing and the particular con-20. ~figuration thereof;
FIG. 5 i5 a vertical sectional view taken onthe broken line ~-5 of FIG. 2 and illustrates the eluid relationship~of the filter with the absorbent means and , the filter and absorbent components with the specific design of the housing and co~er therefor; anfl FIG. 6 is an exploded view illustrating the various components of the test device.
5. Description of the Preferred Embodiments of the Invention:
The chromatic assay device of the present ~n~en-tion may be used to perform sol.id phase immunoassays for the detection of antigens or antibodies, hereinafter referred to as analytes, in biological and non-biological 10. fluids. The device may be non-immunologically used to identify and/or quantitate proteins or substances in biological and non-biologi~al fluids. The device may be : utilized to perform assays such as competitive or non-: competitive enzyme-linked immunoassays, enzyme-multiplied lS. immunoassays, enzyme-inhibition assays, heterogeneous or homogeneous fluorescent immunoassays, chemiluminescent and bioluminescent assays, these assays utilizing various labelled probes, and the like.
Obviously, the particular analyte test to be 20. used will depend upon the chosen sample and the desired result to be achieved.
Referring to the drawings, and particularly to FIG. 1 thereof, we show a test device 10 constructed in accordance with the teachings of our invention which is incorporated in a housing 12, said housing consisting of 5. a lower or bottom component 14 and a cover or closure 16.
The bottom component 14 of the housing may be fabricated by injection molding from suitable synthetic plastic materials such as polyethylene and, as will appear further hereinbelow, is specifical.Ly designed to receive a flat 10. co-planar filter 20.
The closure 16 overlies the filter 20 and is : secured to the housing by pressure-sensitive adhesive or other adhering means and incorporates an application port ~ or openina 22 and a reaction port or opening 24, said ports : 15. communicating, respectively, with the application zone 26 and the Eeaction zone 28~of the filter.
The test device 10 is designed for the performance of a sinqle test, but, as best shown in FI~,. 2 of the draw-ings,~a device 30 incorporating a multiplicity of application 20. ports 22 and reaotion ports 24 can be provided in a closure or cover 32 which overlies a suitabl~ configured, as will be explained in greater detail below, lower housing portion 34 and filter 36. The ports 22 and 24 respectively overlie application and reaction zones~38fand 40 of the filter 36.
.
131~87~
The filters 20 and 36 are constituted by a planar glass fiber matrix which is sandwiched be~tween the lower component of the housing and the cover therefor.
As best shown in FIG. 3 of the drawings, the 5. filter 36 includes a plurality of application zones 38 . and reaction zones 40 which are maintained in fluid .. ..
communication by separation zones 42. The separation zones 4~ are of elongate configuration and establish fluid communication between the application zones 38 and lO. the reaction zones 40.
The application zon~s 36 are shown as being roughly trapezoidal in configuration and provide a rela-tively large area for the application of the test sample.
For a purpose which wlll be expl~ined hereinbelow, the 15. separation zones 42 are of relatively restricted width in comparison with the width of the application zones 38.
Located in fluid communication with the appli-cation zones 38 is first absorbent means 44 constituted . by an elongated strip 46 of absorbent material. Similarly, 20. a second absorbent means 48 constituted by an elongated strip 52 is disposed in fluid communication with the multiplicity of reaction zones 40 provided by the filter 36 .
:
. The lower or bottom portion 34 of the.housing of the test device 30 is configured, as best shown in FIGS. 4 and 5 of th~ drawings, to provide receptacles 55 for the application, separation and reac1:ion zones o.f 5. the filter 36 so that fluid flow is confined in the plane of the filter 36 because of the sandwich created between the closure 32 and the lower portion 34 of the ho~sing.
The receptacles are defined bv integrally molded lobes 58 in the body of the lower por~ion 34 of the housing and lO. stringently confine the relevant portions of the filter in the receptacles 56.
Juxtaposed to the receptacles 56 is a first elongated rectangular well 60 for the reception of the first absorbent means 44, and a corresponding well 62 15. is provided for the reception of the second absorbent means 48. ~
The closure or cover 32 of the device 30 can be fabricated from vinyl or other plastic sheet material and may be adhesively or otherwise secured to the bottom 20. portion 34 of the housing of the test device 30.
The application, separation and reaction zones are contiguous w;ith-in the co-planar surfaces of the glass fiber matrix. A sample or samples applied to the sample application zones 38 will mlgrate laterally by capillary and chromatographic action. As will be described herein-~elow in greater detail, the fundamental result achieved by the test devices constructed in accordance with the teachings of the invention is bilateral flow of the fluid 5. component of suspensions applied to the application zones 38.
During the bilateral migration, particulate matter present within the sample volume, i.e., cellular components of whole blood, salt crystals of urine or protein aggregates of serum or plasma, etc., are filtered from the fluid portion 10. of the applied sample by particle size exclusion dictated by the mean pore size of the glass fiber matrix. Since the mean pore size of the glass fiber matrix is not an absolute value, but, rather, represents a Poisson distribution of a range of pore sizes, the length and width of the separation 15. zone will be influenced and dictated by the mean porosity of the glass fiber matrix. Likewise, since the mean diameter o particulates within the sample will vary, a separation gradient will be realized within the body of the separation zone, with larger particulates remaining closer 20. to the application zone, while smaller particulates will mlgrate some distance from the application zone.
Therefore, the length and width of the separation zone between the reaction and sample application zone~ must be carefully established empirically in order to position the reaction zone at a proper distance from the sample application zone to prohibit an inhibitory quantum of particulates from entering the reaction zone. If the 5. separation zone length is too short, some particulates may enter the reaction one; if too long, the volume of filtered sample fluid containing the desired analyte to be detected may be insufficient for optimal detection.
Bilateral mi~ration of the fluid portion of the lO. applied sample is also channeled in a direction l80 degrees away from the separation zone and, subsequently, the reaction zone, by the tapered constriction in the lateral boundaries of the trapezoidally-shaped glass fiber matrix.
This constriction of the glass fiber matrix favors migra-15. tion of the sample through the separation zone in thedirection of the reaction zone, yet still allows for some migration Qf fluid awa~ from the separation and reaction zones~ facilitating removal of unwanted or interfering debris ~particulates, protein aggregates, unreacted test 20. reagents) from the reaction zone upon subsequent applica-tion of wash solution and/or test reagents to the reaction zone. In essence, this design functions as a safety valve and reduces or eliminates back-washing of unreacted 1317~76 components into the reaction zone which may cause high background gignals.
Sandwich relationship between the filter 36 and the cover 32 with the bottom portion 34 of the housing of 5. the filter and with the associated ahsorbent means 44 and 48 is illustrated in the cross-sectional view of FIG. 5, which, of course, is equally applicable to the test device of FIG. 1, as well as the test device of FIG. 2. It will be noted rom the showings of F~GS. 3 and 5 that a portion 10. of each application zone 38 is in fluid communication with the correspondiny first absorbent means 44, as best shown at 66 in FIGS. 3 and 5 of the drawings. Consequently, the absorbent means 44 is in fluid communication with the application zones 38 and causes a bilateral flow of fluid 15. from the sample being applied to the application zones simultaneously with flow in the opposite direction from the application zones 38 into the separation zones 42.
The bilateral flow through the separation zones 42 is facilitated by the location of the reaction zones 40 20. adjacent to a relatively large area of the filter 36, shown, in the particular embodiment of the test device, as generally rectangular in configuration and overlying the second absorbent means 48.
Consequently, bilateral flow established in this manner reduces the hydraulic pressure in ~he application zones 38 and causes rapid settling of par.ticulates or other inclusions in the sample suspension, thus causing rapid 5. settling out of the particulates or othex detritus before reaching the reaction zones 40.
It will also be noted that the secona absorbent means 48 is of much larger dimensions than the absorbent means 44, causing more rapid absorption of the excess ~luid 10. of the sample and causing the accentuation of the bilateral flow phenomenon achieved b.y the ~ilter design and its association with the first and second absorbent means.
It will be readily apparent to those skilled in . the art that the configuration of the application zones 38 15. can be readily altered to accommodate the needs of the particular samples being tested by the devices 10 and 30 and, furthermore, as specified hereinabove, the length and width of the separation zones be empirically established to con~orm to the bilateral flow patterns to be established 20. ~or the particular sample.
Moreover, the relativ~ dimensions and depth of the absorbent means 44 and 48 can be altered to establish qreater or lesser fluid communication between the application zones 38 and reaFtion zones 40, respectively.
1 3 1 7~76 A typical glass fiber matrix filter has its source in Eaton-Dikeman Division of Filtration Sciences, Mount Holly Springs, Pennsylvania. The weight is 71 gm/m2;
the depth is 0.43 mm; the mèan pore size is 0.6 micron (u~;
5. the mean fiber diameter is 0.7u ~0.25u to 1.5u); and the composition is borosilicate glass.
The dimensions of the application zone are 8 mm ; in diameter, and the separation zone 4 mm X 9mm.
These dimensions are suitable to effect se~aration 10~ of cells, protein aggregates or other debris from a 30-40 ul sample of human whole blood or serum a~plied to the sample application zone followed by a wash volume of S0-60 ul applied to the same. Modification of the preferred embodi-ment is indicated i~ the whole blood sample is from an 15. animal other than a human, such as equine or bovine whole blood, which generally has smaller red blood cell diameters (5.5u or 5.9u, respectively) than human (6.9u-8.1u). In this instance, a glass ~iber matrix of a smaller pore size would be desirable, or, alternately, a longer or shorter 20. separation zone may be re~uired.
` Although the filters 20 and 36 are described as fahricated in accordance with the previously set forth specifications, it will be obvious to those skilled in the .
`
1 3 ~ 7876 art that the filter means can be made of any porous material capable of drawing liquid through its structure by capillary action. The pores of the filter matrix should/ obviously, be sufficiently small to accomplish filter separation of the 5. insolubilized components of the test sample from solubilized components.
The filter may be composed of such materials as glass fiber filter paper, nitrocellulose,-plastic, synthetic polymer, cellulose, cellulose acetate, and various other 10. equivalent materials having the qualities and characteristics described hereinabove.
Of course, it is desirable to utilize materials which are inert and chemically non-reactive with the analytes and washing solvents with which the test device lS to be 15. utilized.
The test devices 10 and 30, of course, have their respective reaction zones 28 and 40 treated with specific analyte reactants. ~ocalized regions o~ the respective filters 20 and 36 are treated to provide the reaction zones 20. 28 and 40 to prepare the test devices 10 and 30 for use with a predetermined test specimen without any preparatory addi~ions to the test devices. For example, a binding protein could be placed in the reaction zones to which an antibody is bound, which antibody is im~unologically 25. reactivé with a specific antigen.
1 31 787~
Consequently, when a specimen is applied to the application zones 26 and 3~ throu~h the application ports 22, the fluid component of the suspension is immediately sub~ected to the bilateral action achieved by ~he specific construction 5. of the test devices alluded to hereinabove.
The absor~ent material utilized in the first and second absorbent means 44 and 48 may be of any suitable material, such as hydrophilic polymers, particulate absor-bents, glass fiber, carbon fiber, cellulose iber, wood pulp 10. or sponge material.
As previously mentioned, the size and shape of the respective absorbent means 44 and 48 is dictated by the volumetric considerations apDlicable to the specific test for which the test devices 10 and 30 are designed, and 15. corresponding diminishment or enhancement of the absorptive capacity of the first and second absorbent means 44 and 48 result from empirical calculations of the needs for the establishment of greater or lesser bilateral flow of the fluid components of the test specimen.
20. Method of the Invention In practicing the method o the invention, a suitable volume of sample is applied directly to the sample application zone of the glass fiber matrix. A suitable :
:
1317~76 volume of wash reasent is then applie~ to the same area of the sample application zone.
Capillary and chromatographic forces within the body of the glass fiber matrix draw the fluid portion of 5. the sample primarily in the direction of the separation zone but, also, secondarily in the opposite direction.
The bilateral flow is defined by the lateral boundaries of the glass fiber matrix and the fluid communication of the application and reaction zones with their respective 10. absorbent means. As the fluid migrates through the separa-tion zone, particles larger than the mean pore size of the glass fiber matrix are restricted in their lateral migration toward the reaction zone, so that only the fluid portion of the sample reaches and flows into and through the reaction 15. zone. The analyte, contained within the fluid portion o the sample reacts and binds with the specific complimentary .
immuno-reagent (antigen or antibody) or chemical test reagents, which have been immobilized to the glass fiber matrix in the area of the reaction zone.
20. Subsequently, a suitable volume of wash reagent is applied directly to th~ ~ This washes away .... ,, ~. _ unreacted sample components which may interfere with subse-quent steps, in bilateral directions, again defined by the ' ' lateral boundaries of the glass fiber matrix, the directions being 1) away from the separation and sample application zones, and 2) toward the separation and sample application zones, reversing the original direc~ion of flow.
5. Wash in the latter direction inhibits or prevents previously filtered particulates from reaching the reaction zone and actually acts as a "counter current" to back flush potential interfering particulates present in the original sample away from the reaction zone. The test analyte 10. present in the fluid portion of the sample is bound to the complimentary ~m~no-reagent or chemical test reagent immobilized to the glass fiber matrix at the reaction zone site.
Subsequently, an immuno-reagent, or chemical test 15. reagent in the case of a biochemical test, complimentary to the test analyte, conjugated with an enzyme or other suitable tracer, such as a radionuclide or fluorescent dye, is applied directly to the reaction zone. Unbound immuno-reagent conjugate or chemical test reagent is washed from 20. the reaction zone in the lateral bi-directional mode outlined above by the application of a suitable wash volume applied directly to the reaction zone~
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Sequentially, a suitable substrate or chromogen is added to the reaction zone. I the analyte was pre.sent in the sample, it will be sandwiched between the immobilized and enxyme conjugate immuno-reagents within the reaction 5. zone. The enzyme conjugated to the analyte bound immuno-reagent acts upon the substrate or chromogen to produce a colored product within the reaction zone which may be viewed or measured with an instrument.
Exem lar Filter Construction and P Y__ 10. Methods of Utilizing Same Example 1. Detection of Antibody to Rubella Virus in Whole Blood Inactivated Rubella virus antigen is immobilized onto the reaction zone of the glass fiber matrix. This is 15. followed by the addition of a blocking agent such as 1.0 - bovine serum albumin or 0.5~ non-fat milk suspension to the same area and allowed to dry.
The use of a blocking agent decreases the non-specific binding of extraneous proteins present in the 20. fluid (serous) portion of whole blood to the reaction zone of the glass fiber matrix.
1 31 787h To perform an assay, approximately 30 microliters of whole blood is applied to the sample application zone of the device. This is followed by 60 microliters of a wash solution consisting of 0.53 non-fat milk in a phosphate 5. buffered saline applied to the same area. Migration of the whole blood sample through the separation zone will filter and separate the cellulax components from the sample within the area of the separation zone. Evidence of separation of the fluid portion of the whole blood sample is observed lQ. in the reaction zone by the appearance of serous fluids wetting the reaction zone area.
60 microliters of the wash solution is then applied to the wetted reaction zone. Upon absorption, the .., ,. _ . . ...
wash step is repeated. It will be noticed that the serous lS. components and pigments contained therein will be eliminated via the lateral bi-directional mode described earlier from the reaction zone by this wash procedure.
However, if the whole blood specimen contains antibodies to the Rubella virus, the antibodies in the 20. serous portion of the blood sample will bind to the Rubella virus antigens immobilized within the reaction zone of the glass fiber matrix. Thenl 60 microliters of an affinity purified rabbit anti-human IgG alkaline phosphatase con~ugate is applied to the reaction zone. This will bind to the antibody of the Rubella virus which may be present in the blood sample and will be trapped by the immobilized an~igen located in the reaction zone of the glass fiber matrix.
5. Unreacted en2yme conjugate is washed away as described above.
Finally, 60 microliters of a suitable`substrate chromogen may be applied to the reaction zone. Appearance of a colored product at the reaction zone is evidence of enzyme activity and, therefore, indicative of antibody to 10. Rubella virus present in the whole blood sample.
Example 2. Determination of Human Choriogonadotropin in Urine by _ a "Sandwich" Technique A polypeptide hormone, human choriogonadotropin 15. (HCG), is secreted into the maternal circulatory system by the trophoblasts of the developing fetus. Ultimately, this hormone is excreted in the maternal urine. Detection of ~CG in the urine is presumptive evidence of pregnancy. HCG
is collected, concentrated and purified by well known pub-20. lished methods. The purified hormone may be used to generateantibodies (polyclonal or monoclonal) in the appropriate species, i.e., rabbits or mice, respectively.
1 31 7~76 The antibody of HCG is immobilized to the reaction zone of the glass fiber matrix. A blocking protein is then applied to the reaction zone as described in the previous example.
5. To determine if a urine specimen contains HC~7, a few drops of the specimen are applied to the sampl~ appli-cation zone of the glass fiber matrix of the device. This is followed by a sufficient volume of a wash solution applied to the same area to cause the sample to migrate 10. throu~h the separation zone towar~ and through the reaction zone of the glass fiber matrix which contains the immobi-lized antibody to HCG. If HC~, is present in the sample, it will ~ind to the i~mobilized antibody located within the reaction zone.
15. Alternately, sufficient volume of urine may be applied to the sample application zone to cause the sample to chromatograph through the reaction zone without the use of a wash. In either case, migration of the urine sample through the separation zone will filter out urine particu-20. lates which may interfere in subsequent testing steps.
Then, a suitable volume of a washing solution is applied to the reaction zone. Lateral, bi directional flow of the wash solution will carry unreacted urine components away from the reaction zone, i.e., away from the separation and sample application zones as well as toward the separa tion and sample application zones, reversing the original dixection of flow. Movement toward the reaction zone of wash fluid in the latter dixection prohibîts further mo~e-5. ment of unwanted particulates by counter flow forces.
Indeed, subsequent addition of any wash or test reagent to : the area of the reaction zone will force any trapped particulates or debris located within the separation zone away from the reaction zone.
lO. Applicatian to the reaction zone of an appropriate enzyme labeled antibody to HCG (either polyclonal or mono-clonal~ will bind to the HCG of the sample which has been trapped by the immobilized antibody bound to the reaction zone of the glass fiber matrix. Again, a wash solution is 15. applied, as indicated above, to wash away, in a lateral, bi-directional mode, any unreacted enzyme conjugated anti-body.
Subsequent addition of a suitable substrate chromogen solution to the reaction zone will indicate the 20. presence of enzyme and, therefore, the presence of HCG, by : the development of a colored product at the reaction zone.
The method described above in this example is typical of a "sandwich technique", whereby the analyte, HC~. in this - .
~317876 case, is sandwiched between two antibodies, one immobilized to the glass fiber matrix of the reaction zone, the other - conjugated to an enzyme or other suitable label. The presence of the HCG analyte is indicated by the development 5. of color within the reaction zone.
Example 3. Determination of Human Choriogonadotropin in Urine by Competitive Inhibition Immunoassa Y
10. The test devices of the invention are not limited to "sandwich" methodoLogy, but ma~ be applied to aompetitive inhibition techniques as described by the following example.
Antibody immobilization, sample application and washing methods and separation/chromatographic principles are as 15. described in the prevlous example. However, instead of application of an antibody enzyme conjugate, one may apply to the reaction zone an enzyme conjugate of the analyte, i.e., HCG coupled to an appropriate enzyme.
If HCG is present in the sample~ it will bind to 20. a finite and limited number of available antibody binding sites located and lmmobilized within the reaction zone of the glass fiber matrix. If the sample contains substantial amounts of HCG, then all available antibody binding sites in the reaction zone will be saturated.
Upon subsequent application of an enzyme conjugated to HCG (instead of enzyme conjugated to an anti-HCG antibody), all available immobilized antibody binding sites are saturated with the HCG from the sample and will not bind to the enzyme-5. HCG conjugate. When a sui~able wash solution is applied,the enzyme-HCG conjugate will be washed away from the reaction zone in a lateral, bi-directional fashion.
Application of a suitable substrate chromo~en solution to the xeaction zone will not develop a color in 10. this ins~ance sinca no enzyme is available. If, howev~r, the sample contains no or insufficient quantities of HCG, to saturate all immobilized antibody bindin~ sites, then HC~
enzyme conju~ate will bind to the available immobilized HCG binding sites and will not be washed away with subse-15. quent washing steps.
Therefore, in this instance, upon subsequentapplication of a suitable substrate chromogen solution to the reaction zone of the glass fiber matrix, some color will develop, indicating the sample had little or no HC&
20. present. In a competitive inhibition assay as just des-cribed, the absence of color development in the reaction zone is indicative of the presence of the analyte (HCG) in the sample, while~he presence of color development in the reaction zone indicates little or no analyte (HC~.) in the sample. The device can also be used for competitive immuno-assays of low molecular weight analytes, such as thyroid hormones, therapeu~ic drugs, steroids and other low 5. molecular weight analytes.
Example 4. Detection of Glucose in Whole Blood __ _ _ _ _ _ The device may be used to perform assays to indicate the presence or quantitation of analytes without 10. employing immunological methods and principles. For example, one may detect the presence of glucose in whole bLood by standard enzyme analytical techniques. In this instance, a mixture of the enzymes glucose oxidase and ~ horseradish peroxidase is immobilized to the reaction 15. zone of the device. Whole blood is then applied to the sample application zone.
A suitable wash solution is then applied to the sample application zone to effect the bi-directional lateral chromatographic separation of the fluid portion of the 20. sample ~rom the ce1lular components as described previously to introduce the fluid portion containing glucose into the reaction zoneO The immobilized oxidase acts upon the giucose of the sample to produce D-glucono-3-lactone and hydrogen peroxide.
~ 3 1 7876 The horseradish peroxidase, also i~mobilized within the reaction zone, catalyses the hydrogen peroxide in situ as it is generated. Subse~uent addition to the reaction zone of a suitable chromogen test reagent will 5. react with the products of catalysis to produce a colored product, the intensity of which is proportional to the amount o glucose present in ~he original sample. The intensity of color development may be observed visually or detected by the use o~ suitable instrumentation. It 10. is evident from this example that the device of the invention accomplishes other than immunoassays with equal efféctiveness.
It will be readily apparent that the utilization of the test devices manufactured in accordance with the ~; 15. teachings of this invention provides both more effective and less time-consumin~ testing of various suspensions in the field by relativel~ inexperienced personnel. ~he bilateral mi~ration of the flui~ components of the various samples applied to the application zones attributable to 20. the unique construction of the test devices prevents the contamination of the reaction zones by the partlculates in the suspension samples and also facilitates the migra-tion of the fluid component of the sample to the reaction zones.
~`
-2~-It is also contemplated by the invention that a plurality of test devices manufactured in accordance with the teachings of the invention and incorporating single application, separation and reaction zones may be snapped 5. together or otherwise associated on a mounting board or the like to permit a series o different tests to be accomplished by juxtaposition of the single test devices.
It will be obvious to those skilled in the art that various modifications of the test devices of the 10. invention may be made without departing from the sco~e of the claims.
.
,
It will be obvious to those skilled in the art that various modifications of the test devices of the 10. invention may be made without departing from the sco~e of the claims.
.
,
Claims (24)
1. In a test device for performing solid phase immunoassays, the combination of: a planar fiber filter matrix, said matrix including a test sample application zone, a separation zone and reaction zone, said separation zone separating said application zone from said reaction zone and being sufficiently long to prevent migration of particulates in said sample into said reaction zone; first absorption means contiguous to said sample application zone; and second absorption means contiguous to said reaction zone whereby bilateral flow of the liquid component of said test sample occurs.
2. The test device of Claim 1 wherein said test sample application zone has an extremity in fluid communication with said first absorbent means to cause the absorption of a portion of the fluid component of said test sample by said first absorbent means.
3. The device of Claim 2 wherein said reaction zone is in fluid communication with said second absorption means to absorb test fluid component of said sample and excess reagent conveying said sample.
4. The test device of Claim 1 in which said test sample application zone is of bulbous configuration, said separation zone being of reduced width to cause bilateral flow of the fluid component of said sample, respectively, from an extremity of said application zone into said first absorbent means and through said separation zone into said reaction zone and, ultimately, into said second absorbent means.
5. The device of Claim 1 wherein said first absorbent means underlies an extremity of said test sample application zone and said second absorbent means is in fluid communication with said reaction zone.
6. The device of Claim 1 in which said matrix is fabricated from glass fiber and said absorbent means from cellulosic material.
7. The device of Claim 1 in which the porosity of said matrix, as determined by the range of pore sizes of said matrix, is insufficient to permit substantial migration of particulates in said sample from said absorption zone and said separation zone into said reaction zone, but sufficient to permit bilateral flow of the liquid component of said test sample and said reagents accompanying the same into said reaction zone and to permit bilateral flow of said fluid component and added reagents into said first and second absorbent means.
8. The device of Claim 7 in which said application zone is of trapezoidal configuration and said separation zone is constituted by an elongated shank portion integral with said application zone and with said reaction zone to permit fluid flow from said application zone through said separation zone into said reaction zone.
9. The device of Claim 8 in which said application zone has an extremity overlying said first absorbent means and said reaction zone is in fluid communication with said second absorbent means to accomplish the aforesaid bilateral flow of said fluid component of said test sample.
10. The device of Claim 1 in which a portion of the perimeter of said application zone is in fluid communication with said first adsorbent means and said reaction zone is in fluid communication with said second absorbent means to establish bilateral flow of the fluid component of said test sample.
11. In a test device for performing solid phase immunoassays, the combination of: a housing; a planar filter matrix located in said housing, said filter matrix incorporating a test sample application zone, a separation zone integral with said test sample application zone and a reaction zone communicating with said separation zone;
a first absorbent means located in said housing in fluid communication with said application zone; and second absorbent means in said housing in spaced relationship with said first absorbent means and in fluid communication with said reaction zone whereby bilateral flow of the fluid component of said test sample occurs.
a first absorbent means located in said housing in fluid communication with said application zone; and second absorbent means in said housing in spaced relationship with said first absorbent means and in fluid communication with said reaction zone whereby bilateral flow of the fluid component of said test sample occurs.
12. The test device of Claim 11 in which the fluid communication of said application zone with said first absorbent means is accomplished by disposing said first absorbent means in contiguity to a portion of the perimeter of said application zone.
13. The device of Claim 11 in which said fluid communication between said first absorbent means and said application zone is accomplished by disposing said absorbent means below a portion of said application zone.
14. The device of Claim 11 in which said second absorbent means is disposed externally of said reaction zone.
15. The device of Claim 11 in which said housing incorporates a cover and said cover has first and second ports communicating, respectively, with said application and said reaction zones.
16. In a chromatographic test device for performing solid phase immunoassays, the combination: a planar filter matrix including a body portion having a plurality of laterally extending integral arms disposed in spaced relationship with one another, each of said arms having an expanded end portion; first absorbent means in fluid communication with said expanded end portions and second absorbent means in fluid communication with said body portion whereby bilateral flow of fluid deposited upon said expanded end portions may be achieved.
11. The device of Claim 16 wherein said expanded end portions provide test sample application zones, said arms provide separation and reaction zones communicating with each of said application zones to accomplish bilateral flow of the fluid component of the test sample deposited upon each of said application zones.
18. The device of Claim 16 in which the fluid communication between said first absorbent means and said expanded end portions is achieved by overlying contiguity of portions of said expanded end portions with said first absorbent means and said liquid communication of said body portion is achieved by corresponding overlying relationship of said body portion with said second absorbent means.
19. The device of Claim 16 in which said filter matrix is provided with a housing, said housing including, respectively, first and second receptacles for said first and second absorbent means and receptacles for receiving said body, arm and expanded portions of said matrix.
20. The device of Claim 19 in which said housing is provided with a cover and said cover incorporates ports corresponding, respectively, with said application and reaction zones of said filter matrix.
21. A method of performing an immunoassay in conjunction with a chromatographic test device which incorporates a planar filter matrix having sample application, separation and reaction zones, and first and second absorbent means in fluid communication, respectively, with said application and reaction zones, the steps of:
applying a suitable volume of liquid sample to said sample application zone; applying a suitable volume of wash reagent to said sample application zone; permitting capillary forces within said filter matrix to bilaterally draw the fluid portion of said sample primarily in the direction of said separation zone, but secondarily in the direction of said first absorbent means; entrapping particulates in said application and separation zones;
permitting the fluid portion of said sample to flow into said reaction zone which is treated with a reagent immobilized to the fiber matrix; applying a suitable volume of wash reagent to said reaction zone to wash away unreacted sample components in bilateral directions away from the reaction zone and toward the separation and sample application zones; applying a tracer reagent to said reaction zone; washing said reaction zone; and applying chromatic-eliciting substrate to said reaction zone to produce a chromatic reaction.
applying a suitable volume of liquid sample to said sample application zone; applying a suitable volume of wash reagent to said sample application zone; permitting capillary forces within said filter matrix to bilaterally draw the fluid portion of said sample primarily in the direction of said separation zone, but secondarily in the direction of said first absorbent means; entrapping particulates in said application and separation zones;
permitting the fluid portion of said sample to flow into said reaction zone which is treated with a reagent immobilized to the fiber matrix; applying a suitable volume of wash reagent to said reaction zone to wash away unreacted sample components in bilateral directions away from the reaction zone and toward the separation and sample application zones; applying a tracer reagent to said reaction zone; washing said reaction zone; and applying chromatic-eliciting substrate to said reaction zone to produce a chromatic reaction.
22. The method of Claim 21 in which said tracer reagent is conjugated with an enzyme.
23. The method of Claim 21 in which said tracer reagent is conjugated with a radionuclide.
24. The method of Claim 22 in which said tracer reagent is conjugated with a fluorescent dye.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/133,804 US5006474A (en) | 1987-12-16 | 1987-12-16 | Bi-directional lateral chromatographic test device |
| US07/133,804 | 1987-12-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1317876C true CA1317876C (en) | 1993-05-18 |
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ID=22460378
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000585262A Expired - Fee Related CA1317876C (en) | 1987-12-16 | 1988-12-07 | Bi-directional lateral chromatographic test device |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US5006474A (en) |
| EP (1) | EP0322340A3 (en) |
| JP (1) | JPH02138870A (en) |
| AU (1) | AU2701388A (en) |
| CA (1) | CA1317876C (en) |
| DE (1) | DE322340T1 (en) |
| DK (1) | DK693188A (en) |
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| US4761381A (en) * | 1985-09-18 | 1988-08-02 | Miles Inc. | Volume metering capillary gap device for applying a liquid sample onto a reactive surface |
| IL79945A (en) * | 1986-09-04 | 1991-06-10 | I D L International Diagnostic | Method for the quantitative determination of total cholesterol in blood |
| US4918025A (en) * | 1987-03-03 | 1990-04-17 | Pb Diagnostic Systems, Inc. | Self contained immunoassay element |
| US4857453A (en) * | 1987-04-07 | 1989-08-15 | Syntex (U.S.A.) Inc. | Immunoassay device |
-
1987
- 1987-12-16 US US07/133,804 patent/US5006474A/en not_active Expired - Lifetime
-
1988
- 1988-12-07 CA CA000585262A patent/CA1317876C/en not_active Expired - Fee Related
- 1988-12-13 DK DK693188A patent/DK693188A/en not_active Application Discontinuation
- 1988-12-14 DE DE198888630224T patent/DE322340T1/en active Pending
- 1988-12-14 EP EP19880630224 patent/EP0322340A3/en not_active Withdrawn
- 1988-12-16 AU AU27013/88A patent/AU2701388A/en not_active Abandoned
- 1988-12-16 JP JP63318365A patent/JPH02138870A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0322340A2 (en) | 1989-06-28 |
| DK693188D0 (en) | 1988-12-13 |
| DE322340T1 (en) | 1989-12-07 |
| JPH02138870A (en) | 1990-05-28 |
| US5006474A (en) | 1991-04-09 |
| AU2701388A (en) | 1989-06-22 |
| EP0322340A3 (en) | 1990-12-05 |
| DK693188A (en) | 1989-06-17 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |