Quantitative lateral flow assays and devices Number:7,144,742 from the United States Patent and Trademark Office (PTO) owispatent This invention provides solid phase specific binding lateral flow assay methods, devices and kits for quantitating high and low molecular weight analytes. The methods and devices of the invention employ labelled reagents which are either analyte analogs or complementary specific binding pair members for the analyte and a novel arrangement of capture zones comprising immobilized specific binding substances for either the analyte or the labelled reagent to effect bound from unbound labelled reagent as a function of analyte concentration. The capture zones are disposed on a non-bibulous matrix defining a flow path from a sample receiving zone to the capture zone. The devices of this invention also include multilane flow paths and multiple capture zones to quantitate analyte.<H Quantitative, devices, Quantitative, la, 7144742.html, Patent, pto, 7144742

Title: Quantitative lateral flow assays and devices

Abstract: This invention provides solid phase specific binding lateral flow assay methods, devices and kits for quantitating high and low molecular weight analytes. The methods and devices of the invention employ labelled reagents which are either analyte analogs or complementary specific binding pair members for the analyte and a novel arrangement of capture zones comprising immobilized specific binding substances for either the analyte or the labelled reagent to effect bound from unbound labelled reagent as a function of analyte concentration. The capture zones are disposed on a non-bibulous matrix defining a flow path from a sample receiving zone to the capture zone. The devices of this invention also include multilane flow paths and multiple capture zones to quantitate analyte.

Patent Number: 7,144,742 Issued on 12/05/2006 to Boehringer, et al.

Inventors: Boehringer; Hans (San Diego, CA), Rowley; Gerald (San Diego, CA), Pronovost; Allan D. (San Diego, CA)
Assignee: Quidel Corporation (San Diego, CA)

Appl. No.: 11/016,550

Filed: December 17, 2004

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
08812616	Aug., 2005	6924153

Current U.S. Class: 436/514 ; 422/55; 422/56; 422/57; 422/58; 435/287.1; 435/287.2; 435/287.7; 435/287.9; 435/7.5; 435/7.92; 435/7.93; 435/7.94; 435/805; 435/810; 435/969; 435/970; 435/973; 435/975; 436/169; 436/513; 436/518; 436/531; 436/533; 436/534; 436/805; 436/808; 436/810

Current International Class: G01N 33/558 (20060101)

Field of Search: 422/55-58,61 435/7.92,7.93,7.94,7.5,287.1,287.2,287.7,287.9,805,810,969,970,973,975 436/169,513,514,518,531,533,534,805,808,810

References Cited [Referenced By]

U.S. Patent Documents


4168146	September 1979	Grubb et al.
4271140	June 1981	Bunting
4435504	March 1984	Zuk et al.
4496654	January 1985	Katz et al.
4517288	May 1985	Giegel et al.
4740468	April 1988	Weng et al.
4806311	February 1989	Greenquist
4861711	August 1989	Friesen et al.
4883688	November 1989	Houts et al.
4943522	July 1990	Eisinger
4959307	September 1990	Olson
5200317	April 1993	Georgevich
5217905	June 1993	Marchand et al.
5229073	July 1993	Luo et al.
5252496	October 1993	Kang et al.
5310650	May 1994	McMahon et al.
5384264	January 1995	Chen
5500350	March 1996	Baker
5521102	May 1996	Boehringer
5559041	September 1996	Kang
5602040	February 1997	May

Foreign Patent Documents


0 191 640	Aug., 1986	EP
WO8808534	Nov., 1988	WO
WO-94/28415	May., 1994	WO

Other References

Allen et al., Clin. Chem. (1990) 36:1591-1597. cited by other .
Brown et al., "Natural family planning," American Journal of Obstetrics and Gynecology Oct. 1997, Part 2. cited by other .
International Search Report for PCT/US98/04441, mailed on Jul. 20, 1998, 2 pages. cited by other .
Lou et al., Clin. Chem. (1993) 39:619-624. cited by other .
Muggio et al., "Enzyme-Immunoassay," CRC Press, 1987, pp. 61, 184, 185. cited by other.

Primary Examiner: Chin; Christopher L.
Attorney, Agent or Firm: Morrison & Foerster LLP

Parent Case Text

This is a continuation application of U.S. Ser. No. 08/812,616, filed Mar. 6, 1997, issued as U.S. Pat. No. 6,924,153 on Aug. 2, 2005, which is hereby incorporated by reference in its entirety.

Claims

What is claimed is:

1. A device for determining an amount of an analyte in a sample, wherein the analyte is a member of a specific binding pair (sbp member), comprising a lateral flow matrix which defines a flow path and which comprises in series: a sample receiving zone; a labeling zone, wherein the labeling zone comprises a diffusively bound labeled first sbp member that is complementary to the analyte; and two or more serially oriented capture zones capable of providing visual quantitation of the amount of analyte in the sample; and wherein each capture zone comprises at least a second sbp member immobilized in the capture zone, the second sbp member being complementary to the analyte.

2. The device of claim 1, wherein the labeled first sbp member is an antibody capable of binding the analyte.

3. The device of claim 1, wherein the first sbp member includes a visually detectable label.

4. The device of claim 3, wherein the visually detectable label comprises a visible particulate label.

5. The device of claim 1, wherein the second sbp member is attached to particles and the particles are immobilized in the capture zones.

6. The device of claim 1, wherein the second sbp member is an antibody capable of binding the analyte.

7. The device of claim 1, wherein the second sbp member is labelled with a ligand and is immobilized on the capture zone by a receptor for the ligand coimmobilized on the capture zone.

8. The device of claim 7, wherein the ligand is a hapten and the receptor is a complement to the hapten.

9. The device of claim 1, wherein the second sbp member is an antibody against a complex formed between the analyte and the first sbp member.

10. The device of claim 1, wherein the analyte is a polyepitopic molecule and the first and second sbp members are antibodies against different epitopes of the analyte.

11. The device of claim 1, wherein the analyte is human IgE.

12. The device of claim 1, wherein the first sbp member is goat anti-human IgE and the second sbp member is mouse monoclonal anti-human IgE.

13. The device of claim 1, wherein the lateral flow matrix comprises two capture zones having the second sbp member uniformly immobilized in the single capture zones.

14. The device of claim 1, wherein the sample receiving zone comprises an amount of a third sbp member immobilized within the sample receiving zone and complementary to the analyte, the amount being sufficient to bind a threshold level of the analyte.

15. The device of claim 1, wherein the device comprises a plurality of discrete lateral flow matrices.

16. The device of claim 15, wherein the plurality of discrete lateral flow matrices have a common sample receiving zone, whereby a sample deposited in the sample receiving zone flows along each of the lateral flow matrices.

17. The device of claim 1 wherein a predetermined amount of the second sbp member is present in each capture zone.

18. A device for determining an amount of an analyte in a sample, wherein the analyte is a member of a specific binding pair (sbp member), comprising a lateral flow matrix which defines a flow path and which comprises in series: a sample receiving zone; a labeling zone, wherein the labeling zone comprises a diffusively bound labeled first sbp member that is complementary to the analyte; and two or more serially oriented capture zones capable of providing visual quantitation of the amount of analyte in the sample; wherein each capture zone comprises at least a second sbp member immobilized in the capture zone, the second sbp member being complementary to the analyte, and wherein the lateral flow matrix comprises a plurality of spatially separated capture zones.

19. The device of claim 18 wherein a predetermined amount of the second sbp member is present in each capture zone.

20. A device for determining an amount of an analyte in a sample, wherein the analyte is a member of a specific binding pair (sbp member), comprising a lateral flow matrix which defines a flow path and which comprises in series: a sample receiving zone; a labeling zone, wherein the labeling zone comprises a diffusively bound labeled first sbp member that is complementary to the analyte; two or more serially oriented capture zones capable of providing visual quantitation of the amount of analyte in the sample; and wherein each capture zone comprises at least a second sbp member immobilized in the capture zone, the second sbp member being complementary to the analyte; and wherein the pattern of binding of labeled analyte within the capture zone is a function of the analyte concentration.

21. The device of claim 20 wherein a predetermined amount of the second sbp member is present in each capture zone.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of solid phase binding assays using ligands and their specific binding receptors. In particular, it relates to the use of such assays to detect in a quantitative or semi-quantitative manner the amount of an analyte in a sample suspected of containing the analyte.

The ability to employ naturally occurring receptors or antibodies directed against specific compounds in assaying for the presence of a compound of interest has created a burgeoning immunoassay business. In these assays, a homologous pair of specific binding pair members ("sbp members"), usually an immunological pair, comprising a ligand and a receptor (antiligand) is involved, wherein one of the sbp members is labelled with a label which provides a detectable signal. The immunoassay methodology results in a distribution of the signal label between signal label bound in a complex of the sbp members and unbound signal label. The differentiation between bound and unbound signal label can be a result of physical separation of bound from unbound signal label or modulation of the detectable signal between bound and unbound signal label.

For the most part, immunoassays that have been directed towards quantitative determinations have required complex instrumentation, relatively sophisticated equipment, careful experimental technique and skilled operators, such as is found in clinical laboratories. Therefore, quantitative and semi-quantitative immunoassays have found less extensive application in settings such as the home, a medical practitioner's office, a health care maintenance organization setting or a primary care setting in a hospital where complex instrumentation is unavailable and testing is typically done by untrained personnel. Even in a clinical laboratory, simple and rapid tests for screening diagnostic, monitoring or prognosis for outcome purposes done by inexperienced personnel could provide substantial economies in terms of timeliness of results and labor costs.

In developing an immunoassay, there are many considerations. One consideration is to provide substantial differentiation between the observed signal resulting from signal label when bound as compared to unbound. Another consideration is to minimize interference from endogenous materials in the sample suspected of containing the compound of interest. A further consideration is the ease with which the observed signal can be detected and serve to differentiate between concentrations in the concentration range of interest. Other factors include the ease of preparation of reagents, the precision with which the samples and reagents must be prepared and measured, the storage stability of the reagents, the number of steps required in the protocol, and the proficiency with which each of the steps must be performed. Therefore, in developing a quantitative assay that can be used by untrained personnel, such as assays to be performed in the home, medical offices and the like, the observed result should be minimally affected by variations in the manner in which the protocol is carried out and the techniques for performing the various steps should be simple. Preferably, one-step protocols should be employed.

Recently, a variety of solid phase binding assays which do not require complex instrumentation have been described for detection of analyte in a sample suspected of containing the analyte. However, such assays typically provide qualitative results. Frequently, such solid phase assays also employ a multiplicity of steps, such as wash steps, to separate unbound label from bound label. Therefore, it would be desirable to provide simple, one-step solid phase non-instrumented methods and devices for quantitating an analyte in a sample suspected of containing the analyte. This invention fulfills that and other needs.

2. Summary of Related Art

A Noninstrumented Quantitative Test System and Its Application for Determining Cholesterol Concentration in Whole Blood; M. P. Allen et al.; Clinical Chemistry, Vol 36 No. 9: 1591 1597 (1990); discloses a noninstrumental solid phase method of quantitating cholesterol using enzymatic conversion of immobilized substrate.

One-Step Competitive Immunochromatographic Assay for Semiquantitative Determination of Lipoprotein(a) in Plasma; S. C. Lou et al.; Clinical Chemistry, Vol. 36 No. 4: 619 624 (1993); discloses semiquantitative measurement of lipoproteins using a solid phase assay with multiple capture bars.

Immunochemical semi-quantitative Assay Method--and appts.; Patent No. WO 94/28415A to H. Manita; discloses a semiquantitative chromatographic immunochemical assay method which consists of passing the sample for assay through a predetermined amount of an immobilized antibody (IAb) which recognizes the substance to be assayed, then allowing the excess to pass to a label (such as a color indicator) which indicates its presence.

Concentrating Immunochemical Test Strip; European Patent No. EP 0 191 640 A2 to D. Calderhead et al. (1986); discloses solid phase methods and devices for detecting analytes involving contacting a test strip containing a first sbp member with a test solution comprising the analyte and a second sbp member complementary to the analyte. The first sbp member is capable of binding the second sbp member.

U.S. Pat. No. 4,861,711; H. Friesen et al. (1989); discloses a solid-phase diagnostic device for the determination of biological substances.

U.S. Pat. No. 4,740,468; L. Weng et al. (1988); discloses a solid phase specific binding method and device for detecting an analyte.

U.S. Pat. No. 4,806,311; A. Greenquist (1989); discloses a multi-zone test device for analyte determination using a labelled reagent and immobilized reagent which are specific binding partners whose binding to each other depends on the amount of analyte present and a detection zone with an immobilized binding substance for the labelled reagent.

U.S. Pat. No. 4,168,146; A. Grubb et al. (1979); discloses a solid phase method and strip with bound antibodies.

U.S. Pat. No. 4,959,307; J. Olson (1990); discloses a solid phase method and device for detecting an analyte involving contacting a test solution containing sample, antibody to the analyte and a labelled analyte with a test strip containing an immobilized first receptor that binds to the labelled analyte and an immobilized second receptor that binds to the antibody.

U.S. Pat. No. 4,435,504; R. Zuk (1984); discloses a chromatographic immunoassay employing a sbp member and a label conjugate.

U.S. Pat. No. 4,883,688; T. Houts et al. (1989) discloses an immunochromatographic device which quantitates analyte as a function of the distance migrated by a sbp member along the device.

SUMMARY OF THE INVENTION

The present invention generally provides methods devices and kits for visually quantifying the amount on an analyte in a sample. For example, in one aspect, the present invention provides a method of determining an amount of an analyte in a sample, wherein the analyte is a member of a specific binding pair (sbp member). The method comprises providing a lateral flow matrix which defines a flow path and which comprises in series, a sample receiving zone, a labeling zone, and one or more serially oriented capture zones. The labeling zone comprises a diffusively bound labeled first sbp member that is complementary to or analogous to the analyte. Each of the one or more capture zones comprises at least a second sbp member immobilized in the capture zone, the second sbp member being complementary to the analyte. The sample is contacted with the sample receiving zone, whereby the sample flows along the flow path. Quantitation is carried out by observing the pattern of label that accumulates at the one or more capture zones and correlating that pattern to the amount of analyte in the sample.

In a related aspect, the methods of the invention are as described above, except that the labeling zone comprises a diffusively bound labeled first sbp member that is complementary to the analyte, and each of the one or more capture zones comprises at least a second sbp member immobilized in the capture zone, the second sbp member being analogous to the analyte. Quantitation is then carried out as above.

In another alternate aspect, the methods of the invention provide a lateral flow matrix which defines a flow path and which comprises in series, a sample receiving zone, a labeling zone, a barrier zone and one or more serially oriented capture zones. In this aspect, the labeling zone comprises a diffusively bound labeled first sbp member that is complementary to the analyte, the barrier zone comprises a second sbp member analogous to the analyte immobilized in the barrier zone, and each of the one or more capture zones comprises at least a third sbp member immobilized in the one or more capture zones, the third sbp member being capable of binding the first sbp member.

In yet another aspect, the labeling zone comprises a diffusively bound labeled first specific binding pair member that is analogous to the analyte, the barrier zone comprises a second specific binding pair member that is complementary to the analyte, and each of the one or more capture zones comprises at least a third specific binding pair member immobilized in the one or more capture zones, the third specific binding pair member being complementary to the analyte.

The methods of the invention also provide lateral flow matrices which define a flow path and which comprise in series, a sample receiving zone, a labeling zone and at least first and second serially oriented capture zones. In this aspect, the labeling zone comprises a diffusively bound labeled first sbp member that is complementary to the analyte whereby the first spb member and the analyte form an analyte-first spb member complex. The first capture zone comprises a second sbp member immobilized therein which is capable of binding the analyte-first sbp member complex with a first affinity, and the second capture zone comprises a third sbp member that is capable of binding the analyte-first spb member complex with a second affinity. In this aspect, the second affinity is typically different from the first affinity.

In addition to providing the methods described above, the present invention also provides devices for practicing these methods, i.e., for use in visually quantifying an amount of an analyte in a sample, and kits incorporating these devices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a single lane lateral flow assay device for visually quantitating analytes in accordance with the principles of this invention with a sample receiving pad 12, a labelling zone 14, a capture zone 16 with capture lines 16a, 16b and 16c and an absorbent zone 18.

FIG. 2 shows a multiple flow path lateral flow assay device in which multiple flow paths emanate from a single sample receiving zone 12 with labelling zones 14 on the flow paths and intermediate barrier zones 16a of varying analyte analogue concentration on the different flow paths are placed upstream of the detection zones 16b.

FIG. 3 shows a multiple flow path lateral flow assay device with multiple matrices in fluid flow isolation from each other, each matrix having a sample receiving pad 12 in which varying amounts of an sbp member for the analyte are diffusively bound, labelling zones 14, intermediate barrier zones 16a and detection zones 16b.

FIG. 4 shows an example of a flow path with detection zones having capture reagents of increasing affinity arranged in downstream sequence. The flow path has a sample receiving zone 12, a labelling zone 14 having a labelled biotinylated anti-analyte mouse IgG antibody, a barrier zone 16a having immobilized analyte or analyte analogue and detection zones 16b1, 16b2 and 16b3. 16b1 has monoclonal anti-mouse IgG of low affinity; 16b2 has polyclonal anti-mouse IgG of high affinity; and 16b3 has streptavidin of very high affinity.

DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention relates to solid phase specific binding pair lateral flow assays for the visual quantitative and semiquantitative determination of high and low molecular weight analytes in samples suspected of containing such analytes.

In describing the various aspects of the present invention, a number of terms will be generally defined as follows:

"Sample suspected of containing an analyte" shall mean any sample that is reasonably suspected of containing an analyte which can be analyzed by the method of the present invention. Such samples can include human, animal or man-made samples. The sample can be prepared in any convenient medium which does not interfere with the assay. Typically, the sample is an aqueous solution or biological fluid as described in more detail below.

The fluid sample may be a biological fluid such as, but not limited to, whole blood, serum, plasma, nasal secretions, sputum, urine, sweat, saliva, transdermal exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid, vaginal or urethral secretions, or the like. Herein, fluid homogenates of cellular tissues such as, for example, hair, skin and nail scrapings, meat extracts and skins of fruits and nuts are also considered biological fluids. Fluid samples also include nonbiological fluids such as, for example, soil extracts and water supplies. Multiple different analytes may be detected from a single fluid sample.

"Specific binding pair member" shall mean a molecule (sbp member) which is one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as being complementary with a particular spatial and polar organization of the other molecule. The two molecules are related in the sense that their binding to each other is such that they are capable of distinguishing their binding partner from other assay constituents having similar characteristics. The members of the specific binding pair are referred to as ligand and receptor (antiligand), sbp member and sbp partner, and the like. A molecule may also be a sbp member for an aggregation of molecules; for example an antibody raised against an immune complex of a second antibody and its corresponding antigen may be considered to be an sbp member for the immune complex. Complementary sbp members bind to each other, as for example, a ligand and its complementary receptor, such as biotin and avidin/streptavidin, antigen and antibody against that antigen. Sbp members will usually be members of an immunological binding pair such as an antigen-antibody, although other specific binding pairs, such as biotin-avidin, hormones-hormone receptors, nucleic acid duplexes, IgG-protein A, and the like are specific binding pairs which are not immunological binding pairs. An sbp member is analogous to another sbp member if they are both capable of binding to another identical complementary sbp member. Such an sbp member may, for example, be either a ligand or a receptor that has been modified by the replacement of at least one hydrogen atom by a group to provide, for example, a labelled ligand or labelled receptor. The sbp members can be analogous to or complementary to the analyte or to an sbp member that is complementary to the analyte. Different combinations of sbp members maybe used in the same assay or test device.

The present assays can be used for the quantitation and semiquantitation of any analyte for which a specific binding partner exists. Analytes may be polyvalent or monovalent. Polyvalent analytes include polypeptides and proteins, polysaccharides, nucleic acids, antibodies, microorganisms, bacteria, viruses and combinations thereof. Monovalent analytes include drugs, haptens, pesticides, pollutants, steroids, vitamins and the like. Descriptions and listings of representative analytes are found in U.S. Pat. Nos. 4,299,916, 4,275,149, 4,806,311, all incorporated by reference.

The term "analyte analog" or "ligand analog" refers to a modified analyte or analyte surrogate or modified ligand or ligand surrogate that can compete with the analyte for binding to an sbp member complementary to the analyte or ligand, such as an antibody against the analyte. The modification typically provides a means for attaching the analyte or ligand to another molecule, such as, for example, a label, or surface. The term "analyte surrogate" or ligand surrogate" refers to a compound that can specifically bind to a receptor complementary to the analyte or ligand. Thus, the analyte surrogate or ligand surrogate binds to the receptor in a manner similar to the analyte or ligand. The surrogate could be, for example, an antibody directed against the idiotype of an antibody to the analyte or ligand. Combinations of ligand analogs may be used.

The term "ligand" as used herein, means any compound for which a receptor naturally exists or can be prepared.

"Antigen" shall mean any compound capable of binding to an antibody, or against which antibodies can be raised.

"Receptor" shall mean any compound or composition capable of recognizing a particular spatial or polar orientation of a molecule, e.g., epitopic or determinant site. Illustrative receptors include: antibodies, enzymes, thyroxine binding globulin, intrinsic factor, lectins, nucleic acids, protein A, complement, complement C1q, and the like. Receptors are also referred to as antiligands.

"Antibody" shall mean an immunoglobulin having an area on its surface or in a cavity that specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be polyclonal or monoclonal. Antibodies may include a complete immunoglobulin or fragments thereof, which immunoglobulins include the various classes and isotypes, such as IgA (IgA1 and IgA2), IgD, IgE, IgM, and IgG (IgG1, IgG2, IgG3, and IgG4) etc. Fragments thereof may include Fab, Fv and F(ab').sub.2, Fab', and the like. Antibodies may also include chimeric antibodies made by recombinant methods.

The terms "impregnated" or "diffusively bound" or "freely suspendible" are meant to refer to a state of permeation or reversible surface adherence. Substances which are impregnated or diffusively bound are not immobilized within or upon the support matrix, but are capable of being mixed or suspended in fluids placed on the support matrix.

"Ancillary materials" shall mean any materials that may be employed in an assay in accordance with the present invention. For example, buffers will normally be present in the labelling means, the neutralization means, as well as stabilizers for the assay medium and assay components. Frequently, in addition to these additives, additional proteins, such as albumins, or surfactants, non-ionic or ionic, binding enhancers, e.g. polyalkylene glycols, or the like, may be present, including free antibodies, analyte analogs, or other unrelated ligands, for the purpose of removing or adding materials or to modify the amount, postition, partitioning, or appearance of the analyte or other compounds used in the invention.

Generally, the devices and methods of the present invention of the present invention employ lateral flow assay techniques and matrices capable of bibulous and/or non-bibulous lateral flow as generally described in U.S. Pat. Nos. 5,424,193, 4,943,522; 4,861,711; 4,857,453; 4,855,240; 4,775,636; 4,703,017; 4,361,537; 4,235,601; 4,168,146; 4,094,647; co-pending application U.S. Ser. No. 07/639,967, European Patent Application Nos. 451,800; 158,746; 276,152; 306,772 and British Patent Application No. 2,204,398; each of which is incorporated herein by reference.

It is a general object of the present invention to provide methods, devices, systems and kits which can be used to visually quantify or semi-quantify an amount of an analyte of interest in a particular sample. Although these systems are generally described in terms of visual quantification, it will be readily appreciated that other detection systems, optical or otherwise, may be used in such quantitation.

The present invention can be practiced in a variety of assay formats including sandwich and competitive modes. In one aspect of a competitive format, a labelled analyte analog and an analyte will compete for binding to a limited amount of binding sites on a complementary specific binding pair member. As explained in more detail below, the complementary sbp member is immobilized on a solid phase in a manner which allows for the direct quantitative or semi-quantitative read out of the amount of analyte present in the sample. In a sandwich assay, the sample suspected of containing the analyte is contacted with a complementary first sbp member to form a complex between the analyte and the first sbp member. Generally, the first sbp member is labelled. This complex is then contacted by a second sbp member, which is generally complementary to the analyte, bound to a solid support, which effects separation of the bound first sbp member from the unbound first sbp member. In the immunometric mode, a labelled antibody to the analyte is used as the first sbp member and analyte or analyte analog is immobilized on the solid phase to capture unbound labelled antibody. This effects separation of bound labelled antibody from unbound labelled antibody. Analyte concentration may be directly determined by detection of the amount of labelled antibody captured by the immobilized analyte. Optionally, a third sbp member which binds to the complex between analyte and labelled antibody may also be immobilized on the solid phase. As described in more detail below, detection of the amount of complex bound to the third sbp member provides an alternative method of quantitating analyte present in the sample. Similarly, combinations of third sbp members or ligands, antiligands, antiglobulins or ligand analogs maybe used to facilitate, define and/or control analyte separation directly or indirectly for quantitation by providing the means of controlling the efficiency of separation. In order to more fully describe and explain the assay formats capable of being employed in the invention, a variety of specific embodiments will now be described in more detail.

Sandwich Assay Format

One aspect of the invention employs a sandwich assay format. The sample is mixed with a labelled first specific binding pair member for the analyte and allowed to traverse a lateral flow matrix, past a series of spatially separated capture zones located on the matrix. The sample may be mixed with the labelled first sbp member prior to addition of the sample to the matrix. Alternatively, the labelled first sbp member may be diffusively bound on the matrix on a labelling zone at a point upstream of the series of capture zones. Sometimes, the sample is added directly to the labelling zone. Preferably, the sample is added to a sample receiving zone on the matrix at a point upstream of the labelling zone and allowed to flow through the labelling zone. The labelled first sbp member located within the labelling zone is capable of being freely suspendible in the sample. Therefore, if analyte is present in the sample, the labelled first sbp member will bind to the analyte and the resulting analyte-labelled first sbp member complex will be transported to and through the capture zones. The extent of complex formation between the analyte and the labeled sbp member is directly proportional to the amount of analyte present in the sample. A second sbp member capable of binding to the analyte-first sbp member complex is immobilized on each of the capture zones. This second sbp member is not capable of binding the labelled sbp member unless the labelled sbp member is bound to the analyte. Thus, the amount of labelled sbp member that accumulates on the capture zones is directly proportional to the amount of analyte present in the sample.

The analyte-labelled first sbp member complex flows sequentially through the series of capture zones and becomes bound to the capture zones. The first capture zone binds to and depletes some of the complex in the sample. Therefore, the concentration of complex which reaches the second capture zone is lower, having been depleted by the quantity of the complex which bound to the first capture zone, and the rate of binding of complex to the second capture zone is lower than the rate of binding of complex to the first capture zone. As such, for a given amount of analyte in the sample, detectable signal takes longer to appear on the second capture zone relative to the first capture zone.

Similarly, the concentration of complex reaching the third capture zone is also depleted relative to the concentration that reached the second capture zone and signal will take even longer to appear. In this fashion, it is apparent that for a given concentration of analyte in the sample, a downstream capture zone will take longer to produce a detectable signal compared to a capture zone that is upstream from it. Similarly, as the concentration of analyte in the sample increases and the amount of analyte-labelled first sbp member complex reaching the capture zones increases, detectable signal will appear earlier at a particular capture zone. Therefore, if the matrix is inspected at a predetermined time after sample addition, different analyte concentrations will produce a different pattern of signal on the series of capture zones. For example, a low concentration of analyte may only produce signal on the two most upstream capture zones, a higher analyte concentration may produce signal on the three most upstream capture zones, an even higher analyte concentration will produce signal on the four most upstream capture zones, and so on. Therefore, the number of lines with detectable signal is proportional to the amount of analyte present in the sample. The pattern of lines observed constitutes the composite signal that is correlated to the analyte concentration. Uniform color development on each line may be obtained by modifying the concentration or binding affinity of the sbp member binding reagent on each capture zone. As shown in the Examples, one may also inspect the strip at different time points and correlate the number of lines at which color is produced at different times with the amount of analyte present in the sample. The pattern of lines which produce signal can be correlated with the analyte concentration with the aid of a chart or other tabulation which allows the user to visually determine the analyte concentration by comparison to the chart; for example signal on the two most upstream lines indicates a certain analyte concentration, whereas signal on the three most upstream lines indicates another higher analyte concentration etc. Such charts or tables can be used with all embodiments of this invention described herein which correlate the number or pattern of lines showing signal with analyte concentration.

Adjustment of the concentrations of the labelled sbp member and the immobilized second sbp member allows one to control the quantity and rate at which color is produced at the capture zones for a given analyte concentration and thus to quantitate analyte within specific ranges. Typically, the amount of labelled specific binding pair member used is sufficient to bind all of the analyte that is expected to be present. For example, IgE is typically measured at 20 100 IU/ml urine; hCG at 10 200 mIU/ml urine and PDG at 1 20 .mu.g/ml urine. The concentration, affinity and combination of binding reagents may be experimentally determined to facilitate separation. Increasing the amount of second specific binding pair member immobilized in the capture zone will increase the efficiency of capture and produce signal more rapidly. The amount of second sbp member on the capture zone(s) can be adjusted to allow for discrimination between various ranges of analyte concentration. It is also possible to increase the discrimination between different analyte concentrations and provide sharper "cut offs" between those concentrations by immobilizing second sbp members of increasing affinity for the analyte on the capture zones as one proceeds in downstream sequence. One can also provide intermediate capture zones masked off from visual observation facilitating the efficiency of separation and color development.

Alternatively, combinations of reagents may be immobilized on the capture zone(s). For example, the first capture line may employ an antiglobulin and the second capture line may employ avidin both as a means to differentially capture labelled biotinylated globulins specific to an analyte and their complexes with analyte. Similar considerations apply to other assay formats and devices disclosed herein which rely on capturing a labelled sbp member or complex thereof by one or more multiple capture zones arranged in downstream sequence.

In this format, the analyte is generally a polyvalent analyte, such as protein or hormone, with multiple binding sites for the sbp members, wherein binding of the first sbp member does not interfere with the binding of the second sbp member. For example, when the analyte is IgE, the first sbp member may be an antibody against one epitope of the IgE and the second sbp member may be an antibody against a different epitope. Alternatively, the second sbp member may be an antibody specifically against the complex formed between IgE and the first sbp member.

This invention also provides a device for quantitating analyte concentrations as shown in FIG. 1. With reference to FIG. 1, the devices comprise a matrix capable of lateral non-bibulous flow comprising a sample receiving zone 12, an labelling zone 14, a capture zone 16 and optionally an absorbent pad 18 in fluid contact and arranged in downstream sequence as shown. The labelling zone has diffusively bound therein a labelling reagent, which in the case of the sandwich assay described above is an sbp member complementary to the analyte. Analyte in the sample flows to the labelling zone 14, binds to the labelled complementary sbp member therein and flows to the capture zone 16. The capture zone has immobilized therein a second sbp member complementary to the analyte as described above. The capture zone will comprise a series of spatially separated capture lines 16a, 16b, 16c and so on each of which has the second sbp member immobilized therein. The density of the second sbp member on the capture zones can vary or different second sbp members of increasing affinity for the analyte can be present in the sequence 16a<16b<16c and so on. Combinations of different ligand anti-ligand pairs may be used, such as second sbp members (e.g., antibodies) recognizing different epitopes of the analyte, the binding constants for which are varied. In this arrangement, there is no second sbp member immobilized on the segments of the matrix between the capture lines. The second sbp member binds the analyte-labelled first sbp member complex thus producing a visually detectable signal. As described earlier, when multiple capture lines are used as the detection zone, the pattern on lines on which signal is detected may be used to visually quantitate the analyte concentration, e.g., by comparing the pattern to a calibrated chart.

Competitive Formats

Another aspect of the invention employs a competitive format. The sample is mixed with a labelled analyte analog capable of binding to a first specific binding pair member complementary to the analyte and allowed to traverse a bibulous or non-bibulous matrix capable of lateral flow (also termed "a lateral flow matrix"), past a series of spatially separated capture zones located on the matrix. The sample flows sequentially past the series of capture zones. The sample may be mixed with the labelled analyte analog prior to addition of the mixture to the matrix. Alternatively, the labelled analyte analog may be diffusively bound to the matrix on a labelling zone at a point upstream of the series of capture zones. Sometimes, the sample is added directly to the labelling zone. Preferably, the sample is added to a sample receiving zone on the matrix at a point upstream of the labelling zone and allowed to flow through the labelling zone. The labelled analyte analog located on the labelling zone is capable of being freely suspendible in the sample.

A first specific binding pair member complementary to the analyte is immobilized on the capture zones. Sample and labelled analyte analog flow to and through the capture zones and compete for binding to the immobilized first sbp member. The rate of capture of labelled analyte analog by the capture zones is inversely proportional to the analyte concentration. At high analyte concentrations, the upstream capture zones are preferentially bound by analyte and detectable signal appears later on these capture zones. Labelled analyte analog competes effectively for binding at the downstream capture zones only after analyte has been depleted by binding at the upstream capture zones. This can be used to control the timing and rate of bound/free separation. Viewed alternatively, high analyte concentrations result in visually detectable signal appearing at the downstream capture zones as well as the upstream capture zones. Whereas at low analyte concentrations, labelled analyte analog is able to compete more effectively for binding to the upstream capture zones and is captured at those capture zones. As a result, at low analyte concentrations, the labelled analyte analog either does not produce visually detectable signal at, or takes longer to produce visually detectable signal at the more downstream capture zones. As explained above, the number of capture zones showing detectable signal provides a visual direct readout of the analyte concentration. Higher analyte concentrations lead to detectable signal appearing at more capture zones. Concentrations of the labelled analyte analog and the amount of immobilized sbp member as well as the number and type of capture zones can be adjusted by one of skill on the art to allow for quantitation of analyte in the desired range.

In another embodiment, the sbp member is immobilized uniformly on a single capture zone on the matrix instead of being located on a series of spatially separated zones. Analyte competes with labelled analyte analog for binding to the immobilized sbp member. In this embodiment, the distance traversed by the labelled analyte analog prior to capture is directly proportional to the analyte concentration. The binding constants of the analyte and the labelled analyte analog to the sbp member and the gradient of the capture reagent provides for differential separation under non-chromatographic conditions. The signal detected from the labelled analyte analog serves as a "footprint" on the matrix of the distance traversed by the labelled analyte analog and this distance can be read off directly. Therefore, the analyte concentration is directly measurable by comparing the distance travelled by the labelled analyte analog to a suitable calibration curve obtained by using known quantities of analyte.

Another aspect of a competitive assay format employs an immobilized sbp member on an intermediate "barrier" zone located on the matrix downstream of the labelling zone and upstream of a detection zone. The barrier zone, whether a labelled analyte analog, or a labelled sbp member complementary to the analyte, serves as a means of preventing a labelled species from migrating further along the matrix unless the analyte concentration exceeds a certain threshold level.

In one aspect of this embodiment, labelled analyte analog, such as a labelled antigen, is provided, for example, by binding it to dyed latex beads either directly or through a carrier protein. Alternatively, the target specific labelling complex disclosed in PCT publication WO 94/01775 can be used. The target specific antigen complex is preferably used when the analyte is an antibody. The labelled antigen may be deposited onto the matrix in a labelling zone downstream of the sample receiving zone and upstream of the detection zone or premixed with the sample. A barrier zone comprising an sbp member complementary to the analyte, such as an antibody, is deposited onto the matrix downstream of the labelling zone. The third zone, the detection zone, which is downstream of the barrier zone, comprises a binding substance for the labelled antigen. Sample mixes with labelled antigen and passes first through the barrier zone. When no antigen is present in the sample, all the labelled antigen will bind to the barrier zone. The amount of antibody on the barrier zone must be sufficient to bind all the labelled antigen when antigen is not present in the sample. Usually, this zone will be masked off from view and will not be visible in the test device. If antigen is present in the sample, labelled antigen competes with sample antigen for the antibody immobilized in the barrier zone. If the sample contains antigen above a threshold level, the barrier zone antibody is unable to capture all of the labelled antigen. This level is controlled by the relative ratios of antigen and labelled antigen as well as the concentration and affinity of the barrier zone antibody. For a given level of labelled antigen, as barrier zone antibody density increases higher levels of sample antigen will be required for the barrier zone threshold level to be exceeded. The density, concentration, amount and/or affinity of barrier zone reagent (antibody in this example) is adjusted such that 100% bound/free separation is effected at antigen concentrations below the desired threshold level. These parameters can be varied by one of skill in the art to quantitate sample analyte in the desired range.

Thus, when the breakthrough threshold analyte concentration is exceeded, some of the labelled antigen evades capture at the barrier zone antibody and flows through to the detection zone. The detection zone contains a binding substance for the labelled species (in this case, labelled antigen) that evades capture at the barrier zone when the threshold analyte concentration is exceeded. This binding substance can be a specific binding pair member for the antigen, the label or the carrier protein linking the antigen to the latex bead, or a receptor for a ligand on the labelled antigen. Frequently this sbp member is an antibody. If there is a sufficient amount of free antigen in the sample, then the detection zone will be visible indicating a positive test result. The amount of the antibody deposited on the detection zone can be adjusted in such a way that labelled antigen which does not bind to the barrier zone is in excess at the detection zone. Then, if multiple detection zones (second, third, fourth and so on) are present in downstream sequence, labelled antigen will migrate to the successive detection zones depending on the amount of labelled antigen present thus allowing a semi-quantitative assay with multiple lines. As described earlier, the visual detection zones can have antibodies of increasing affinity arranged in downstream sequence to provide for good discrimination between analyte concentrations. Alternatively, the detection zones can have in downstream sequence combinations of reagents, e.g., antibodies to different epitopes, followed by streptavidin, a capture zone of higher binding affinity or enhanced binding affinity, to efficiently remove finite quantities of labelled antigen even though labelled antigen concentration diminishes as the sample flows downstream.

Immunometric Formats

Other embodiments of the present invention employ a format of the immunometric type. In these embodiments, analyte in the sample is first allowed to bind to labelled sbp member complementary to the analyte to form an analyte-labelled sbp member complex. The amount of unbound labelled sbp member remaining is inversely proportional to the amount of analyte in the sample. The labelled sbp member may be premixed with the sample or it may be diffusively bound to the matrix in the labelling zone upstream of the capture zone and downstream of the sample receiving zone. The analyte-labelled sbp member complex is then allowed to flow through a capture zone which comprises an immobilized analyte analog which is capable of binding to the unbound labelled sbp member. Generally, the immobilized analyte analog is not able to bind to the labelled sbp member bound to the analyte, thereby effecting a separation between bound and unbound label.

In one aspect of this embodiment, the immobilized analyte analog is located on a series of spatially separated capture zones arranged in downstream sequence and the test solution comprising the sample and labelled sbp member flows sequentially through the series of capture zones. As analyte concentration increases, color appears on a greater number of capture zones. Immobilized analyte analog competes with the analyte for binding to free labelled sbp member. Free labelled sbp member, if present, is bound at the more upstream capture zones. As analyte concentration increases, more labelled sbp member reaches the downstream capture zones in the form of analyte-labelled sbp member complex. As a result, greater amounts of labelled sbp member reach the downstream capture zones. Without being bound by any one particular theory, it is believed that as the complex flows downstream analyte dissociates from the labelled sbp member complex and is bound at the capture zone. Immobilized analyte analog on the downstream capture zones is thus able to compete for binding to the labelled sbp member. Since different antibodies typically have different on/off rates, quantitation is antibody dependent and one uses an antibody with the correct off rate for the analyte in the concentration range being quantitated. Off rates are determined empirically and the correct antibody selected experimentally for each case. Increasing analyte concentration thereby correlates with the appearance of detectable signal on an increasing number of downstream capture zones. As described earlier, and shown in more detail in the Examples, the number of capture zones on which signal can be detected due to capture of the labelled sbp member can be correlated with varying ranges of analyte in the sample by varying the number of capture zones, and the concentrations of labelled sbp member and immobilized analyte analog by protocols known to those skilled in the art.

In another embodiment of this type of assay, an analyte analog is immobilized on a intermediate zone (hereinafter the "barrier zone") downstream of the sample receiving zone and labelling zone and upstream of a detection zone. Analyte analog on the barrier zone is able to bind free labelled sbp member. Generally, there is sufficient analyte analog on the barrier zone to bind all the labelled sbp member in the absence of analyte. Bound labelled sbp member flows through the barrier zone in the form of an analyte-labelled sbp member complex which is unable to bind to the immobilized analyte analog. The detection zone contains an immobilized sbp member that is capable of binding the analyte-labelled sbp member complex. The concentration of sbp member on the labelled sbp member can be controlled to effect 100% bound/free separation on the barrier zone thus providing a sharp cut off at the analyte threshold level. The immobilized sbp member on the detection zone may be a specific binding substance for the label, an antibody against an exposed epitope of the analyte in the analyte-labelled sbp member complex, an antibody against a carrier protein that links the label to the sbp member, an antibody against the species of the labelled sbp member (when the labelled sbp member is a labelled antibody), a receptor for a ligand on the labelled sbp member, and the like. Higher analyte concentrations lead to higher concentrations of analyte-labelled sbp member complex and thus more detectable signal at the detection zone(s). The amount of signal is directly proportional to the sample analyte concentration. Either the intensity of signal at the detection zone, the time at which a detectable signal appears on the detection zone or the number of detection zones can be used to visually quantitate the analyte concentration in the sample by comparison to a calibration curve obtained by using known amounts of analyte.

In this format, the analyte may be either a polyvalent analyte such as a protein or a hormone, or a monovalent analyte such as a small drug, steroid or hormone. For example, when the analyte is HCG, the labelled sbp member can be latex-labelled mouse (IgG) anti-HCG, the barrier zone can contain immobilized HCG and the detection zone will contain immobilized goat anti-mouse IgG (Fc). The goat anti-mouse IgG will be able to capture the HCG-mouse (IgG) anti-HCG immune complex. Using an anti-mouse IgG directed against the Fc portion of the mouse antibody increases the efficiency of capture because there is less likelihood of interference to binding from the HCG already bound to the mouse IgG at its antigen binding region. As shown in the Examples, a similar scheme can be employed for quantitating a small molecule, as is shown for progesterone. In this case, a progesterone-BSA conjugate is immobilized on the barrier zone and goat anti-mouse IgG (Fc) on the detection zone. As with the previous embodiments, one skilled in the art will be able to readily quantitate analyte in a desired range by varying the concentrations of labelled sbp member and immobilized analyte analog. An advantage of this approach is that one can use just one sbp member for either high or low molecular weight analytes providing improved specificity and simpler reagent development.

Optionally, the detection zone may be a series of spatially separated zones arranged in downstream sequence as described earlier. Again, as described earlier, the rate at which signal appears on the downstream zones and the number of such zones on which signal is detected after a predetermined time can be correlated with the amount of analyte in the sample. The downstream detection zones can have binding substances of greater affinity for the analyte-labelled sbp member complex than the upstream detection zones to provide good discrimination as described earlier. FIG. 4 shows a representative example. Variations in both the binding affinities and the concentrations of the reagents immobilized on the multiple detection zones are used to control the kinetics of capture during liquid flow along the matrix:

1) Antibodies of varying affinity, e.g., 10.sup.-6, 10.sup.-7, 10.sup.-9, 10.sup.-10 and so on as determined by Scatchard analysis are used on different detection zones.

2) A high affinity antibody may generally be adjusted to have a lower affinity by chemical derivatization. For example, a small hapten may be conjugated to the antibody at varying ratios, 100:1, 10:1, 1:1, etc., to affect binding affinity (usually, increased derivatization results in decreased affinity). Alternatively, chemical substitution of the antibody may also affect affinity.

3) Antibodies to high frequency, intermediate frequency and rare frequency occurring antigen epitopes (all on the same antigen molecule) are used on separate capture zones to selectively partition antigens, labelled antigens or complexes thereof.

4) High affinity antibody is mixed in varying proportions with irrelevant antibody, e.g., high affinity mouse anti-analyte or anti-globulin mixed with normal mouse IgG.

5) Different ligand-receptor binding pair capture reagents of varying affinity, e.g., anti-mouse IgG and streptavidin-biotin as in FIG. 4. The arrays of binding substances immobilized on the various capture zones disclo