WO1987007384A1

WO1987007384A1 - Enzyme immunoassay device

Info

Publication number: WO1987007384A1
Application number: PCT/US1987/000198
Authority: WO
Inventors: Michael J. Walsh
Original assignee: Quidel
Priority date: 1986-05-30
Filing date: 1987-02-02
Publication date: 1987-12-03
Also published as: AU7033787A

Abstract

A device for concentrating enzyme immunoassay sandwich product in a very narrow band (14) in a column (10) for development and visual reading. The device may be constructed using a clear or transparent vessel or column (10), with a fluid inlet, in which there is disposed a band (14) or a plurality of bands (14) of small particles to which one of an immunochemical couple is bound, the other member of which couple is the species to be determined. The immunochemical band (14) is defined by bands (16, 18) of non-immunochemically active particles. An absorbent pad (23) may support the bands.

Description

ENZYME IMMUNOASSAY DEVICE

Field of the Invention This invention relates to immunology, and, more particularly to immunoassays generally and to enzyme immunoassays specifically.

Background of the Invention Enzyme immunoassays have become widely known and described in numerous tests, treatises, scientific papers and patents since the pioneering work begun by Dr. Eva Engvall et al., Engvall, E. and Perlmann, P., Enzyme-linked immunosorbent assay (ELISA), immunochem. 8:871-874, 1971; and the work of Schuurs and coworkers, see, e.g. Van Weemen Antigen-enzyme conjugates, FEBS Letters, 15:232-236, 1971, and several U.S. Patents naming Schuurs, et al., as inventors, see, e.g. U. S. Patents Nos. RE 31,006, 3,654,090, 3,839,153, 3,850,752, 3,862,302, 3,862,928, 3,879,262, and 4,016,043. Monoclonal antibodies in enzyme immunoassays are well- known, see the patent to David, U.S. 4,376,110, which has been held to be invalid, and the work of Herzenberg and of Engvall, Rouslahti and ϋotilla.

ENZYME IMMUNOASSAY, Ishikawa, M.D., Tadashi, Kawai, and Kiyoshi, Miyai, Editors; IGAKU-SHOIN, New York 1981 describes in considerable fundamental detail the principles and practices involved in enzyme immunoassays. Reference is also made to other texts and treatises in the field, such as IMMUNOCHEMICAL METHODS

IN THE BIOLOGICAL SCIENCES: ENZYMES AND PROTEINS, Mayer, R.J. and Walker, J.H., Academic Press, New York 1980, and OUANTITATIVE ENZYME IMMUNOASSAY, Engvall, E. and

Pesce, A.J., Blackwell Scientific Publications, London, (Scandinavian Journal of Immunology, 1978) and the reference cited therein for a comprehensive disclosure of the principles and usual practices involved in enzyme immunoassay. Various approaches have been described for carrying out enzyme immunoassays. The early ELISA's were what is commonly called a "competitive" assay in which the enzyme labelled antigen or antibody competed with the antigen or antibody to be determined for a reaction site on a bead, pad or surface to which one member of an immunologically coupling pair was attached. Later, the "sandwich" assay became popular. In the sandwich assay, the antibody or antigen to be determined was "sandwiched" by an immunochemical reaction between a solid surface treated with an immunological species reactive with the species to be determined and the same or a different reactive immunological species which has been coupled to an enzyme label. The principles of these types of ELISAs are discussed by Belanger, L., Alternative Approaches in Enzyme Tmmunoassays, Scand. J. Immunol., Vol 8, Suppl. 7, 33-41, 1978 (Chapter 4 in

QUANTITATIVE ENZYME IMMUNOASSAY, supra) . Many forms of solid supports to which one member of an immunochemical couple, e.g. antigen-antibody or hapten-antibody couple, have been disclosed. A common early form of solid support was a plate, tube or bead of polystyrene which was well-known from radioimmunossay (RIA) work to bind certain immunological species.

Filter paper, glass, various plastics (chemical polymers), and other solid support surfaces have been used for many years. Examples of such a system which used antibody (or antigen) coated polystyrene beads are described by Bohn. et al., in U. S. Patent No.

4,424,279, Jan. 3, 1984; and U.S. Patent No. 4,458,020, July 3, 1984, in which the coated balls are utilized in unique configurations.

While a great many patents and publications have described various solid binding surface materials for use in enzyme immunoassays, no disclosure of an immunoassay column of the type described hereinafter and claimed in this patent is known.

It is known to use columns per se in immunoassays generally. One such procedure is disclosed in U.S. Patent No. 4,508,829, April 2, 1985, in which a pregnancy test is described wherein HCG antibody is bound to a solid lectin support. Urine to be tested is first reacted with anti-HCG antibodies bound to a colored particle, e.g. stained staphylocci bacteria or colored latex, which forms a colored conjugate with the HCG antigen in the test urine. The urine containing the colored conjugate is passed through the column of lectin bound anti-HCG which binds the colored complex onto the column, resulting in a 'coloration on the column related to the presence or absence and amount of HCG present in the Urine sample.

Not withstanding the numerous approaches which have been made to developing more sensitive and more reliable, simpler enzyme immunoassays, most immunoassays suffer from various disadvantages in poor sensitivity, low reliability, difficulty in reading, overly complicated procedures, etc., and there continues to be a need for improvements in simplicity, reliability, and sensitivity. It is, accordingly, an important feature of this invention to provide a highly sensitive, reliable and relatively simple enzyme immunoassay procedure, apparatus and kit.

Summary of the Invention In one form, the invention is embodied in a kit for carrying out an enzyme immunoassay to determine the presence, absence or amount of an immunochemically bindable substance. The kit includes, along with packaging, information, protective packing, etc., a soluble immunochemically binding substance which is reactive with the immunochemically bindable substance which is to be determined, said soluble immunochemically binding substance being conjugated to an enzyme or other label which is capable, upon reaction with a developer, of developing a distinctive color and which is in a form which will dissolve in a liquid sample suspected of containing the bindable substance to be determined. The kit also includes a column comprising an elongate at least partially transparent tube having a proximal end adapted to receive liquid sample and a distal end adapted to discharge the liquid sample when it has passed through the tube, solid particle packing filling at least a portion of the tube intermediate the proximal and distal ends thereof, at least one mutually contiguous assay group of the solid packing particles having bound on the surface thereof an insoluble immunochemically binding substance which is reactive with the immunochemically bindable substance to be determined and which upon reaction with the bindable substance to the surface of the solid particles and a developing reagent which is reactive with the conjugated enzyme or other label to form upon reaction with said enzyme or other label a distinctive color.

The immunochemically bindable substance to be determined is frequently an antigen but may be an antibody or other immunological substance. If the substance to be determined is an antigen, then the soluble and insoluble immunochemically binding substances may be either antibodies or parts of antibodies to the antigen to be determined and immunochemically reactive species of any type e.g. virus or virus fragments, bacterial cell constituents, haptens on a carrier, etc. While reference is made here to antigens and antibodies, any suitable immunochemical couple may be used. Polyclonal antibodies or parts thereof may be used in insoluble form and in soluble form, but it has been found to be advantageous in some instances that at least one of the soluble and insoluble immunochemically binding substances is a monoclonal antibody, and in some instances there are advantages in having both the soluble and insoluble immunochemically binding substances in the form of different monoclonal antibodies or parts thereof to the antigen to be determined.

Virtually any solid particulate matter, including fibrous material, may form the solid support in the column. It has been found convenient, however, to provide solid packing particles in the form of microbeads of, for example, polystyrene, nylon, agarose, cellulose or a combination of these. At least a portion of the beads, or other particles, such as cellulose or modified cellulose fibers, fritted glass, etc., have the insoluble immunochemically binding substance bound to the surface thereof.

A mutually contiguous reference group of the solid packing particles having bound to the surfaces thereof a biochemical substance which does not bind the bindable substance to be determined is very convenient and increases the confidence level in the assay. Such a control is not fundamental to the invention, however, and is merely a desired option. The biochemical substance is preferably bound to the surface of the reference solid packing particles immunochemically equivalently to the binding of the insoluble immunochemically binding reagent to the assay packing particles. An immunochemically equivalent binding means, as the phrase is used here, that the reagents and the procedures for binding are the same or are sufficiently similar that the binding reagent system is immunochemically inert to or reacts with all significant components of the sample in substantially the same way and degree regardless of which immunochemical substance is bound. In one of many alternative forms, the column comprises at least two assay groups of solid packing particles having bound to the respective surfaces thereof immunochemically different insoluble immunochemically binding substances for substantially simultaneously detecting, in the respective assay groups, at least two immunochemically different bindable substances in a single sample liquid. In one purely optional form, not at all necessary to the practice of the invention, the column comprises means of concentrating the particles having bound thereto immunochemically binding substances adjacent the interior surface of the tube to increase the visibility of such particles throughout the wall of the tube. This means is, in one form, a short cylinder closed at both ends with projections extending outwardly. The cylinder resides in the center of the column, by reason of the spacers which keep the cylinder surface approximately equidistant from the column walls, and concentrates the immunochemically coated particles adjacent the column walls where they are more visible than would be the case if the concentrating means were not present.

The invention also encompasses a method for determining the presence, absence or amount of an immunochemically bindable substance in a liquid sample. The method steps may include, in any of many desired sequences of steps, the step of incubating in the liquid sample suspected of containing the immunochemically bindable substance to be determined a conjugate comprising a soluble immunochemical binding substance for the bindable substance and a label which is capable of reacting with a developer to form a colored product, the binding substance being specifically immunochemically reactive with the bindable substance.

Perfect specificity is not essential, but the binding substance must be sufficiently specific in its immunochemical reaction that it will bind with the substance to be determined preferentially or that competing immunochemical binding reactions, if any, may be distinguished or reduced. The product of the incubation, if the bindable substance to be determined is present in the sample, is a conjugate of the binding substance, the bindable substance and the label. The product of the preceding step is passed through a column comprising an elongate at least partially transparent tube having a proximal end adapted to discharge the liquid sample when it has passed through the tube, solid particle packing filling at least a portion of the tube intermediate the proximal and distal ends thereof, at least one mutually contiguous assay group of the solid packing particles having bound on the surface thereof an insoluble immunochemically binding substance which is reactive with the immunochemically bindable substance to be determined, and which upon reaction with the bindable substance, binds the bindable substance to the surface of the solid particles. A principal reaction product, if the bindable substance is present in the sample, is a sandwich of insoluble binding substance on the particles, the bindable substance, the soluble binding substance and the label. A developing solution is then passed through the column. The developing reagent which is reactive with the label to form upon reaction with said label a distinctive color. The result of this series of steps is a colored band or portion in the column the intensity of which is proportional to the amount of bindable substance in the sample.

It is to be understood that the assay can, in many circumstances, be carried out as a one-step assay in which all of the reagent and the sample are simply introduced into the device of this invention substantially simultaneously. In a preferred form of the method the immunochemically bindable substance to be determined is an antigen, the soluble immunochemically binding substance is an antibody which is conjugated to an enzyme label, and the insoluble immunochemically binding substance is an antibody. At least one of the soluble and insoluble immunochemically binding substances may be a monoclonal antibody, or both the soluble and insoluble immunochemically binding substances may be, respectively, different monoclonal antibodies to the antigen to be determined.

The various facets of the invention are described in more detail hereinafter. The examples and specific listing of materials, steps, etc. are given to more fully enable the reader to practice the invention in all its facets, and are not limiting of the scope of the invention. The drawing which follows is a somewhat simplified depiction of only one of a myriad of forms in which the invention may be embodied.

Brief Description of the Drawings Figure 1 is a side view of a device constructed and arranged in accordance with the principles of this invention. Figure 2 is a cross section of the device as shown in Figure 1 taken on the diameter thereof.

Figure 3 is a cross-section of an alternative configuration for the device of this invention.

Figure 4 illustrates the inclusion of solutions in a kit assembled in accordance with this invention.

Description of the Preferred Embodiment The following descriptions are of the presently preferred embodiment, and is exemplary but not limiting of the invention. The concept of the present invention involves a rapid immunoassay based .on the flow-through, one- direction properties of a small chromatographic column. Specific "sandwiching" antibodies are bound to a solid support of microbeads or microspheres, or other solid particles, as one-half of the antibody-antigen-antibody- enzyme complex. A solution phase antibody-enzyme conjugate forms the other half of the complex and effectively sandwiches and is thereby immobilized by any antigen present in the sample. Excess Ab-enzyme conjugate is then rinsed away using a wash solution. Finally, a developer or substrate solution is added, in this case utilizing a precipitating chromophore as a byproduct of the antibody-enzyme conjugate - substrate reaction. After a short developing time, the presence of the precipitated chromophore or color on the microbeads indicates the presence of the specific enzyme. This immobilization of the antibody-enzyme conjugate, on the microbeads, signals specific "sandwiching" of the antigen found in the patient's sample. Color formation on the beads therefore indicates the presence of the antigen in the patient sample, or a positive result. When no antigen is present, the antibody-antigen - antibody-enzyme complex is unable to form, and no enzyme substrate reaction occurs and the beads remain colorless. A wash step rinses away any residual "non-sandwiched" antibody enzyme conjugate from the microbeads, and there is no substrate color formation on the microbeads.

A color-forming enzyme assay is described but only as an example. Fluorescent labels, etc., may be used. The result is a rapid, simple enzyme immunoassay system which requires very little manipulation and an easy-to-read end-point determination. Note here also the possible inclusion of a negative control region of microbeads or plastic filter as an additional advantage - ease of readability.

As a specific example, the Ab-enzyme conjugate is preincubated with the patient sample and, when antigen is present, one half of the "aandwich" occurs - the antibody-enzyme conjugate binds to the antigen in solution. The system is tailored so that this reaction takes place very quickly, usually in less than five minutes.

This reaction fluid, sample and antibody-enzyme conjugate, is then introduced onto the top of the enzyme immunoassay column and allowed to flow through the column by gravity force, though suction could be used.

It is at this time that the actual "sandwiching" occurs. Conjugate-bound antigen is recognized by the antibodies located on the microbeads and bound by them as the sample flows through the column. The sample may be allowed to incubate in place for a. short time to assure an efficient reaction. The presence of antigen in the sample allows for the immobilization of the antibody- enzyme conjugate on the surface of the microbead. Without antigen present, the antibody-enzyme conjugate flows through the column unaffected.

The column is then rinsed with a wash buffer to remove any remaining antibody-enzyme conjugate which might otherwise react with the substrate and produce a false positive result. After the wash, only that antibody-enzyme conjugate which is specifically sandwiched through the antigen-antibody solid surface remains. Due to the one-direction flow of this device, this wash step proves to be simple and very efficient in removing unbound antibody-enzyme conjugate. The developer is then loaded onto the column and flows down through the column which includes the potential antigen sandwiching region. Should this substrate contact immobilized enzyme (from the antibody- enzyme conjugate), a reaction occurs which results in a precipitating chromophore and color formation on the beads. If no antigen was present and no sandwich formation has occurred, no reaction will occur as the substrate passes through this region of the column, and no color forms on the microbeads. In this example, color formation signals a positive result, and lack of color a negative result; other signals may also be used to detect a positive reaction.

The column is constructed in a transparent tube to aid visualization. The column could consist of two zones - a test or indicator region, and a control region. Antibody specific to the antigen being tested for is bound to the solid surface of the test or indicator region. Nonspecific antibody or protein is bound to the solid surface of the control region. The surfaces are stacked vertically in the enzyme immunoassay column to accommodate an easy visual reference of the test layer to the control layer.

In addition, this column concept can be used as a series of stackable units, each to determine a single analyte or its control. These units are stacked during assembly to form one column which would, as the sample percolated through, run several different assays. A mixture of conjugates could then be poured through, followed by the substrate solution.

Presently the solid surfaces of both regions are microbeads composed of, but not limited to, polystyrene, nylon, agarose, cellulose, or a combination thereof. Alternative solid surfaces include various inserts, filters, fritted surfaces, paper, etc. The key property is a surface to which antibody can be bound, either covalently or otherwise, through any number of biochemical reactions and through which fluids can pass freely, subject to the physical constraints of the system. Another property is the transparency of the column to allow for the visual end-point readability. The column also allows for a one-direction flow of reaction fluid through and by a potential sandwich forming region, thereby enabling localization. immobilization, and concentration of enzyme-containing complexes, and subsequent intensification of specific color formation. It has been shown to be extremely sensitive (25 mlU/ml) and fast. Work has also been done with the detection of Strep Group A in this format. It can be utilized in any system where the detection of very small quantities of specific antigen is required in a relatively short period of time.

The design of this invention also includes the possibility of antibody detection. For example, a specific antigen or an antibody specific for an antibody may be bound to the solid surface and facilitate detection of another antibody. Certain disease states are monitored by the immunological spikes or sudden titer increases in specific antibody types or class.

The general advantages of this invention as compared with many dipstick, bead, or plate ELISA assays, or for that matter conventional detection systems such as RIA, IFA, Immunodiffusion, etc., include the following:

This assay design permits convective mixing of the sample and/or other test reagents as they percolate through the column matrix solid phase. This is an advantage over assay systems where no stirring or mixing occurs as the test runs. Mixing leads to greater sensitivity or a faster assay at an equivalent sensitivity. Plus, the mixing that is accomplished, as the sample flows, requires no hands-on time, unlike the vortexing needed in a coated tube assay, or the rocking required in a latex hemagglutination-type test.

As an example of the invention, and not by way of restriction or limitation, a device may be constructed according to this invention using a clear or transparent walled vessel of some kind, such as the cylindrical vessel 10, with a fluid inlet 12, shown in Figure 1 in which there is disposed a band, or a plurality of bands, of small particles, e.g. microbeads, to which one or an immunochemical couple is bound, thus forming a solid phase having thereon one immunochemically active member of a couple, the other member of which couple would normally be the species to be determined. (It is also possible, using various known permutations, to operate within the principles of the invention with a solid phase having bound thereto a material having the same immunochemical reactivity as the species to be determined.) This band of immunologically reactive surfaced particles is identified at 14, and may comprise, among the many possible configurations and c ompo s it i ons , g l a s s , p o l y s t y r e n e , polymethylmethacrylate, silica or other beads, of porous particles or fibers, which have bound on the surface antibodies, antigens, haptens, etc. The band is shown as developed, e.g. blue compared with white or off-white before development. It is desirable to define the immunochemical band by a band of non-immunochemically active particles (or particles of different immunochemical activity) shown at 16 and 18. It is also desirable to provide an absorbent pad 20 which may also support the layers just mentioned. The absorbent pad may be of cotton, paper or other absorbent material.

Figure 3 depicts an alternative embodiment wherein the container 110 with fluid inlet 112 contains a plurality of immunochemically active solid particle bands separated by non-immunochemically active bands.

Thus band 114 is immunochemically active and is defined by bands 116 and 118 which are of different or no immunochemical activity. Band 118 also defines the top of immunochemically active band 120. Band 122 is immunochemically non-reactive and defines the bottom of band 120 and the top of immunochemically active band 124. Band 126 is immunochemically non-reactive and defines the bottom of band 124 and the top of immunochemically active band 128 the bottom of which is defined by immunochemically nonreactive band 130. An absorbent body 132 may be provided.

The invention may be embodied in a kit which includes one or more solutions, such as, for example a container 134 of antibody-enzyme conjugate, for example, or antigen-conjugate solution, etc. or even of wash solution 136 and container 138 of developer solution 138. Suitable boxes, supports, etc. may also be provided.

Because of the use of well-characterized monoclonal and polyclonal antibodies, the degree of specificity can be easily quantified.

The design of this invention is such that there is a minimum of hands-on manipulation, a common source of error, and no external equipment is required. Its simplicity of operation and ease of readability are two of its greatest assets. Also, because the invention is self contained, it lends itself readily to automation and batching of samples.

A major advantage over related technology, is the potential to run multiple simultaneous assays on the same sample. The assay column may be envisioned as stackable units which may be interconnected so that sample flows from one to the next below, etc., eventually flowing into a reservoir. This permits testing for multiple analytes as well as multiple controls in a single specimen, with no significant increase in time or assay complexity. Furthermore, the column, when viewed from the side, appears to intensify the color, compared to known systems having a filter with color viewed from above. The concentration of bindable substance, sandwich and colored enzyme product into a very thin layer increases the sensitivity of the assay. Another advantage is ease of use. The assay column may be freestanding. Samples are simply added and then allowed to flow through at a controlled rate, which can be optimized for each test. Again, while the sample flows, no hands-on time is required.

Because the column may be self-contained, with a sample and wash collecting reservoir, running the assay becomes simpler and neater, with no additional sample handling or complicated disposal of spent reagents or

"used" sample. This is particularly important for samples which are potentially infectious. Because the assay unit is self-contained, potentially infectious specimen handling and disposal is greatly simplified. Assay columns may have a device attached to the top, a reagent/ sample distribution chamber which would permit all the test samples, reagents, and wash solutions to be added initially to the chamber at one time. The reagents would then flow out of the chamber in the proper order to permit the test to run with no additional manipulation.

The device is at least partially transparent plastic or glass and is inert.

Any porous physically and chemically stable material with the ability to bind, covalently or noncovalently, a variety of substances, including proteins - antibodies, allergens, antigens, drugs and their metabolites, bacteria and digested protein from bacteria, polypeptides, steroids, hormones, enzymes, cofactors, vitamins may be used. Low nonspecific binding of proteins, including labelled antibody, antigen, drugs, allergens is desired. Both a high surface area and a good flow-through rate are desirable and advantageous. Polystyrene, nylon, agarose, cellulose, polyvinyl chloride, paper, glass fiber, scintered glass, or a combination of particles of any of the above substances may be used within the device. Nitrocellulose, metal of some type, other polymers (plastics) cotton, wool, are other examples. Thick rolled paper, cellulose, cotton, having the ability to absorb approximately two times the total reaction volume and sample volume, may be used to aid in speed of reaction. Wicking the reaction liquids through the column by negative pressure through capillary action and liquid surface tension may be used in addition to gravity flow.

A number of groups of particles containing bound substances, e.g. different antigens, different drugs, or a mixture thereof, etc. to accomplish a number of analyte tests in one procedure is contemplated. Liposomes containing chromophores, enzyme substrates, antibody-enzyme conjugates or any other potential reactant may be included.

Liquid Enzyme-Conjugates and Labelled Components include any items listed above, but not so limited, may contain any of a variety of enzymes used in EIA technology such as Alkaline Phosphatase, Horseradish Peroxidose, B-Galactosidase, but any color forming label may be used. Alternatively, this invention would also work well with fluorescent conjugates, such as Fluorescein Isothiocyanate (FITC) , Rhodamine T, Rhodamine M, Rhodamine ITC, etc. In some cases, histological stains could be used to localize the presence of certain enzymes, whether they be in the sample itself or part of a conjugate.

In terms of chromophores, the properties should include - a visual end product, either by eye or with a fluorimeter a source of exciting light, as well as immobility, either through insoluble precipitation at the reaction site, or the property of being bound to the labelling molecule (protein, etc.) itself. For alkaline phosphatase enzyme conjugates, some of the possibilities include 5-Bromo - 4-Chloro - 3-Indolyl Phosphate, 3- Indoxyl Phosphate, Napththol AS Phosphate, and Nitroblue Tetrazolium. For Peroxidase-enzyme conjugates, some possibilities include 3-3 Diaminobenzidine

Tetrahydrochloride and 4-Chloro - 1-Napthol. For B- Galactosidase, some possibilities include 5-Bromo - 4- Chloro - 3-Indolyl B-D Galactopyranaside (X-GAL).

Wash solutions may be simple biological buffer systems, such as a phosphate buffered saline, for example, as required for the assay system and may include ionic or non-ionic detergents as necessary. Detergents could include Polyoxy ethylene Sorbitan monolaurate (Tween-20), Nonidet P-40 (NP-40) and Triton X-100.

The following steps, in any of several orders, may be included in the invention, without leaving the principle of operation thereof.

A. Reconstituting lyophilized enzyme conjugate with patient sample, swirled to mix completely.

B. Loading sample on to Enzyme Immunoassay Column, and allowing the column to stand after complete sample load. This step may not always be necessary.

C. Loading the wash buffer onto the column and allowing it to completely enter the column before adding developer.

D. Loading developer onto the column. E. Positives will give a clear colored band separate and distinguishable from the control solid surface, which should show no color. No distinction of regions on the basis of color indicates a negative result, or the lack of antigen in the sample.

The process of this invention may be carried out in any of a great variety of types of apparatus or kits. The kit shown in the drawing is merely exemplary of one form of a kit which may be used.

One important feature of this invention is that it is adaptable to a number of analytes which may be tested for simultaneously sensitive assays for a number of components. It is possible, for example, to provide an immunochemical assay column of the type described in this invention which can be used to detect the presence or absence of HCG antigen, strep-A, drugs of abuse, allergens, specific IgE antibodies, or any other combination of non-interfering immunochemical materials. In some instances, it may be desirable to test the same sample for two or more different antibodies or antigens. As pointed out above, the invention permits a very rapid immunochemical assay, which is more sensitive than many previous commercially available assays, which has a very high degree of reliability and repeatability, and which is inexpensive to manufacture and simple to use. Industrial Application

This invention is suitable for use in clinical laboratories, hospitals, and by individuals to aid in the diagnosis of disease or in the early detection of pregnancy, or in the detection of the presence of drugs of abuse, or other immunochemical materials.

The scope of the invention is not limited by the preceding examples. References made to the following claims which particularly point out and distinctly define the invention.

Claims

WHAT IS CLAIMED IS;

1. Apparatus for performing an immunoassay comprising the combination of a vessel having an at least partially transparent wall , a band of immunochemically reactive particles in the vessel visible through said transparent wall for concentrating of observable reactant of the immunoassay in a narrow visible band, immunochemically reactive reagent bound to the surface of said immunochemically reactive particles, and immunochemically nonreactive means defining the upper and lower surfaces of said band of immunochemically reactive particles.

2. A kit for performing an immunoassay comprising the combination of a vessel having an at least partially transparent wall, a band of immunochemically reactive particles in the vessel visible through said transparent wall, immunochemically reactive reagent bound to the surface of said immunochemically reactive particles, immunochemically nonreactive means defining the upper and lower surfaces of said band of immunochemically reactive particles, and a solution of conjugate of reagent immunochemically equivalent to the aforesaid immunochemically reactive reagent coupled to a detectable label.

3. A kit for performing a ELISA immunoassay comprising the combination of a vessel having an at least partially transparent wall, a band of immunochemically reactive particles in the vessel visible through said transparent wall, immunochemically reactive reagent bound to the surface of said immunochemically reactive particles, immunochemically nonreactive means defining the upper and lower surfaces of said band of immunochemically reactive particles, a solution of conjugate of reagent immunochemically equivalent to the aforesaid immunochemically reactive reagent coupled to an enzyme, and a solution of developer substrate for the enzyme.

4. A device for performing an ELISA immunoassay comprising the combination of a generally cylindrical vessel having a transparent wall, a layer of immunochemically reactive particles in the vessel having bound on the surface thereof one member of an immunochemically reactive couple, at least one layer of immunochemically non-reactive particles defining the layer of immunochemically reactive particles, the vessel forming an opening for introducing a sample solution into the vessel, enzyme labelled conjugate of the aforesaid member of the aforesaid immunochemically reactive couple, and developer substrate for said enzyme.

5. The device of claim 4 further comprising means in the vessel for absorbing liquid and for supporting the layer of immunochemically reactive particles.

6. A device for performing an ELISA immunoassay to detect or determine the presence or absence or amount of one member of the immunochemically reactive couple consisting of an antigen and an antibody thereto, an antibody and an antigen or antibody thereto, or a hapten and an antibody thereto, comprising the combination of a generally cylindrical vessel having a transparent wall, a layer of immunochemically reactive particles in the vessel having bound on the surface thereof one member of one of the aforesaid immunochemically reactive couples, the other member of said couple being the species to be determined in the assay, at least one layer of immunochemically non-reactive particles defining the layer of immunochemically reactive particles, the vessel forming an opening for introducing a sample solution into the vessel, enzyme labelled conjugate of the aforesaid member of the aforesaid immunochemically reactive couple, and developer substrate for said enzyme.

7. A method for performing an ELISA immunoassay to detect or determine the presence or absence or amount of one member of the immunochemically reactive couple consisting of an antigen and an antibody thereto, an antibody and an antigen or antibody thereto, or a hapten and an antibody thereto, comprising the steps of defining in a generally cylindrical vessel having a transparent wall a layer of immunochemically reactive particles having bound on the surface thereof one member of one of the aforesaid immunochemically reactive couples, the other member of said couple being the species to be determined in the assay, introducing a sample solution suspected of containing the member of the immunochemically reactive couple to be detected into the vessel and causing said sample solution to flow through the aforesaid layer of immunochemically reactive particles, introducing a solution containing enzyme labelled conjugate of the aforesaid member of the aforesaid immunochemically reactive couple and causing said solution containing enzyme labelled conjugate to flow through the aforesaid layer of immunochemically reactive particles, and introducing a solution containing a developer substrate for said enzyme into said vessel and causing said developer substrate solution to flow through the aforesaid layer of immunochemically reactive particles to thereby form a colored layer in said vessel visible through a wall thereof if the member of the immunochemically reactive couple to be determined is in the sample.