WO1994024315A1

WO1994024315A1 - Specific binding agent test apparatus and method

Info

Publication number: WO1994024315A1
Application number: PCT/US1994/004453
Authority: WO
Inventors: Buck A. Rhodes; Paul O. Zamora
Original assignee: Rhomed Incorporated
Priority date: 1993-04-22
Filing date: 1994-04-22
Publication date: 1994-10-27
Also published as: AU6711894A

Abstract

A method and apparatus for determining the immunoreactive or bioreactive fraction of labeled Specific Binding Agents is provided, in which a separating strip, containing a zone including the complimentary member of the Specific Binding Pair, is employed. The total amount of label in the labeled Specific Binding Agent applied to the strip and the amount of label at the zone associated with labeled Specific Binding Agent forming a Specific Binding Pair is detected, thereby permitting a measure of the immunoreactive or bioreactive fraction. In an alternate embodiment, a complimentary member of the Specific Binding Pair which is particulate-associated is employed. In yet another embodiment, particulate-associated analytes, which are capable of forming a specific binding pair, may be detected and quantified using a separating strip material. To conduct the assay, a labeled specific binding agent is introduced to the particulate-associated analyte, and incubated to allow formation of specific binding pairs. A quantity of the combination is then introduced to the separating strip material, the particulates allowed to immobilize, and the separating strip material contacted with solvent. The analyte is then detected by examining the zone for the presence of a label.

Description

SPECIFIC BINDING AGENT TEST APPARATUS AND METHOD

BACKGROUND OF THE INVENTION Field of the Invention (Technical Field):

This invention relates to a method and apparatus for determination of the fraction of a signal generating label or tag, such as a radioactive isotope, an enzyme, a toxin, a fluorochrome, or other marker, which is associated with a functionally active specific binding agent. This method and apparatus is particularly useful for the rapid, quality control assessment of the immunoreactive fraction of a radiolabeled antibody intended for use as a radiopharmaceutical or for use in radioimmunoassays. It is also useful for assessing the functional purity of other types of binding reagents such as enzyme-, fluorescence-, or toxin-tagged antibodies, antibody fragments, peptides or other ligands which are to be used as analytical reagents or as drugs. This invention also relates generally to solid phase methods for conducting specific binding assays upon sample fluids, and more specifically to the use of immobilized analytes on separating strips in conducting such assays.

Background Art:

Labeled specific binding agents have found wide utility in conducting immunoassays and similar specific-binding assays, and in the diagnosis and treatment of disease. The immunologically or biologically active specific binding agent, which can be an antibody, peptide, polypeptide, or other similar substance, forms a specific binding pair with its complementary receptor, forming a ligand and receptor (anti-ligand) system. The functional utility of these labeled binding agents depends on the ability of the binding agent to bind to its specific substrate after labeling. The degree (fractional percent) to which it retains biological binding post- labeling is critical in the subsequent use of the labeled specific binding agent in a clinical or analytical setting. In in vitro immunoassays, for example, the formation of an antibody-antigen complex formation is monitored with a signal generating tag conjugated to an antibody. If the tagged antibody has lost functional binding capacity, or the tag disassociates from the antibody, the antibody will not effectively function in the quantitative determination of an antibody-antigen complex.

In addition to in vitro assays, there are also numerous antibody- and peptide- based pharmaceutical preparations in development, in which the antibody or peptide serves as a specific binding agent, with target antigen or other receptors located at the site of disease or pathology. These products can be administered to the human body to visualize or monitor functioning of various parts of the body or to determine the presence and location of particular antigens, antibodies, hormones or the like; and can be used in the treatment of various disease states. For example, radiolabeled antibodies have been experimentally employed to both diagnose, through imaging modalities, and to treat, through radiotherapy, cancers and other neoplasms. Other antibody-based specific binding pair agents have been experimentally employed, for example, for diagnosis of thrombosis (using fibrin- or platelet-related antigenic receptors), extent of heart damage (using myosin antigenic receptors), and localization of sites of infection or inflammation (using white blood cell-related antigenic receptors, or specific pathogen-related antigenic receptors). Peptide-based radiopharmaceuticals have also been explored for imaging of sites of inflammation or infection atherosclerotic plaque formation, and other such conditions. In these peptide-based radiopharmaceuticals, the peptide is a member of a specific binding pair, with the receptor located at the site of disease or pathology.

As in the example of in vitro applications of radiolabeled antibodies, the in vivo diagnostic or therapeutic utility of tagged antibodies or other specific binding agents depends on the ability of the tagged specific binding agent to bind to its target. If the tagged specific binding agent has diminished binding capability post- labeling, it will not effectively localize to its target in the body, with concomitant decreased efficacy. For immunoassays and related types of tests, it is typical to employ an enzyme-based chromogen label, but other labels are frequently employed, including radionuclide labels, fluorescent labels, and chemiluminescent labels. For pharmaceutical use, it is most common to employ radiolabels, either for radioimmunoimaging or radioimmunotherapy, but the use of toxins for therapy has also been explored.

The clinical and diagnostic utility of labeled, immunologically or biologically active specific binding agents depends on the purity of the specific binding agent as well as the biological reactivity of labeled specific binding agent. If the purity of the specific binding agent is compromised, or if the immunological or biological reactivity of the labeled specific binding agent is compromised, the targeting selectivity of the complex is altered, resulting in loss of functional and clinical utility.

There are numerous methods well-known in the art for the labeling or tagging of antibodies, peptides and other specific binding agents with radioisotopes, as well as enzymes, toxins, fluorochromes, and other markers. Methods for radiolabeling generally use either a bifunctional chelate, which essentially bridges the substrate to be labeled and the radiolabel, or use means of direct labeling, whereby the radiolabel is incorporated into the substrate. A wide variety of radionuclides can be used, including commonly ^,25I, ^,3II, "" c, ^U1ln, ⁶⁷Ga, ³H, ³²P,

¹⁴C, ³⁵S and less commonly ⁹⁰Y, ^l88Re, ¹⁸⁶Re, ¹⁹⁹Au, ^{1 3}I and ⁶⁷Cu, among others.

These and other radionuclides and methods of conjugation to biologically active materials are known to those skilled in the art. Radiolabeled members of specific binding pairs can be used in immunoassays and similar such tests, as well as used in radiopharmaceuticals.

Enzymes and toxins are other classes of tags that can be bound to antibodies, proteins, peptides, and the like for use in vivo or in vitro. As with radiolabeled antibodies, the utility of such conjugates depends on their ability to bind to a substrate after tagging. For example, if the antigen binding capacity of an enzyme-antibody complex is compromised as a consequence of labeling with the enzyme, it will have reduced efficacy when applied to immunoassays. Numerous methods of tagging or labeling specific binding pair substances with enzyme labels, fluorescer-quencher labels, fluorescer labels, chemiluminescent labels, and other detectable labels are known in the art, most for the purpose of producing detectable signals in immunoassays. Similarly, if the antigen binding capacity of a toxin- antibody complex is compromised as a consequence of labeling with the toxin, it will have reduced efficacy when used as a therapeutic agent in vivo.

With radiolabeled antibody or peptide specific binding agents, the radiochemical purity, in terms of radiolabel associated with immunologically or biologically active specific binding agents, can be critical to clinical efficacy. The degree of radiological immunoreactivity of radiolabeled antibodies used in vivo can significantly affect biodistribution and targeting. Yokoyama et al

(Immunoreactivity affects the biodistribution and tumor targeting of radiolabeled anti-p97 Fab fragment. J Nucl Med 28:651 , 1987) and Temponi et al (A role for antiidiotypic antibodies in immunoscintigraphy and radioim unotherapy. Eur J

Nucl Med 14:293, 1988) have shown, in animal biodistribution studies using radiolabeled antibodies, that both the absolute amount of radioisotope uptake by tumor and the target-to-non-target ratio increase as the immunoreactive fraction of the radiolabeled preparation increases.

As with other radiopharmaceuticals, the biodistribution and subsequent image quality of a radiolabeled antibody, peptide or other specific binding agent depends on radiochemical purity. Radiochemical purity of a specific binding agent preparation can be defined as the percentage of the radionuclide which is bound to immunoreactive or biologically active specific binding agent. This percentage value is usually referred to as the immunoreactive fraction with radiolabeled antibodies. Peptides and other receptor specific agents also have a reactive fraction, which may be referred to as the bioreactive fraction. The terms immunoreactive fraction and bioreactive fraction can, for most purposes, be used interchangeably. If the net radioactivity associated with the immunoreactive fraction is compromised, then clinical efficacy of the radiopharmaceutical is also compromised. This is true even if the radiolabeled member of a specific binding pair is radiochemically pure in the sense that all or substantially all radioactivity is associated with the member of a specific binding pair. The key measure is the immunoreactive fraction; the percent of radioactivity associated with a member of a specific binding pair which is biologically or immunologically functional.

Radiolabeling methods can easily damage the immunoreactivity of antibodies. Particularly with iodination and labeling via bifunctional chelates, it is possible to label the antibody within the hypervariable region of the molecule, altering the structure of the binding site, and thereby interfering with the antibody- antigen reaction. Radiolysis and denaturation of the protein during manufacturing are other causes of damage.

Previous methods for measuring the immunoreactive fraction of a radiolabeled antibody preparation have been complicated, generally involving laborious preparation steps, multiple steps and a significant length of time to complete. These methods include use of affinity column chromatography, as described by Petit et al (Iodination and Acceptance Testing of Antibodies, in Tumor Imaging: The Radioimmunochemical Detection of Cancer, SW Burchiel, BA Rhodes, eds. , Masson Publishing USA Inc., New York, 1982, pp 99-110) and Breslow et al (Quality Control of Radiolabeled Antibodies in Tumor Imaging: The Radioimmunochemical Detection of Cancer, SW Burchiel and BA Rhodes, eds. , Masson Publishing USA Inc. , New York, 1982, pp 157-166); measurement of binding to a series of decreasing number of live or formalin-fixed cells (the extrapolation method) such as the method described by Lindmo et al (Determination of the immunoreactive fraction of radiolabeled antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Methods 72:77-89, 1984) and used for radiolabeled anti-melanoma antibodies by Beaumier et al (Immunoreactivity assay for labeled anti-melanoma monoclonal antibodies. J Nucl Med 27:824-828, 1986); and semiquantitative binding studies using cell membranes (Watanabe Y et al: Semiquantitative in vitro binding assay of indium-l l l-labeled monoclonal antibodies to human cancer and normal tissues. J Nucl Med 29: 1436-1442, 1988). The extrapolation method requires the maintenance and growth of live cells, multiple measurements, and graphic analysis of the measurements to extrapolate to the theoretical value at infinite antigen excess. The cell membrane method requires processing of the cell membranes or microsomes (including the collection of tissue or cells, homogenization of the materials, and the isolation of the membrane fractions by differential centrifugation); separation of the bound radiolabeled material from the unbound radiolabeled material by centrifugation or precipitation; and, wash steps to insure removal of the unbound radiolabeled material. Frequently, considerable care needs to be taken with membrane preparations to reduce or eliminate spurious proteases or other degrading enzymes.

Rhodes, in U.S. Patent No. 4,940,670, Method for Compounding and Testing Patient Specific Monoclonal Antibodies and Monoclonal Antibody Fragments for In Vivo Use, discloses a method and apparatus for measurement of the immunoreactive fraction of a radiolabeled monoclonal antibody or antibody fragment. This quality control method provides for both a negative control test and a positive control test, containing antigen which is a member of the specific binding pair. To perform the method, a quantity of the radiolabeled preparation is added to the negative control and to the positive control, the associated radioactivity measured; both controls washed to remove any unbound antibody, and a final measure of the associated radioactivity made. Binding to the positive control and negative control is calculated by dividing the final measure of radioactivity by the initial measure of radioactivity.

In Rhodes et al (Quality control test for immunoreactivity of radiolabeled antibody. BioTech iques 8:70-74, 1990), a bead-type, solid-phase assay is disclosed for measurement of the immunoreactive fraction of a radiolabeled monoclonal antibody or antibody fragment. The test, available under the tradename "RhoChek" (RhoMed, Albuquerque, New Mexico), employs solid phase antigens, bound to beads, and involves multiple wash and centrifugation steps. Radioactivity is counted after addition of the radiolabeled preparation to the beads, and again after completion of the wash and centrifugation steps. The percentage radioactivity remaining after washing is a measure of the radioactivity associated with immunoreactive antibody.

With radiolabeled preparations, there are methods known in the art for determining radiochemical purity in terms of amount of uncomplexed radionuclide, amount of radiocolloid, and similar parameters. For example, Thrall et al (Clinical comparison of cardiac blood pool visualization with Technetium-99m red blood cells labeled in vivo and with Technetium-99m human serum albumin. J Nucl Med 19:796, 1978) and S. Kahn (U.S. Patent No. 4,095,950, Method for the Chromatographic Analysis of a Technetium-Containing Mixture) disclose chromatographic methods for the determination of radiochemical purity of technetium-labeled preparations, by determination of bound versus unbound technetium, reduced versus unreduced technetium, and percentage colloidal technetium. However, such measures of radiochemical purity do not determine the immunoreactive fraction, which is to say the percentage of the total radioactivity associated with an immunologically or biologically active specific binding agent. At most, such methods can determine the percentage radioactivity associated with the specific binding agent, but cannot measure the functional immunological or biological activity of the specific binding agent.

It is also useful to be able to determine the percentage of other signal or tag, such as enzymes, fluorochromes, or toxins, associated with immunologically or biologically functional specific binding agents. Toxin delivery systems, for example, in which a toxin is conjugated to a specific binding agent targeting a cancer or other site of interest, have been evaluated. For such a system to effectively work, it is essential that a high percentage of the total toxin be associated with the immunologically or biologically functional specific binding agent. Such binding agent may be an antibody, antibody fragment, peptide or similar such substrate. A number of immunoassays, for determining the presence or quantitating a member of a specific binding pair in a substance, have been developed. In these assays, members of a specific binding pair, usually an immunological pair, and frequently referred to as a ligand and receptor or anti-ligand, are involved. One of the members of the specific binding pair is detectably labeled, by any number of means, including those set forth above. The immunoassay methodology results in a distribution of the detectable label between detectable label bound in a complex of the specific binding pair, and unbound detectable label. The differentiation between bound and unbound detectable label can be as a result of either physical separation or modulation of the detectable signal between bound and unbound detectable label.

Among the immunoassay methodologies employed, certain immunoassays employ an immunoseparating strip, which is a bibulous test strip to which one member of the specific binding pair is bound. The sample suspected of containing the analyte is applied to the strip, allowed to migrate by capillary or similar means, and then detected by suitable means. Examples of such methods include those set forth in U.S. Patent No. 4, 168, 146 to Grubb et al, Immunoassay with Test Strip Having Antibodies Bound Thereto; U.S. Patent No. 4,435,504 to Zuk et al, Immunochromatographic Assay With Support Having Bound "MIP" and Second Enzyme; U.S. Patent No. 4,879,215 to Weng et al, Concentrating Immunochemical Test Strip; U.S. Patent No. 4,959,307 to Olson, Immunoseparating Strip; U.S. Patent No. 4,963,468 to Olson, Immunoseparating Strip; and U.S. Patent No. 5,075,078 to Osikowicz et al, Self- Performing Immunochromatographic Device, among others. All such methods, and the provided devices, are for determining the presence or amount of an analyte present in a sample, generally a patient sample, but cannot directly determine the immunoreactive fraction of the tagged specific binding agent.

Other immunoassay methods employ solid phase substrates which are porous. Generally, a specific binding agent is bound to the solid phase substrate, the analyte introduced, and concurrently or following an incubation period, reagents introduced for detection and washing. Detection can be accomplished by numerous methods, including those of sandwich type assays, utilizing a second specific binding agent with specificity for the analyte. All these methods are characterized by the solid phase being porous, so that unbound substances can pass through the solid phase, or be otherwise sequestered so as to not interfere with the immunoassay procedure. Such devices and methods are provided for in U.S. Patent No. 4,912,034 to Kaira et al, Immunoassay Test Device and Method; U.S. Patent No. 4,990,442 to Del Campo, Assay for an Analyte on a Solid Porous Support; and U.S. Patent No. 5,008,080 to Brown, III et al, Solid-Phase Analytical Device and Method for Using Same, among others.

SUMMARY OF THE INVENTION (DISCLOSURE OF THE INVENTION.

In one embodiment, the methods and apparatuses of the present invention are useful for determining an amount of label associated with a labeled specific binding agent in a specific binding agent preparation capable of forming a specific binding pair, thereby permitting determination of the immunoreactive or bioreactive fraction of a labeled specific binding agent. The labeled specific binding agent may be a peptide, protein, glycoprotein, carbohydrate, or lipid. If the specific binding agent is a protein or glycoprotein, then representative labeled specific binding agents include labeled antibodies, which may be polyclonal antibodies, whole monoclonal antibodies or fragments thereof, hormones, lectins, and enzymes.

The specific binding pair may be an antibody-antigen pair, hormone-receptor pair, peptide-receptor pair, enzyme-receptor pair, carbohydrate-protein pair, carbohydrate-fat pair, or lectin-carbohydrate pair. The label on the specific binding agent to be tested may be radioactive, enzymatic, chromogenic, fluorescent, magnetic, reflective, conductive or chemiluminescent, and may be detected by any means. If the label is a radioactive agent, then detection may be using any radiation detector. One radiation detector particularly suited for the invention is a radiochromatogram scanner. A material with capillarity and capacity for solvent transport is employed. This material will generally be a bibulous material, capable of being traversed in at least one direction by a solvent through capillary migration. The material may be a strip, which will have a first end at which solvent transport begins and a second end at which solvent transport ends. If a strip, it may be silica impregnated. The material may also be a sheet, or other format suitable for the method.

In one method, the material includes a first zone with a first reagent, with the first reagent immobilized against solvent transport and capable of forming a specific binding pair with the labeled specific binding agent. The first reagent may be immobilized by impregnating the first zone with the first reagent. The first reagent, which is capable of a forming a specific binding pair with the labeled specific binding agent, is in excess relative to the quantity of the specific binding agent preparation to be introduced to the material. The material may also include a second reagent immobilized against solvent transport which is not capable of forming a specific binding pair with the labeled specific binding agent, thereby substantially blocking non-specific binding of the labeled specific binding agent to the material. In this method, a quantity of the specific binding agent preparation is introduced to the material at a location on or downstream from the first zone, and the material contacted with a solvent, whereby the solvent traverses the locus of introduction of the specific binding agent preparation and at least a portion of the first zone. The amount of label at the first zone associated with labeled specific binding agent forming a specific binding pair is then detected.

It is possible to have a second zone for receiving the specific binding agent preparation, coincident with or upstream from the first zone. It is also possible to have the first zone proximal to the first end and distal to the second end. In situations in which the solvent traverses the locus of introduction of the specific binding agent preparation and the first zone, all that is required is to detect the amount of label at the first zone. In this case, in which the amount of label at the first zone is detected, if the total quantity of label in the quantity of the specific binding agent preparation introduced to the material is determined, then the percentage of the label detected at the first zone of the total quantity of label introduced to the material may be determined. This permits quantification of the amount of label associated with labeled specific binding agent capable of forming a specific binding pair. The total quantity of label introduced to the material may be determined either prior to or subsequent to introduction of the label.

An apparatus and method of determining non-specific binding of label is also provided, allowing a determination of net immunoreactive or bioreactive fraction. To make this determination, a similar material having capillarity and the capacity for solvent transport is provided. This material includes a separate zone with a separate reagent immobilized against solvent transport, the separate reagent being characterized, in part, by not being capable of forming a specific binding pair with the labeled specific binding agent. In use, a quantity of the specific binding agent preparation is introduced to the material at a location on or downstream from the separate zone, and the material contacted with a solvent, whereby the solvent traverses the locus of introduction of the specific binding agent preparation and at least a portion of the separate zone. The amount of label at the separate zone not transported by the solvent means is detected, thereby giving a measure of non¬ specific binding of the label. The separate reagent means may additionally substantially block non-specific binding of the labeled specific binding agent to the material. Here, too, the total quantity of label in the quantity of the specific binding agent preparation introduced to the material may be determined, so that the percentage of the label not transported by the solvent of the total quantity of label introduced to the material may be determined.

In another embodiment, a material as above-described is provided. The material has a first end at which solvent transport begins, a second end at which solvent transport ends, and a first zone proximal to the first end and distal to the second end. A first reagent is provided, capable of forming a specific binding pair with the labeled specific binding agent, and which can be immobilized against solvent transport when introduced to the material. The first reagent can be immobilized against solvent transport by molecular sieving or by chemical means. The material may also include a second reagent immobilized against solvent transport which is not capable of forming a specific binding pair with the labeled specific binding agent, thereby substantially blocking non-specific binding of the labeled specific binding agent to the material. A quantity of labeled specific binding preparation to be tested is introduced to the first reagent, and incubated for a period sufficient to allow formation of specific binding pairs. A quantity of the combination of the specific binding agent preparation and first reagent is then introduced to the first zone, and the material contacted with a solvent, whereby the solvent traverses at least the first zone. The amount of label at the first zone associated with labeled specific binding agent forming a specific binding pair is then detected. In one embodiment, the quantity of first reagent is in excess relative to the quantity of the specific binding agent preparation. Provided the total quantity of label in the quantity of the combination of the specific binding agent preparation and first reagent introduced to the first zone is determined, the percentage of the label detected at the first zone of the total quantity of label introduced to the material may be determined.

The first reagent employed includes cells in which there are cell surface receptors capable of a forming a specific binding pair with the labeled specific binding agent. Representative types of such cells include platelets, red blood cells, and lymphocytes. The first reagent also includes particulates, including particles which are coated with yet another reagent which is itself capable of forming a specific binding pair with the labeled specific binding agent.

In this embodiment, determining non-specific binding of label is also provided, allowing a determination of net immunoreactive or bioreactive fraction. To make this determination, a similar material having capillarity and the capacity for solvent transport is provided. This material includes a separate zone with a separate reagent immobilized against solvent transport, the separate reagent being characterized, in part, by not being capable of forming a specific binding pair with the labeled specific binding agent. In use, a quantity of the specific binding agent preparation is introduced to the material at a location on or downstream from the separate zone, and the material contacted with a solvent, whereby the solvent traverses the locus of introduction of the specific binding agent preparation and at least a portion of the separate zone. The amount of label at the separate zone not transported by the solvent is detected, thereby giving a measure of non-specific binding of the label. The separate reagent may additionally substantially block non¬ specific binding of the labeled specific binding agent to the material. Here too the total quantity of label in the quantity of the specific binding agent preparation introduced to the material may be determined, so that the percentage of the label not transported by the solvent of the total quantity of label introduced to the material may be determined.

In yet another embodiment, the material includes a first zone, proximal to the first end, and a second zone, downstream from the first zone. The second zone further comprises a first reagent immobilized against solvent transport and capable of forming a specific binding pair with the labeled specific binding agent. The first reagent may be immobilized by impregnating the second zone with the first reagent. The first reagent, which is capable of forming a specific binding pair with the labeled specific binding agent, is in excess relative to the quantity of the specific binding agent preparation to be introduced to the material. The material may also include, in the first zone, a second reagent immobilized against solvent transport which is not capable of forming a specific binding pair with the labeled specific, binding agent, thereby substantially blocking non-specific binding of the labeled specific binding agent to the material in the first zone. The entire material may also include the second reagent, or another reagent similarly characterized, so that the entire material, other than as provided by the first reagent, is not capable of forming a specific binding pair with the labeled specific binding agent, and non¬ specific binding of the labeled specific binding agent to the material is substantially blocked. In this method, a quantity of the specific binding agent preparation is introduced to the material at a location on or downstream from the first zone, and the material contacted with a solvent, whereby the solvent traverses at least the first zone and second zone. The amount of label at the second zone associated with labeled specific binding agent forming the specific binding pair is then detected.

In this embodiment, it is possible to detect the total quantity of label in the quantity of the specific binding agent preparation introduced to the material, whereby the percentage of the label in the second zone of the total quantity of label introduced to the material may be determined, providing a measure of the amount of label associated with a labeled specific binding agent in a specific binding agent preparation capable of forming a specific binding pair. The total quantity of label introduced to the material may be determined either prior to or subsequent to introduction of the label.

Determining non-specific binding of label, including formation of label associated with particles or colloids, is also provided, by measuring the amount of label at the first zone. Provided the total quantity of label is determined, the percentage of non-specific binding can be determined.

The method and device of the present invention are also useful for determining the presence of a particulate-associated analyte, the analyte being capable of forming a specific binding pair, in a sample suspected of containing the analyte, using a labeled specific binding agent. Representative types of specific binding pairs which may be employed in this invention include antibody-antigen pairs, hormone-receptor pairs, peptide-receptor pairs, enzyme-receptor pairs, carbohydrate-protein pairs, carbohydrate-fat pairs, and lectin-carbohydrate pairs.

The particulate-associated analyte may be a particulate, such as a cell. Cell particulate-associated analytes include cells with a cell surface receptor capable of forming a specific binding pair with the labeled specific binding agent. These include cells such as platelets, red blood cells, and lymphocytes. Other particulate- associated analytes include particles, in which the particulate may include polyvinylidine fluoride. These particulate-associated analytes include particulates coated with the analyte. The method employs a separating strip material, which material has capillarity and capacity for solvent transport, and is capable of immobilizing particulates. The separating strip material includes a first end at which solvent transport begins, a second end at which solvent transport ends, and a zone, proximal to the first end and distal to the second end, for introduction of the particulate-associated analyte and labeled specific binding agent combination. The separating strip material which may be employed includes bibulous chromatographic material, which may be a strip, sheet or other format which allow conduct of the assay method. One type of bibulous chromatographic material employed is silica gel impregnated material. The separating strip material may include a reagent immobilized against solvent transport which is not capable of forming a specific binding pair with the labeled specific binding agent, and which may additional substantially block non-specific binding of the labeled specific binding agent to the material. A variety of reagents may be employed, including various proteins.

The labeled specific binding agents employed include peptides, proteins, glycoproteins, carbohydrates, and lipids. Among suitable proteins and glycoproteins are included labeled antibodies, hormones, lectins, and enzymes. Antibodies may be polyclonal or monoclonal, and include fragments thereof. The label for the labeled specific binding agent includes use of radioactive, enzymatic, chromogenic, fluorescent, magnetic, reflective, conductive and chemiluminescent agents.

In the method, a labeled specific binding agent capable of forming a specific binding pair with the analyte is first provided. A quantity of the labeled specific binding agent is introduced to the particulate-associated analyte, and incubated for a period sufficient to allow formation of specific binding pairs. A quantity of the combination of the labeled specific binding agent and particulate-associated analyte is then introduced to the zone of the separating strip material, and the particulates allowed to immobilize in the separating strip material against solvent transport. The first end of the separating strip material is contacted with a solvent, and the solvent allowed to traverse the zone. The analyte is then detected by examining the zone for the presence of a label. The presence of particulate-associated analyte may be quantified, by detecting the total quantity of label in the quantity of the combination of the labeled specific binding agent and particulate-associated analyte introduced to the zone, and detecting the total quantity of label at the zone, whereby the percentage of the label .detected at the zone of the total quantity of label introduced to the material may be determined. If the label is a radioactive agent, the amount of label can be quantified by radiation detection systems, including systems which produce a radiochromatogram.

A variety of means may be employed to immobilize the particulates in the separating strip material against solvent transport. These include molecular sieving and chemical means.

Accordingly, it is an object of the present invention to provide a method and apparatus for determining the immunoreactive or bioreactive fraction of labeled specific binding agents.

A further object is to provide a method for determining the immunoreactive or bioreactive fraction of labeled specific binding agents which eliminates the need for use of live, formalin fixed or otherwise fixed cells.

Another object is to provide an apparatus and method for determining the immunoreactive or bioreactive fraction of labeled specific binding agents which does not require wash or centrifugation steps.

Another object is a method and apparatus which can be used with any of a range of labels including radionuclides, enzymes, toxins, fluorochromes, chemiluminescent agents, magnetic, and other detectable labels. Another object of the invention is a method and apparatus which minimizes the generation of hazardous waste fluids and solids, particularly as applied to probes such as toxins and radionuclides.

Another objective of the invention is to provide a method and apparatus for employing immunoaffinity for the rapid determination of the immunoreactive or bioreactive fraction of a labeled specific binding agent.

It is a further object of the present invention to provide a method and apparatus for determining the immunoreactive or bioreactive fraction of a labeled specific binding agent which permits simple single-point measurements, and does not require multiple measurements or extrapolation to theoretical values.

Still another object is a method for determining immunoreactive fraction of labeled specific binding agents which can use a crude antigen homogenate, and does not require purified antigen.

Yet another object is a method for measuring immunoreactivity of radiolabeled antibody which also demonstrates antigen excess, and which provides for a negative control.

A still further object of the present invention is a method for determining the immunoreactive or bioreactive fraction of a labeled specific binding agent which does not require rinsing or wash steps; which can be conducted within a brief period of time; which can be quantitative, semi-quantitative, or qualitative; and which can be made in a kit format for on-demand use.

In another embodiment, it is a further object of the present invention to provide a method based on use of a separating strip for detecting cell surface antigens, receptors, and other targets with labeled antibodies or other targeting agents. It is a further object of the present invention to provide a method which is based on use of a separating strip for separating antigen/antibody aggregate complexes from unreacted antibody.

It is a further object of the present invention to provide a method for estimating the amount of ligand being bound to a particulate analyte without the use of rinse, centrifugation, precipitation, or filtration steps.

It is a further object of the present invention to provide a method which allows for the estimating of the amount of bound ligand and unbound ligand within the same medium.

Other objects, advantages, and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description and drawings to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combination particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The drawings are only for the purpose of illustrating a preferred embodiment of the invention and are not to be construed as limiting the invention. In the drawings: FIGS. 1 and 2 illustrate a separating strip apparatus, containing a zone with either positive or negative material, suitable for use in determining the bioreactive or immunoreactive fraction of labeled specific binding agent. FIGS. 3 and 4 illustrate a separating strip apparatus, containing both negative and a positive material zones, suitable for use in determining the bioreactive or immunoreactive fraction of labeled specific binding agent. FIGS. 5 and 6 illustrate a separating strip apparatus, containing a zone for introduction of a combination of labeled specific binding agent and a particulate-associated complimentary member of the specific binding pair, suitable for use in determining the bioreactive or immunoreactive fraction of labeled specific binding agent.

FIGS. 7 and 8 illustrate results obtained by radiochromatogram using the apparatus of FIGS. 1 and 2. FIGS. 9 and 10 illustrate results obtained, measured as percent binding at the origin, under varying experimental conditions using the apparatus of FIGS. 5 and 6.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF

THE INVENTION (BEST MODES FOR CARRYING OUT THE INVENTION.

Before proceeding with the description of the specific embodiments of the present invention, a number of terms will be defined.

A "Specific Binding Pair" ("SBP") comprises at least two different molecules, where one molecule has an area on the surface or in a cavity which specifically binds to a particular spatial and polar organization of the other molecule. Frequently, the members of a SBP are referred to as ligand and receptor or anti-ligand. Examples of SPBs include antibody-antigen pairs, hormone-receptor pairs, peptide-receptor pairs, enzyme-receptor pairs, carbohydrate-protein pairs (glycoproteins), carbohydrate- fat pairs (glycolipids), lectin-carbohydrate pairs and the like. Some such pairs may also be referred to as immunological pairs, but there is no requirement that the relationship be "immunological" in the sense of classical immunology. The "label" may be any detectable molecule conjugated or otherwise bound to a member of a SBP. As used herein, label most commonly refers to "radiolabel", in which a radionuclide is conjugated or otherwise bound to a member of a SBP. However, other detectable labels may be employed, including enzymes, enzyme-based chromogens, fluorescent labels, chemiluminescent labels, magnetic labels, and reflective labels.

This invention can be used with any detectably labeled "Specific Binding Agent". As used herein, a Specific Binding Agent ("SBA") is any labeled biologically or immunologically functional substance which is a member of a Specific Binding Pair. Representative SBAs include labeled peptides, antibodies and monoclonal antibody components, enzymes, lymphokines, cytokines, hormones and fusion proteins.

The "peptides" of the invention can be: a) naturally-occurring, b) produced by chemical synthesis, c) produced by recombinant DNA technology, d) produced by biochemical or enzymatic fragmentation of larger molecules, e) produced by methods resulting from a combination of any of a-d, or f) produced by any other means for producing peptides.

The peptides can also include peptide fragments, oligopeptides, polypeptides and other like structures, generally consisting of a sequence of amino acids. Representative types of peptides include those derived from laminin, fibronectin, cytokines, lymphokines, hormones, serum albumin, fibrinogen, enzymes, hormones, somatostatin, urokinase, tissue plasminogen activator, and protease inhibitors. The term "peptide" as used throughout the specification and claims is intended to include, but is not limited to, all of the foregoing.

The terms "antibody" or "antibodies", and the phrase "monoclonal antibody component" , as used throughout the specification and claims is intended to include all antibodies, or antibody fragments, of any species, and including both polyclonal and monoclonal antibodies made by any means, as well as chimeric and genetically engineered antibodies, and fragments of all of the foregoing. This includes immunoglobulins of any class, such as IgG, IgM, IgA, IgD or IgE, of any species origin, including human beings, chimeric antibodies or hybrid antibodies with dual or multiple antigen or epitope specificities, and fragments of all of the foregoing, including F(ab')₂, F(ab)₂, Fab', Fab and other fragments, including hybrid fragments, and further includes any immunoglobulin or any natural, synthetic or genetically engineered protein that functionally acts like an antibody by binding to a specific antigen to form a complex.

The terms "separating strips" or "strip" is intended to include any bibulous paper or sheet material, which bibulous paper or sheet material may include a zone or area containing material further composed, in part or whole, of the non-labeled member of a Specific Binding Pair, and which is otherwise made or blocked, to eliminate or limit non-specific binding by the labeled Specific Binding Agent. Separating strips may also include bibulous paper or sheet material which is made or blocked to eliminate or limit non-specific binding by the labeled Specific Binding Agent, but which can act as a cell, particle or particulate trap or sieve. Such trapping may be by any means, including chemical binding, but will most commonly be by molecular sieving. Other means of trapping can include attachment or conjugation by various means, such as adsorption, absorption, ionic attraction, and any other type of binding.

The terms "particulate" or "particle" is intended to include cells, coated particles, and colloidal aggregates, which include, either natively or through subsequent conjugation or other attachment, the non-labeled complimentary member of the Specific Binding Pair. Cells are intended to include any animal, plant, or bacterial cell and includes blood cells such as leukocytes, platelets, and red blood cells. The term "cell" is also used herein to mean membranous fractions derived from cells such as microsomes or enriched subcellular components such as plasma membranes, endoplasmic reticulum, nuclear membranes, and the like. Coated particles are intended to include porous and non-porous particles composed of, for example, polyvinylidine fluoride, agarose, cellulose, glass, vinyl, and others known to those skilled in the art to which is attached, by adsorption, absorption, conjugation or other methodologies, the complimentary member of the Specific Binding Pair. The term "colloid" is intended to mean any aggregate which has a physical size large enough to be sedimented by centrifugation, and generally larger than 0.2 microns in average diameter.

A method and apparatus, is described for analysis of labeled Specific Binding Agents, such as radiolabeled ligands intended for use as radiopharmaceuticals and analytical reagents to be employed in binding assays. In one embodiment, samples of the labeled Specific Binding Agent are spotted onto a pair of separating strips made of dry bibulous material, such as silica-gel- impregnated glass-fiber paper or cellulose-based thin layer chromatography paper. One such strip is pre-treated with a complimentary member of the Specific Binding Pair, and the other such strip is pre-treated with a comparable ligand which does not form a Specific Binding Pair with the Specific Binding Agent. The strips are then blocked with a material to eliminate or limit non-specific binding by the labeled Specific Binding Agent. The bibulous strip is developed using a solvent. When the ascending solvent front reaches a predetermined place near the distal end of the strip, the strip is removed from the developing solution and air dried. The distribution of the label along the strip is determined using the label itself as the means of measurement. The fraction of the label present at the origin of the binding strip and at the origin of the non-binding strip is determined. The difference in the percentage of the tag at the origin of the binding and the non- binding strips estimates the immunoreactive fraction of the labeled Specific Binding Agent. FIGS. 1 and 2 provide an illustrative configuration of the apparatus.

FIGS. 1 and 2 are, respectively, top and side views of the same apparatus.

In these figures, the separating strip material 25 is provided, which may be any dry bibulous material, such as silica-gel-impregnated glass-fiber paper or cellulose- based thin layer chromatography paper. The zone 12 and 22 is impregnated with complimentary member of the Specific Binding Pair, or alternatively, for determining non-specific binding, a comparable ligand which does not form a Specific Binding Pair with the Specific Binding Agent. The zone 11 and 21 is the origin, at which the labeled Specific Binding Agent is spotted. The zone 13 and 23 does not contain complimentary member of the Specific Binding Pair, and may be impregnated with a ligand which blocks non-specific binding of the labeled Specific Binding Agent to the material 25. The arrows 15 and 26 represent the direction of solvent flow, with the separating strip immersed in a solvent up to about mark 10 and 20. The solvent is allowed to ascend to about mark 14 and 24, and is then removed from the solvent. The fraction of label present in zone 12 and 22 is determined; for instances in which zone 12 and 22 is impregnated with complimentary member of the Specific Binding Pair, this provides a measure of total positive binding. For instances in which zone 12 and 22 is impregnated with a comparable ligand which does not form a Specific Binding Pair with the Specific Binding Agent, this provides a measure of non-specific binding. The different in the percentage of label at zone 12 and 22 of a binding and non-binding strips estimates the immunoreactive fraction of the labeled Specific Binding Agent.

In another embodiment, strips comprising dry bibulous material, such as silica-gel-impregnated glass-fiber paper or cellulose-based thin layer chromatography paper, are provided. These strips have at least three areas or zones; the first area is for receiving the labeled Specific Binding Agent, and is proximal to the end at which solvent transport begins. A separate, adjacent area, the second area, is provided which is downstream from the first area, and which is treated with a complimentary member of the Specific Binding Pair. The third area is comprised of the remainder of the strip, downstream from the second area.

The strips are then blocked with a material to eliminate or limit non-specific binding by the labeled Specific Binding Agent. Labeled Specific Binding Agent is spotted onto the strip at the first area, and the bibulous strip is developed using a solvent. When the ascending solvent front reaches a predetermined place near the distal end of the strip, the strip is removed from the developing solution and air dried. The distribution of the label along the strip, and within each area or zone, is determined using the label itself as the means of measurement. The amount of label in the second area, as a percentage of the total label applied to the strip, estimates the immunoreactive fraction of the labeled Specific Binding Agent. The amount of label in the first and third areas estimates label not associated with immunoreactive fraction; the first area measures colloidal and other particulate associated label, while the third area measures the soluble forms of label not associated with immunoreactive Specific Binding Agent. FIGS. 3 and 4 provide an illustrative configuration of the apparatus.

FIGS. 3 and 4 are, respectively, top and side views of the same apparatus. In these figures, the separating strip material 47 is provided, which may be any dry bibulous material, such as silica-gel-impregnated glass-fiber paper or cellulose- based thin layer chromatography paper. The zone 32 and 43 is impregnated with a ligand which does not form a Specific Binding Pair with the Specific Binding Agent. The zone 33 and 44 is impregnated with complimentary member of the Specific Binding Pair. The zone 31 and 42 is the origin, at which the labeled

Specific Binding Agent is spotted. The zone 34 and 45 does not contain complimentary member of the Specific Binding Pair, and may be impregnated with a ligand which blocks non-specific binding of the labeled Specific Binding Agent to the material 47. The zone 41 similarly does not contain complimentary member of the Specific Binding Pair, and may be impregnated with a ligand which blocks non-specific binding of the labeled Specific Binding Agent to the material 47. The arrows 36 and 48 represent the direction of solvent flow, with the separating strip immersed in a solvent up to about mark 30 and 40. The solvent is allowed to ascend to about mark 35 and 46, and is then removed from the solvent. The fraction of label present in both zone 32 and 43, in zone 33 and 44 and in zone 34 and 45 is determined, as is the total label applied to the separating strip FIGS. 3 and 4. The amount of label in zone 33 and 44, as a percentage of the total label applied to the strip, estimates the immunoreactive fraction of the labeled Specific Binding Agent. The amount of label in the zone 32 and 43 and in zone 34 and 45 estimates label not associated with immunoreactive fraction; the amount of label in zone 32 and 43 measures colloidal and other particulate associated label, while the amount of label in zone 34 and 45 measures the soluble forms of label not associated with immunoreactive Specific Binding Agent.

In another embodiment, samples of labeled Specific Binding Agent are mixed with cells, particles or other particulate matter including thereon the complimentary member of the Specific Binding Pair. Examples include cells wherein the complimentary member is a cell surface antigen or receptor, and the like. After allowing incubation for a period, an aliquot of the labeled Specific Binding Agent and cells or particulate matter are spotted onto a blocked separating strip made of dry bibulous material, such as silica-gel-impregnated glass-fiber paper or cellulose-based thin layer chromatography paper. The strip may be blocked with serum, other proteins or any substance which will substantially eliminate binding of the labeled Specific Binding Agent to the strip itself. A negative control may be used, consisting of cells, proteinaceous compositions or other compositions which do not contain the complimentary member of the Specific Binding Pair. This negative control is similarly spotted onto a blocked separating strip. The bibulous strips are then developed using a solvent. The cells or other particulate matter are trapped at or near the origin, and labeled Specific Binding Agent forming a Specific Binding Pair with the complimentary Specific Binding Agent comprising a part of the cells or other particulate matter is similarly trapped at or near the origin. Label not associated with Specific Binding Agent, and labeled Specific Binding Agent which is not immunologically or biologically active, migrates with the solvent front. When the ascending solvent front reaches a predetermined place near the distal end of the strip, the strip is removed from the developing solution and air dried. The distribution of the label along the strip is determined using the label itself as the means of measurement. The fraction of the label present at the origin of the strip spotted with the matter containing complimentary member of the Specific Binding Pair, and the fraction of the labeled present at the origin of the non-binding strip, is determined. The difference in the percentage of the tag at the origin of the binding and the non-binding strips estimates the immunoreactive fraction of the labeled Specific Binding Agent. FIGS. 5 and 6 provide an illustrative configuration of the apparatus. FIGS. 5 and 6 are, respectively, top and side views of the same apparatus. In these figures, the separating strip material 66 is provided, which may be any dry bibulous material, such as silica-gel-impregnated glass-fiber paper or cellulose- based thin layer chromatography paper. The zone 52 and 63 is impregnated with a ligand which does not form a Specific Binding Pair with the Specific Binding Agent, and which substantially eliminates binding of the labeled Specific Binding Agent to the strip material 66. Zone 61 and zone 53 and 64 are similarly block or impregnated, with the same or a different ligand which does not form a Specific Binding Pair with the Specific Binding Agent, and which substantially eliminates binding of the labeled Specific Binding Agent to the strip material 66. The zone 51 and 62 is the origin. A combination of the labeled Specific Binding Agent and cell or particulate matter including thereon the complimentary member of the Specific Binding Pair is allowed to incubate, and an aliquot spotted onto origin 51 and 62 of the positive strip. For measuring non-specific binding, a combination of the labeled Specific Binding Agent and cell or particulate matter not including thereon the complimentary member of the Specific Binding Pair is allowed to incubate, and an aliquot spotted onto origin 51 and 62 of a negative strip of FIGS. 5 and 6. The arrows 55 and 67 represent the direction of solvent flow, with the separating strip immersed in a solvent up to about mark 50 and 60. The solvent is allowed to ascend to about mark 54 and 65, and is then removed from the solvent. The cells or other particulate matter is trapped, by molecular seiving or other means, at or near origin 51 and 62, but in any case within zone 52 and 63. The fraction of label present in zone 52 and 63 is determined, as a fraction or percentage of the total label applied to origin 51 and 62. The difference in the percentage of the label in zone 52 and 63 of the separating strip with positive cell or particulate matter and the separating strip with negative cell or particulate matter estimates the immunoreactive fraction of the labeled Specific Binding Agent.

These means can be used to measure the immunoreactive fraction of a radiolabeled antibody or other radiolabeled Specific Binding Agent, and can thus be used for rapid quality control testing of radiopharmaceuticals. Quality controlling of immunoconjugates intended for use as therapeutic drugs is also possible. These means can further be used for quality control testing of reagents intended for use in various assays, such as radioimmunoassays and enzyme-linked immunoassays.

This method improves upon previous thin layer chromatography procedures for purity testing or quality controlling testing of radiopharmaceuticals. Previous methods are restricted to measuring radiochemical impurities, such as unbound radioisotope or radio-colloid contaminants. This method incorporates affinity-based binding, so that the fraction of radioisotopic tag or other marker which is associated with functionally useful Specific Binding Agents can also be determined by a simple, rapid test.

When used for the assessment of the quality of reagents, this method is not to be confused with affinity thin layer chromatographic methods which are used to determine the concentration of an analyte in a solution. The prior art involves the use of affinity thin layer chromatography for concentrating and measuring unknown quantities of analytes in a solution. In contrast, the method and apparatus described provides a procedure for the assessment of the quality of reagents, including those which might be employed in prior art assays. The method provided herein is distinct from those affinity thin layer chromatography systems in that it permits differentiation between specific and non-specific binding of the labeled Specific Binding Agent itself, rather than isolation or quantitation of an analyte. This invention provides means to determine the fractional amount of the label or other signal generating moiety which is bound to the functionally active Specific Binding Agents.

Another method of the invention is provided in which samples of labeled Specific Binding Agent are mixed with analyte containing cells or other particulate matter to permit determination of the presence or absence, and relative concentration, of the complimentary analyte member of the Specific Binding Pair. Examples include cells in cases in which the complimentary analyte member is a cell surface antigen or receptor, and the like. After allowing incubation for a period, an aliquot of the labeled Specific Binding Agent and cells or particulate matter are spotted onto a blocked separating strip made of dry bibulous material, such as silica-gel-impregnated glass-fiber paper or cellulose-based thin layer chromatography paper. The strip may be blocked with serum, other proteins or any substance which will substantially eliminate binding of the labeled Specific Binding Agent to the strip itself. A negative control may be used, consisting of cells, proteinaceous compositions or other compositions which do not contain the complimentary member of the Specific Binding Pair. This negative control is similarly spotted onto a blocked separating strip. The bibulous strips are then developed using a solvent. The cells or other particulate matter are trapped at or near the origin, and labeled Specific Binding Agent forming a Specific Binding Pair with the complimentary analyte Specific Binding Agent comprising a part of the cells or other particulate matter is similarly trapped at or near the origin. Labeled Specific Binding Agent which does not form a Specific Binding Pair with the complimentary analyte Specific Binding Agent, due to a lack or insufficiency of the complimentary analyte Specific Binding Agent, migrates with the solvent front. When the ascending solvent front reaches a predetermined place near the distal end of the strip, the strip is removed from the developing solution and air dried. The distribution of the label along the strip is determined using the label as the means of measurement. By means of radioactivity, color change or other signal generation, the presence of the unknown in the analyte may be determined by the amount of signal present on cells or particulate matter on or near the origin. If the concentration of labeled Specific Binding Agent, and concentration of cells or other particulate matter in the analyte, is known, then the signal generating system may be employed quantitatively, to determine the relative or absolute amount of complimentary Specific Binding Agent present in the analyte.

The invention is further illustrated by the following non-limiting examples. EXAMPLE 1. SEPARATING STRIPS Tumor Tissue

Tumor tissue was obtained from xenografts of the human colon carcinoma cell line LS-174T grown in athymic rodents. Tumors were visible and palpable after 7 days. The animals were maintained for 3 weeks in order to obtain mucinous ascitic fluid from the tumors. About 30% of the animals developed fluid- filled soft spots within the tumors during this period. These animals were anaesthetized and the fluid withdrawn. The tumor fluid was withdrawn and centrifuged at 10,000 x g and stored frozen until used. The tumor tissue was harvested when the tumors were approximately 1-4 grams. The tumors were homogenized in ice cold water (2: 1 , volume: weight), and residual particulate material removed by centrifugation at 10,000 x g. The resulting tumor homogenate was then stored frozen.

When ready for use the tumor materials were thawed, centrifuged to remove residual particulate material, and filtered through a 0.22 micron filter.

Coating of Strips

Silica-impregnated paper cellulose-based sheets were cut into 1.5 x 10 cm strips and activated by heating for 30 minutes at 110°C. After heating, the strips were stored at room temperature until coating.

Various amounts (0.01-0.6 ml) of tumor fluid or tumor homogenate were applied to a region approximately 3 cm from the intended bottom of the strips and allowed 5 seconds to bind. Typically, from 0.10 to 0.15 ml was applied, resulting in an antigen-coated zone extending from roughly 1.5 to 4.5 cm with application of 0.15 ml, and covering the width of the strip. The strips were then soaked for an additional 5 seconds in fetal bovine serum to block non-specific binding sites, rinsed for 10 seconds in running tap water, and air-dried. The air dried strips were stored refrigerated until used in experiments. In some cases, the tumor fluid was serially diluted in fetal bovine serum prior to application to the strips. Strips intended to act as "negative control strips" were prepared by applying various amounts (0.01-0.6 ml) of fetal bovine serum or newborn calf serum to a region approximately 3 cm from the intended bottom of the strips and allowed 5 seconds to bind. Typically, 0.15 ml was applied. The strips were then soaked for an additional 5 seconds in fetal bovine serum, rinsed for 10 seconds in running tap water, and air-dried. The air dried strips were stored refrigerated until used in experiments.

Chromatography and Analysis of Binding

All radiolabeled antibody preparations were diluted in either fetal bovine serum or newborn calf serum prior to application. No significant difference in results was noted using either serum type. Aliquots of 10 μl were applied to the strips, typically at 2 cm from the bottom of the strip. The antibody sample was diluted to contain between 100,000 and 500,000 CPM. The antibody solution was allowed a short period of time (approximately 5 seconds) to enter the matrix of the chromatography paper, and without drying the strip was placed in an aqueous solvent solution. The aqueous solvent solution was composed of 10 mM phosphate buffer, pH 7.0, containing 150 mM NaCl and 4% ethanol. The chromatogram was developed until the solvent front was within 0.5 cm of the top of the strip. The strip was then removed from the solvent, excess fluid removed by wicking from the bottom of the strip, and the strip air dried.

The percent immunoreactive fraction (IF) was determined as the ratio of the CPM in the origin half of the strip less background (O) divided by the total CPM applied (T) to the strip, multiplied by 100. Total CPM (T) applied to the strip was determined as the total of the CPM in each half of the strip, less background: %IF = (O/T) x 100. EXAMPLE 2. EVALUATION OF IMMUNOREACTIVE FRACTION OF RADIOLABELED ANTIBODY PREPARATIONS

Antibody Labeling with ⁹⁹ⁿTc

Murine monoclonal antibodies B72.3, A5B7, TNT-1, anti-SSEA-1 (MCA- 480 cell line), B37.2.1, SP-5514, 50H.19, BrE-3 and A6H and human gamma globulin (hGG) (Gamimune^®, Cutter Biological, Elkhart, IN) were prepared for

Tc labeling following the methods of U.S. Patents 5,078,985 and 5, 102,990. Use of these methods resulted in essentially quantitative reduction of pertechnetate as determined by thin layer chromatography (TLC), and 95-100% binding of the reduced technetium to the antibody.

Antibody Labeling with ¹⁸⁶Re

Murine monoclonal antibody B72.3 was prepared for ^l86Re labeling following the methods of U.S. Patents 5,078,985 and 5,102,990.

Results with "Tc Labeled Antibody Antigen coated strips were made as set forth in Example 1. Table 1 illustrates the results obtained using strips coated with either a 1: 1 homogenate of LS-174T tumor fluid (antigen positive) or newborn calf serum (antigen negative). The overall binding characteristics observed for the antibodies were consistent with known binding characteristics established by other means, including immunohistochemistry. The binding to the antigen negative strips was consistently low, less than 10%. The binding of colon cancer-positive antibodies to the antigen positive strips was at least 4 times higher than binding to negative control strips.

TABLE 1.

Antibody binding to positive (LS-174T zone) and negative strips. All antibodies surveyed were murine monoclonal antibodies with the exception of hGG, which is clinical-grade human gamma globulin. % RADIOACTIVITY AT THE ORIGIN

ANTIBODY SPECI- POSITIVE NEGATIVE

FICITY STRIP STRIP

REACTIVE WITH COLON TUMORS B72.3 tumor-associated glycoprotein 72 IgG, 54.2 3.6

TNT-1 nuclear histone protein IgG_2a 62.7 5.7

SP-5514 colon specific antigen "protein" IgG, 67.5 5.7

B37.2.1 stage-specific antigen- 1 IgM 52.3 9.9

MCA-480 stage-specific antigen- 1 IgM 53.0 6.2 50H.19 carcinoma/platelet antigen IgG₂, 41.7 4.1

NOT REACTIVE WITH COLON TUMORS

BrE-3 breast tumor antigen IgG, 10.3 2.6

A6H renal cell carcinoma IgG, 9.7 3.3 hGG unknown polyclonal 6.5 1.5

Results with ¹⁸⁶Re Labeled Antibody

¹⁸⁶Re-labeled B72.3 was used with antigen coated strips as described above. FIG. 7 shows the results with a positive separating strip, with a graphic representation of a radiochromatogram of the illustrated strip, showing a substantial portion of the total radioactivity associated with the positive LS-174T zone at the origin. Label at the solvent front presumptively represents label not associated with immunoreactive, labeled B72.3. FIG. 8 shows the results with a negative separating strip, with a graphic representation of a radiochromatogram of the illustrated strip, showing a small portion of the total radioactivity associated with the negative newborn calf serum zone at the origin. Label at the origin represents colloidal or other label which does not advance with the solvent front, representing non-specific binding of the labeled preparation. In FIG. 8, the label associated with the solvent front includes labeled, immunoreactive antibody. The total immunoreactive fraction is determined by subtracting the origin-associated label of FIG. 8 from the origin-associated label of FIG. 7, thereby yielding the net immunoreactive fraction. EXAMPLE 3. QUALITY CONTROL TESTING OF

RADIOLABELED PEPTIDE

Preparation of Peptide Kits and ^{9 m}Tc Labeling

A laminin Bl -chain (Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg) peptide was obtained commercially (Bachem, Inc. , Torrance, CA), and used to prepare labeling kits. The peptide was dissolved to a final concentration of 1.4 mg/ml in chilled, nitrogen-purged 10 mM tartrate/40 mM phthalate buffer, pH 5.6 (P/T buffer) containing 2% maltose. The peptide solution was mixed (7:3) with P/T buffer containing 1.25 mM stannous tartrate. Aliquots of peptide were sterile filtered, dispensed into nitrogen-purged vials, with the vials then stoppered, crimped, and stored frozen at -70°C. To radiolabel, a vial was removed from the freezer and allowed to come to room temperature. Labeling was accomplished by the addition of 0.5 - 2.0 mCi of ^99mTc as sodium pertechnetate in saline and a thirty minute incubation.

Preparation of Separating Strips

Negative control strips were prepared by dipping heat-treated silica- impregnated paper cellulose-based strips in an aqueous solution containing 0.5% gelatin. The strips were allowed to stay in the solution for approximately 10 seconds after which they were rinsed under a running stream of tap water and air- dried at 37°C.

Positive control strips, heated treated as above, were prepared using a stock solution of brain microsomes. Brain tumors, such as gliomas, and normal brain tissues are known to contain receptors specific for the laminin B-l chain peptide used in this example. Murine brain tissue was collected after euthanasia of mice. The brain tissue was pooled, weighed, and to it was added distilled water (4: 1 , volume: weight). The tissue was then homogenized on ice using a sonicator. The homogenate was cleared by low speed centrifugation at approximately 75 x g for 15 minutes. The supernatant was collected and recentrifuged at 15,000 x g for 20 minutes at 4°C. After aspirating the supernatant, the pellet containing brain microsomes was suspended in water (1:6) and stored frozen. For use in preparing positive control strips, the stock solution was thawed and mixed by inversion. Aliquots of 125 μl were applied to heat-treated strips at approximately 3 cm from the bottom of the strip, resulting in a zone of antigen from approximately 1.5 to 4.5 cm from the bottom of the strip, and covering the width of the strip. After the suspension had entered the strip matrix, the strip was dipped in a 0.5% gelatin solution for 10 seconds, rinsed, and dried.

For use in experiments, 10 μl of the radiolabeled peptide was diluted into 1 ml of phosphate buffered saline, pH 7.4, containing 0.5% gelatin. After mixing by vortexing, 10 μl of the radiolabeled peptide was spotted onto each of a positive and negative strip at 2 cm from the bottom of the strip. The strips were immediately placed into chromatography chambers containing phosphate buffered saline containing 4% ethanol. The chromatograms were allowed to develop until the solvent front was within 1 cm of the top of the strip. The strips were removed, wicked from the bottom to remove excess water, and air-dried. The strips were then cut in half and the percent radioactivity in each half determined by use of a gamma counter. 11.7% of the radioactivity was associated with the origin half of the negative control strip, and 59.0% of the radioactivity was associated with the origin half of the positive strip which contained homogenized brain microsomes.

EXAMPLE 4. SEPARATING STRIPS EMPLOYING NEUTROPHIL CELL SURFACE ANTIGEN

Preparation of Materials

Silica-gel impregnated cellulose sheets (ITLC-SG, Gelman Sciences) were cut into 1.5 x 10 cm strips and activated by heating for 30 minutes at 110°C, as per the manufacturer's instructions. The strips were soaked for 5 seconds in newborn calf serum, rinsed for 10 seconds in running tap water, and air-dried.

Following air drying, the strips were further dehydrated by incubation at 37 °C for at least 2 hours. The dried strips were stored at room temperature under desiccation until used. Human neutrophils were obtained from a normal adult donors, separated by elutriation, and stored on wet ice in Dulbecco's phosphate buffered saline containing EGTA. 4 volumes of PBS, pH 7.4, containing 50% serum, was added to the cell suspension and the cells collected by gentle centrifugation. The cell pellet was resuspended in PBS, pH 7.4, containing 50% serum, and the cell concentration adjusted to 1 x 10⁷ cells/ml. Some cell suspensions, in protein-free solutions, were fixed in buffered formalin; these suspensions were rinsed by repeated centrifugation in buffered saline containing 100 mM glycine. Fixed cells were resuspended in PBS, pH 7.4, containing 50% serum and 0.02% sodium azide, and adjusted to a concentration of 1 x 10⁷ cells/ml.

Radiolabeled Preparation

"Tc-labeled anti-SSEA-1 , a murine monoclonal IgM antibody produced by the MCA-480 cell line, and "Tc-hGG, "Tc-labeled human polyclonal, non¬ specific gamma globulin, used as a negative control, were labeled by the methods of Example 2. "Tc-anti-SSEA-l binds to a cell surface antigen found on human neutrophils, and also, as in Example 2, cross-reacts with a colon cancer associated antigen.

Assay Procedure and Results

Aliquots of cells were added to individual wells of 96-well cluster plates and the volume adjusted to 200 μl with PBS, pH 7.4, containing 50% serum. Aliquots of 20 μl of "Tc-anti-SSEA-l was added to each well and the samples mixed. The samples were incubated at 37°C for 30 minutes, with mixing by pipetting. After mixing, aliquots of 10 μl (containing between 100,000 and 500,000 CPM) were applied to the strips, typically at the 2 cm mark. The solution was allowed to enter the matrix of the chromatography paper (approximately 5 seconds), and without drying the strip was placed in an aqueous solvent solution composed of 10 mM phosphate buffer, pH 7.0, containing 150 mM NaCl, and optionally containing various concentrations of ethanol. The chromatogram was developed until the solvent front was at 9.5 cm (within 0.5 cm of the top of the strip). The strip was then removed from the solvent, excess fluid removed by wicking from the bottom of the strip, and the strip air dried. The strips were then cut in half and the radioactivity in each half determined.

The percent immunoreactive fraction (IF) was determined as the ratio of the CPM in the origin half of the strip less background (O) divided by the total CPM applied (T) to the strip, multiplied by 100. Total CPM (T) applied to the strip was determined as the total of the CPM in each half of the strip, less background: %IF = (O/T) x 100.

Table 2 presents data from experiments in which varying numbers of human neutrophils (live and fixed) were used to bind ""Tc-anti-SSEA-l. The percentage cell binding with ""Tc-anti-SSEA-l increased as a function of cell concentration.

"Tc-hGG was the negative control, and nearly all of it migrated with the solvent front regardless of the cell concentration. Using fixed neutrophils in conditions of antigen excess, the amount of "Tc-anti-SSEA-l binding was greater than 60% while the amount of "Tc-hGG binding was less than 5%. TABLE 2.

Comparison of binding of "Tc-anti-SSEA-l and "Tc-hGG (negative control) to increasing numbers of live and formalin-fixed cells. After 30 minute incubation of a fixed quantity of radiolabeled antibody and cells, an aliquot was used for with separating strips as described above. % RADIOACTIVITY AT THE ORIGIN

CELL NUMBER ANTI-SSEA-1 x 10⁶ LIVE FIXED hGG

0 2.7 2.0 4.8

0.25 23.6 17.5 2.6

0.5 33.3 27.3 2.8

1 47.8 33.6 5.5

2 49.6 48.1 0.1

4 58.7 53.6 0.1 EXAMPLE 5. CELL SURFACE ANTIGEN SEPARATING STRIPS EMPLOYING STIMULATED NEUTROPHILS

Cell surface antigen separating strips were prepared as in Example 4, using normal human neutrophils which were not fixed. A quantity of freshly-isolated neutrophils was selected which was below the threshold of plateau-binding for a fixed concentration of "Tc-anti-SSEA- 1. Plateau binding was approximately 52 % . 2 μg of a chemotactic peptide (f-Met-Leu-Phe-Gly-Gly-His-Trp), which bound to human neutrophils as determined by flow cytometry, or serum as a negative control, was added to the cells. After 5 minutes ""Tc-anti-SSEA-l was added, the preparations mixed and incubated at 37°C for 30 minutes. The preparations were then analyzed using the methods of Example 4. Without stimulation by a chemotactic peptide, the origin half of the strip containing the cells had 42.7% ± 1.1 % (S.E., n=3) of the total radioactivity. With chemotactic peptide stimulation, the origin half of the strip containing these cells had 49.7% ± 4.8% of the total radioactivity. One set of stimulated cells also received approximately 1 mM sodium ascorbate to act as a free radical scavenger; the origin half of the strip containing these cells had 51.3% ± 1.3% of the radioactivity.

EXAMPLE 6. SEPARATING STRIPS EMPLOYING PLATELET CELL SURFACE ANTIGEN Separating strips and cells were prepared as in Example 4, using normal human platelets collected by centrifugation from platelet-rich plasma and resuspended in phosphate buffered saline, pH 7.0, containing 25% bovine serum and 0.5 mM EDTA. "Tc- 50H.19, an IgG _a murine monoclonal antibody reactive with platelet determinants, was used. The assay procedures of Example 4 were used.

Using freshly isolated platelets, the measured immunoreactive fraction of "TC-50H.19 was 74.6% ± 0.9% (S.D. , n=4), while the corresponding value for "Tc-hGG was 3.6% ± 1.6% . Similar results were obtained with formaldehyde- fixed platelets, with an immunoreactive fraction for "Tc-50H.19 antibody of 75.6% ± 1.4% , and a negative control "Tc-hGG value of 7.3% ± 2.5% (S.D., n=3). EXAMPLE 7. VARYING PLATELET CONCENTRATION Using the methods and reagents of Example 6, the platelet concentration was varied, using doubling dilutions of platelets. The amount of "Tc-50H.19 used with each cell suspension was held constant, so that approximately 0.3 μg of "Tc-50H.19 was added to each cell suspension. The radiolabeled antibody preparation was incubated with the cell suspension for 30 minutes at 37°C, following which an aliquot of the cell suspension was applied to separating strips and analyzed as in Example 4. The binding of "Tc-50H.19 to fixed platelets was found to be dependent on platelet concentration, and is illustrated in FIG. 9. FIG. 9 shows that as the concentration of platelets increases, the binding at the origin, measured as a percentage of the total label applied, increased, and appears to reach a plateau level at approximately 70% to 80% .

EXAMPLE 8. TIME KINETICS OF BINDING TO PLATELET CELL SURFACE ANTIGEN The time dependent kinetics of "Tc-50H.19 binding to platelets was evaluated using formalin-fixed platelets and separating strips, prepared as in Example 6. At a constant platelet concentration, the maximum amount of binding was observed 15 minutes after addition of the labeled antibody to the platelets, with no increase in binding observed at later time periods. At 15, 30 and 60 minutes, the results were very consistent, with measured label at the origin of 75.5 ± 0.5%, 75.2 ± 0.8% and 75.3 ± 1.1 % , respectively, where n = 3. With incubations of 0.5, 2 and 5 minutes, the apparent immunoreactive fraction was decreased, and the results were significantly less consistent, with measured label at the origin of 24.4% ± 5.7%, 49.2% ± 5.6% and 66.5% ± 2.3%, respectively.

EXAMPLE 9. COMPETITION WITH UNLABELED SPECIFIC

BINDING AGENT USING PLATELET CELL SURFACE ANTIGEN

The binding of "Tc-50H.19 to formalin-fixed platelets was found to be specifically blocked by high concentrations of unlabeled 50H.19, but not unlabeled BrE-3, a murine antibody reactive with breast epithelial mucin. Reagents and materials were prepared as in Example 6. "Tc-50H.19 with no addition of unlabeled antibody exhibited an immunoreactive fraction of 73.2% and served as the control and reference point. A series of incubations were performed in which various concentrations, using doubling dilutions starting at 25 μg of unlabeled antibody, was added to 1.5 μg of "Tc-50H.19. Separate experiments were performed adding unlabeled 50H.19 and unlabeled BrE-3. BrE-3 antibody did not reduce the binding of "Tc-50H.19 at any concentration of BrE-3 used. Unlabeled 50H.19 did compete with "Tc-50H.19 and markedly reduced the binding of "Tc- 50H.19. Results are shown in FIG. 10. In FIG. 10, the addition of BrE-3 did not affect binding of labeled 50H.19, as is shown by the constant plateau. The addition of unlabeled 50H.19 did have a drastic effect in high concentrations, with substantial competitive inhibition. Plateau binding was achieved with addition of 0.8 μg of unlabeled 50H.19, and is shown at all lesser amounts.

EXAMPLE 10. VARYING ANTIGEN CONCENTRATION ON SEPARATING STRIPS Strips were prepared as in Example 1, using tumor homogenate of the carcinoma cell line LS-174T. Concentrations were varied, such that weight to volume ratios of tumor to water varied from 1: 1 to 1:8, with no homogenate applied as a negative control. 100 μl of each homogenate, following centrifugation, was applied to each strip, and the strips then blocked with 50% newborn calf serum and washed. Two antibodies were employed, B72.3 and TNT-1 , radiolabeled with "Tc by the method of Example 2. Chromatography and analysis was performed as in Example 1 , and the results are shown in Table 3.

TABLE 3.

Effect of increasing homogenate load on the detection of immunoreactive fraction of "Tc-labeled murine antibodies. The experimental values shown are the average of two separate, closelv agreeing determinations.

LS-174T HOMOGENATE W/V OF % IMMUNORl__ACTIVEFRACnON

mg APPLIED HOMOGENATE B72.3 TNT-1

0 3.5% 2.7%

1.0 1:8 35.0 50.0

2.0 1:4 38.3 60.8

3.5 1 :2 49.0 60.4

8.0 1 : 1 49.7 62.7

This method, employing strips with varying antigen concentrations, or similarly employing varying concentrations of cells or other particulate antigenic material, may be employed to determine that the antigen or receptor is in functional infinite excess, by determining the plateau level of binding.

EXAMPLE 11. VARYING AMOUNT OF LABELED SPECIFIC BINDING AGENT APPLIED TO SEPARATING STRIPS

Strips were prepared as in Example 1 , using tumor homogenate of the carcinoma cell line LS-174T applied to the strips. Antibody B72.3 was labeled as in Example 2. The amount of labeled antibody applied was varied, so that from

0.03 to 4.0 μg of "Tc-B72.3 was applied to the strips. Strips were then developed and the immunoreactive fractions calculated using the methods of

Example 1. Table 4 shows the results.

TABLE 4.

10 μl of "Tc-B72.3 was applied to each strip, so that the total antibody applied ranged from 4.0 μg to 0.03 μg.

SERIAL ANTIBODY % BINDING AT

DILUTION PER 100 ul ORIGIN

0 40.0 μg 28.3

1 20.0 48.4

2 10.0 56.5

3 5.0 58.2

4 2.7 54.2

5 1.5 54.3

6 0.8 54.4

7 0.5 54.2

8 0.4 54.9

9 0.3 61.9

This method, employing varying amounts of labeled Specific Binding Agent, may be employed to determine that the unlabeled Specific Binding Agent, generally an antigen or receptor, is in functional infinite excess relative to the amount of labeled Specific Binding Agent, by determining the plateau level of binding. In practice, if one or more serial dilutions of the labeled Specific Binding Agent are employed, and if the immunoreactive or bioreactive fraction at all dilutions are substantially similar, then the unlabeled Specific Binding Agent is in functional infinite excess relative to the labeled Specific Binding Agent. This method may be employed both where unlabeled Specific Binding Agent is bound to separating strips, and where cells or particulate materials are employed.

EXAMPLE 12. CANCER CELL SURFACE ANTIGEN PARTICLE SEPARATING STRIPS

Cells and separating strips are prepared as in Example 4, using colorectal cancer cells from the cell line LS-174T grown using normal cell culture technique. Using "Tc-B72.3, labeled by the method of Example 2, an assay is conducted as in Example 4. Binding in the origin half of the strip containing cells from the cell line LS-174T is higher than that observed on negative control strips. EXAMPLE 13. COMPARISON OF METHODS

Separating strips, containing a zone of LS-174T tumor homogenate, were prepared as in Example 1. Neutrophil cell surface antigen cells and blocked separating strips were prepared as in Example 4. RhoChek™ (RhoMed Incorporated, Albuquerque, New Mexico) was used as a control; this is a solid- phase antigen homogenate bound to plastic particles, which in use is repeatedly washed and centrifuged to remove radioactivity not associated with bound immunoreactive antibody. The RhoChek™ product uses LS-174T tumor homogenate as a positive control. "Tc-anti-SSEA-l was used, and three separate preparations were radiolabeled. For analysis by RhoChek™ and by separating strips prepared by the method of Example 1 , the net immunoreactive fraction is reported. For separating strips by the method of Example 1 , the net immunoreactive fraction is the immunoreactive fraction obtained using an antigen positive strip minus the immunoreactive fraction obtained using an antigen negative strip. The data is shown in Table 5. TABLE 5.

"Live Cell Assay" and "Fixed Cell Assay" employed separating strips by the method of Example 4; "Antigen Bound Strips" employed separating strips with a zone of tumor homogenate by the method of Example 1; the "Rhochek™ Assay" is as described above. "RhoChek™ Assay" and "Antigen Bound Strips" values are net immunoreactive fraction. In this Table. "ND" means not determined.

Antigen

Live Cell Fixed Cell RhoChek™ Bound

Anti-SSEA-1 Assav Assav Assav Strips

Exp. 1 49.32% ND 35.39% 45.18%

Exp. 2 48.17% ND 33.85% 41.58%

Exp. 3 ND 44.60% 50.32% 49.79%

Exp. 4 ND 48.80% 44.09% 44.99%

Exp. 5 ND 44.30% 43.42% 48.81 %

EXAMPLE 14. SEPARATING STRIPS WITH POSITIVE AND

NEGATIVE CONTROL AREAS

Positive strips were prepared as in Example 1, using LS-174T tumor homogenate for the positive material, with changes as noted herein. To heat activated 1.5 x 10 cm strips, 0.10 ml of tumor homogenate was applied to a region approximately 4.5 cm from the intended bottom of the strips, resulting in an antigen-coated zone extending from roughly 3.5 to 5.5 cm, and covering the width of the strip. The strips were then soaked in newborn calf serum for 5 seconds to block non-specific binding sites, rinsed in running tap water, and air-dried. Three areas were designated: area one, to which the sample was applied and which served to measure colloidal and other non-specific binding of the labeled specific binding agent, being the area from about 0.5 to 3.0 cm; area two, containing the positive tumor homogenate, extending from about 3.0 to 6.0 cm; and, area three, extending from about 6.0 cm to the end of the strip.

"Tc-labeled B72.3 antibody was used, labeled as in Example 2. An aliquot of 10 μl of radiolabeled antibody preparation, diluted to contain between 100,000 and 500,000 CMP, was applied at about 1.5 cm from the bottom of the strip. The strip was then placed in an aqueous solvent solution, and the chromatogram developed until the solvent front was within 0.5 cm of the top of the strip. The strip was then dried, and radioactivity measured using a radiochromatogram imaging scanner (Imaging Scanner System 200; BIOSCAN, Inc., Washington, DC). The total CPM for areas one, two and three were determined. The radioactivity in area one, the negative control area, as a percentage of the total radioactivity applied, correlated with results obtained using negative control strips as in Examples 1 and 2. The radioactivity in area two, the positive control area, as a percentage of the total radioactivity applied, correlated with results obtained using positive control strips as in Examples 1 and 2. This thus allows three measures to be made using a single separating strip: colloidal and other particulate associated radioactivity is measured in area one, the immunoreactive fraction is measured in area two, and the soluble forms of radioactivity not associated with immunoreactive SPA is measured in area three.

EXAMPLE 15. IMMUNOREACTIVE FRACTION OF ENZYME LABELED SPECIFIC BINDING AGENT

Negative and positive strips were prepared as in Example 1, using LS-174T tumor homogenate for the positive material, and serum as the negative material. The murine monoclonal antibody SP-5514 was labeled with horseradish peroxidase by standard methods. The enzyme and antibody conjugate was diluted 1 : 100 into PBS, pH 7.4 containing 50% serum. An aliquot of 10 μl was spotted onto both the positive and negative strips, at approximately 2 cm from the bottom. The strips were chromatographed in PBS, pH 7.4, containing 4% ethanol until the solvent front was near the top of the strips. The strips were removed from the chromatography solution, and while still damp overlayed with paper towels lightly soaked in a chromogen-containing solution (TMB One-Step, Pierce Chemical Company, Rockville, IL). The immunoreactive fraction was estimated visually. On the negative control strip essentially all of the chromogen was deposited at a position slightly behind the solvent front. On the strip with antigen, virtually all the chromogen, indicative of the location of the enzyme and antibody conjugate, was concentrated as a circle which corresponded to the point of application at the origin.

EXAMPLE 16. DETECTION OF ANTIGEN/ANTIBODY

COLLOID COMPLEXES

Antibodies

Murine monoclonal antibodies B72.3 (reactive with tumor-associated glycoprotein 72, murine origin), T84.66 (reactive with carcinoembryonic antigen), and BrE-3 (reactive with milk fat globule protein) were prepared for "Tc labeling following the methods of U.S. Patent Nos. 5,078,985 and 5,102,990. Use of these methods resulted in essentially quantitative reduction of pertechnetate as determined by thin layer chromatography (TLC), and 95-100% binding of the reduced technetium to the antibody.

Tumor Tissue Analyte

Tumor tissue was obtained from xenografts of the human colon carcinoma cell line LS-174T grown in athymic rodents. Tumors were visible and palpable after 7 days. The animals were maintained for 3 weeks in order to obtain mucinous ascitic fluid from the tumors. About 30% of the animals developed fluid- filled soft spots within the tumors during this period. These animals were anaesthetized and the fluid withdrawn. The fluid was then centrifuged at 10,000 x g and stored frozen. The tumor tissue was harvested when the tumors were approximately 1-4 grams. The tumors were homogenized in ice cold water (2: 1, volume: weight), and the residual particulate material removed by centrifugation at 10,000 x g. The resulting tumor homogenate was then stored frozen.

Preparation of Separating Strips

Silicone-impregnated paper cellulose-based sheets were cut into 1.5 x 10 cm strips and activated by heating for 30 minutes at 110°C. After heating, the strips were stored at room temperature until coating. To coat, strips were soaked in either bovine serum albumin (BSA) or human serum albumin (HSA), at a concentration of 5 mg/ml in aqueous 0.9% NaCl, for 30 minutes, rinsed in distilled water for 5 seconds, and air-dried overnight. The air dried strips were stored refrigerated until used in experiments.

Chromatography and Analysis of Binding

An aliquot of 2 μl of dilute radiolabeled antibody, containing between 5,000,000 and 500,000 CPM of high specific activity ""Tc, was added to 100 μl of analyte. The analyte was composed of tumor fluid from the LS-174T tumor or serial dilutions of the tumor fluid in phosphate buffered saline, pH 7.0, containing 0.5% bovine serum albumin. For negative control analyte samples, bovine submaxillary mucin was added to phosphate buffered saline, pH 7.0 containing 0.5% bovine serum albumin at a final mucin concentration of 2%. The antibody and analyte mixture was incubated at 37 °C for 1 hour. 10 μl aliquots of the mixture containing the radiolabeled antibody were applied to the strips at 2 cm from the bottom of the strip. The mixture was allowed approximately 5 seconds to enter the matrix of the separating strip, and without drying the strip was place in an aqueous solvent solution. The aqueous solvent solution was composed of 10 mM phosphate buffer, pH 7.0 containing 150 mM NaCl. The chromatogram was developed until the solvent front was within 0.5 cm of the top of the strip. The strip was removed from the solvent, excess fluid removed by wicking from the bottom of the strip, and the strip air dried. The strips were then cut in half, and the radioactivity in each half measured and compared.

Results of Antigen Binding

Binding, determined by the percentage of total radioactivity associated with the origin half of the strip, was found to be dependant on the presence or absence of the complimentary Specific Binding Agent in the analyte. Results are shown in Table 6.

TABLE 6.

Percent binding in the origin half of strip. Bovine submaxillary mucin in a bovine serum albumin mixture was used as a negative control.

% 9 "9πTv c at Origin

ANALYTE

ANTIBODY SERUM LS-174T

T84.66 1.1 % 13.5%

BrE-3 1.0% 6.8%

B72.3 1.8% 29.3%

EXAMPLE 17. CHROMOGENIC DETECTION OF ANTIGENS IN TUMOR HOMOGENATES

Antibodies and Conjugation

Murine monoclonal antibodies SP-21 (reactive with colon ovarian tumor antigen) and 5514 (reactive with carcinoembryonic antigen) were conjugated with horseradish peroxidase (HRP) using a peroxidase coupling method. An aliquot (5 mg) of HRP was dissolved in 1.2 ml of water, and 0.3 ml of freshly prepared 0.1 M sodium periodate in phosphate buffered saline, pH 7.0, was added. After a 20 minute incubation, the solution was dialyzed against 1 mM acetate buffer, pH 4.0, under refrigeration. Antibody (10 mg/ml) was prepared in 20 mM carbonate buffer, pH 9.0, and 0.5 ml of the antibody mixed with the dialyzed solution. The mixture was incubated for 2 hours, after which 0.1 ml of sodium borohydride (4 mg/ml) in water was added. After 2 hours under refrigeration, the coupled antibody was dialyzed against several changes of phosphate buffered saline. The antibody-HRP complex was separated from unbound HRP by selective precipitation in polyethylene glycol followed by ion exchange chromatography on DEAE- Sephadex. The antibody conjugates were stored at -20°C in 50% glycerol until used.

Analyte

Tumor tissue was obtained from xenografts of the human colon carcinoma cell line LS-174T as in Example 16, or from human cancer specimens obtained as biopsies or during elective surgery. All tumor and tissue specimens were homogenized in 5 volumes of water, cleared by centrifugation, and the soluble material stored frozen until use in experiments.

Preparation of Separating Strips

Silicon-impregnated paper cellulose-based sheets were cut into 1.5 x 10 cm strips and activated by heating for 30 minutes at 110°C. After heating, the strips were stored at room temperature until coating. To coat, strips were soaked in either bovine serum albumin (BSA) or human serum albumin (HSA) at a concentration of 5 mg/ml in aqueous 0.9% NaCl for 30 minutes, rinsed in distilled water for 5 seconds, and air-dried overnight.

Chromatography and Analysis of Binding

An aliquot of 2-5 μl of HRP-conjugated antibody was added to 100 μl of analyte. The analyte was composed of tumor homogenate. The antibody/analyte mixture was incubated at 37 °C for 1 hour. 10 μl aliquots of the mixture were applied to the strips at 2 cm from the bottom of the strip. The mixture was allowed approximately 5 seconds to enter the matrix of the separating strip, and without drying the strip was placed in an aqueous solvent solution. The aqueous solvent solution was composed of 10 mM phosphate buffer, pH 7.0, containing 150 mM NaCl. The chromatogram was developed until the solvent front was within 0.5 cm of the top of the strip. The strip was then removed from the solvent, excess fluid removed by wicking from the bottom of the strip, and while the strip was wet the chromatogram was stained by dropwise coating with a buffered solution of diamino benzidine. After the color developed, the strips were rinsed in tap water and air dried.

The tumor specimen chromatograms developed intense colors which could easily be scored by visual examination. The antibody-antigen complexes were retained at the origin, resulting in a color change at the origin, while unbound antibody migrated with the solvent front, resulting in a color change at the solvent front. The intensity of the color change at the origin was proportional to the antigen concentration, as determined by immunohistochemistry, with results shown in Table 7. Both colon ovarian tumor antigen and carcinoembryonic antigen were detected to varying degrees in the specimens.

TABLE 7.

Comparison of the detection of carcinoembryonic antigen by use of horseradish peroxidase-conjugated SP-558, a murine monoclonal antibody, by methods of the invention and compared to results obtained by conventional immunohistopathology of the same tumors with both SP-558 and an antibody reactive with carcinoembryonic antigen. The tumor specimen chromatograms were scored from negative "-" to highly positive " + + + + ", using a five-step increment scoring system.

DETECTION BY

SAMPLE INVENTION IMMUNOHISTOPATHOLOGY STAINING

One + + + + Liver metastasis of a moderately differentiated adenocarcinoma of large bowel origin. Sharp, heavy staining at 3-4+ throughout the entire specimen, cytoplasm, mucus, and necrotic debris. Hepatocytes negative.

Two + Moderately differentiated mucogenic carcinoma of large bowel origin, strong positive in mucus, cytoplasmic stain very weak, large majority of cells were negative

Three + + + Mucus-rich adenocarcinoma of large bowel origin, poor/moderate differentiation, virtually all cells stain strongly, mucus stain moderate

Four + + + + Moderately differentiated adenocarcinoma of large bowel origin, poor mucus production, trace to strong staining of almost every tumor cell, brush borders accentuated

LS-174T + + + Well differentiated adenocarcinoma of the colon, positive control, ascitic fluid fetal bovine Negative control serum EXAMPLE 18. DETECTION OF ANTIGENS ON NEUTROPHIL WHOLE CELLS USING RADIOLABELED ANTIBODY

Preparation of Separating Strips

Silica-gel impregnated cellulose sheets (ITLC-SG, Gelman Sciences) were cut into 1.5 x 10 cm strips and activated by heating for 30 minutes at 110°C, as per the manufacturer's instructions. The strips were soaked for 5 seconds in newborn calf serum, rinsed for 10 seconds in running tap water, and air-dried. Following air drying, the strips were further dehydrated by incubation at 37°C for at least 2 hours. The dried strips were stored at room temperature under desiccation until used.

Human neutrophils were obtained from normal adult donors, separated by elutriation, and stored on wet ice in Dulbecco's phosphate buffered saline containing EGTA. 4 volumes of PBS, pH 7.4, containing 50% serum, was added to the cell suspension and the cells collected by gentle centrifugation. The cell pellet was resuspended in PBS, pH 7.4, containing 50% serum, and the cell concentration adjusted to 1 x 10⁷ cells/ml. Some cell suspensions, in protein-free solutions, were fixed in buffered formalin; these suspensions were rinsed by repeated centrifugation in buffered saline containing 100 M glycine. Fixed cells were resuspended in PBS, pH 7.4, containing 50% serum and 0.02% sodium azide, and adjusted to a concentration of 1 x 10⁷ cells/ml.

Radiolabeled Preparation

"Tc-labeled anti-SSEA-1 , a murine monoclonal IgM antibody produced by the MCA-480 cell line, and "Tc-hGG, ""Tc-labeled human polyclonal, non¬ specific gamma globulin, used as a negative control, were labeled by the methods of Example 16. "Tc-anti-SSEA-l binds to a cell surface antigen found on human neutrophils.

Assay Procedure and Results

Aliquots of cells were added to individual wells of 96-well cluster plates and the volume adjusted to 200 μl with PBS, pH 7.4, containing 50% serum. Aliquots of 20 μl of ""Tc-anti-SSEA-l was added to each well and the samples mixed. The samples were incubated at 37 °C for 30 minutes, with mixing by pipetting. After mixing, aliquots of 10 μl (containing between 100,000 and 500,000 CPM) were applied to the strips, typically at the 2 cm mark. The solution was allowed to enter the matrix of the separating strip (approximately 5 seconds), and without drying the strip was placed in an aqueous solvent solution composed of 10 mM phosphate buffer, pH 7.0, containing 150 mM NaCl, and optionally containing various concentrations of ethanol. The chromatogram was developed until the solvent front was at 9.5 cm (within 0.5 cm of the top of the strip). The strip was then removed from the solvent, excess fluid removed by wicking from the bottom of the strip, and the strip air dried. The strips were then cut in half and the radioactivity in each half determined and compared.

Table 8 presents data from experiments in which varying numbers of human neutrophils (live and fixed) were used to bind ""Tc-anti-SSEA-l. The percentage cell binding with ""Tc-anti-SSEA-l increased as a function of cell concentration.

""Tc-hGG was the negative control, and nearly all of it migrated with the solvent front regardless of the cell concentration. TABLE 8.

Comparison of binding of "Tc-anti-SSEA-l and ""Tc-hGG (negative control) to increasing numbers of live and formalin-fixed cells. After 30 minute incubation of a fixed quantity of radiolabeled antibody and cells, an aliquot was used introduced to the separating strips, and the strips developed.

% RADIOACTIVITY AT THE ORIGIN C CEELLLL NNUUMMBBEERR ANTI-SSEA-1

X 10⁶ LIVE FIXED hGG

0 2.7% 2.0% 4.8%

0.25 23.6 17.5 2.6

0.5 33.3 27.3 2.8

1 47.8 33.6 5.5

2 49.6 48.1 0.1

4 58.7 53.6 0.1 EXAMPLE 19. ASSAY FOR CELL SURFACE ANTIGEN WITH STIMULATED NEUTROPHILS

Cell surface antigen cells and strips were prepared as in Example 18, using normal human neutrophils which were not fixed. A quantity of freshly-isolated neutrophils was selected which was below the threshold of plateau-binding for a fixed concentration of "Tc-anti-SSEA- 1. Plateau binding was approximately 52 % .

2 μg of a chemotactic peptide (f-Met-Leu-Phe-Gly-Gly-His-Trp), which bound to human neutrophils as determined by flow cytometry, or serum as a negative control, was added to the cells. After 5 minutes ""Tc-anti-SSEA-l was added, the preparations mixed and incubated at 37°C for 30 minutes. The preparations were then analyzed by the methods of Example 18. Without stimulation by a chemotactic peptide, the origin half of the strip containing the cells had 42.7% ± 1.1 % (S.E., n =3) of the total radioactivity. With chemotactic peptide stimulation, the origin half of the strip containing these cells had 49.7% ± 4.8% of the total radioactivity. This assay method thus provided means for determining the increase in receptors due to addition of the chemotactic peptide, permitting relative quantification.

EXAMPLE 20. DETECTION OF PLATELET CELL SURFACE ANTIGEN Cell surface antigen cells and strips were prepared as in Example 18, using normal human platelets collected by centrifugation from platelet-rich plasma and resuspended in phosphate buffered saline, pH 7.0, containing 25% bovine serum and 0.5 mM EDTA. "Tc-50H.19, an IgG_2a murine monoclonal antibody reactive with platelet determinants, was used. The assay procedures of Example 18 were used.

Using freshly isolated platelets, 74.6% ± 0.9% (S.D., n=4) of the "Tc- 50H.19 bound to the platelets, while the corresponding value for the negative control radiolabeled antibody, "Tc-hGG, was 3.6% ± 1.6%. Similar results were obtained with formaldehyde-fixed platelets, with 75.6% ± 1.4% of the "Tc- 50H.19 antibody bound to the platelets, and a negative control "Tc-hGG value of 7.3% ± 2.5 % (S.D. , n =3). EXAMPLE 21. VARYING PLATELET CONCENTRATION IN ASSAY METHODS

Using the methods and reagents of Example 20, the platelet concentration was varied, using doubling dilutions of platelets. The amount of "Tc-50H.19 used with each cell suspension was held constant, so that approximately 0.3 μg of

"Tc-50H.19 was added to each cell suspension. The radiolabeled antibody preparation was incubated with the cell suspension for 30 minutes at 37°C, following which an aliquot of the cell suspension was applied to separating strips and analyzed as in Example 19. The binding of "Tc-50H.19 to fixed platelets was found to be dependent on platelet concentration.

EXAMPLE 22. CANCER CELL SURFACE ANTIGEN ASSAY

Cells and separating strips are prepared as in Example 16, using colorectal cancer cells from the cell line LS-174T grown using normal cell culture technique.

Using "Tc-B72.3, labeled by the method of Example 16, an assay is conducted as in Example 16. Binding in the origin half of the strip containing cells from the cell line LS-174T is higher than that observed on negative control strips.

EXAMPLE 23. DETECTION OF ANALYTES COATED ONTO PARTICLES

Polyvinylidine particles were prepared for coating by washing in methanol. The particles were then collected by centrifugation and rinsed in distilled water. The particles were adjusted to a 1 : 10 w/v ratio, and a 1:5 solution of cleared tumor homogenate added to the slurry. The resulting slurry was incubated at 4° C overnight and subsequently the coated particles were collected by centrifugation. The particles were then washed twice in phosphate buffered saline, and suspended to a final concentration of 10% (v/v) using phosphate buffered saline containing 50% serum and 0.05% sodium azide.

For use in assays the particle slurry was first mixed well, and then a test aliquot of 200 μl placed in a tube. An aliquot of radiolabeled antibody, typically containing 100,000 to 500,000 CPM, was added to the tube. The radiolabeled antibody and particle slurry was mixed, and the solution allowed to incubate for 30 minutes at room temperature. At the end of the incubation period, the radiolabeled antibody and particle slurry was mixed by vortexing, and 10 μl of the radiolabeled antibody and particle slurry mixture applied to a serum-coated separating strip at 2 cm from the bottom of the strip. Without drying, the bottom of the strip (up to 1 cm) was immersed in phosphate buffered saline solvent containing 4% ethanol. The mobile phase was allowed to migrate to within 0.5 cm of the top of the strip. The strip was removed from the solvent, excess fluid removed by wicking from the bottom of the strip, and the strip air dried. The strips were then cut in half, and the radioactivity in each half measured.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

Although the invention has been described in detail with particular reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited above, and of the related application filed concurrently herewith, are hereby incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A method for determining an amount of label associated with a labeled specific binding agent in a specific binding agent preparation capable of forming a specific binding pair, which method comprises the steps of: a) providing material comprising capillarity and capacity for solvent transport, the material comprising first zone means with first reagent means immobilized against solvent transport and capable of forming a specific binding pair with the labeled specific binding agent; b) introducing a quantity of the specific binding agent preparation to the material at a location on or downstream from the first zone means; c) contacting the material with solvent means, whereby the solvent means traverses the locus of introduction of the specific binding agent preparation and at least a portion of the first zone means; and d) detecting the amount of label at the first zone means associated with labeled specific binding agent forming the specific binding pair.

2. The method of claim 1 , wherein the material further comprises second reagent means immobilized against solvent transport which is not capable of forming a specific binding pair with the labeled specific binding agent and wherein the second reagent means immobilized against solvent transport substantially blocks non-specific binding of the labeled specific binding agent to the material.

3. The method of claim 1 , further comprising the following additional steps: e) providing material having capillarity and the capacity for solvent transport, the material comprising second zone means with the second reagent means;

0 introducing a quantity of the specific binding agent preparation to the material at a location on or downstream from the second zone means; g) contacting the material with solvent means, whereby the solvent means traverses the locus of introduction of the specific binding agent preparation and at least a portion of the second zone means; and h) detecting the amount of label at the second zone means not transported by the solvent means.

4. A test apparatus for determining an amount of label associated with a labeled specific binding agent in a specific binding agent in a specific binding agent preparation capable of forming a specific binding pair, said apparatus comprising: bibulous material comprising capillarity and a capacity for solvent transport, said material comprising: first end means at which solvent transport begins; second end means at which solvent transport ends; and solvent transport pathway means; zone means positioned between said first and second end means, said zone means comprising: first zone means for receiving the specific binding agent preparation; and second zone means, coincident with or downstream from said first zone means, said second zone means comprising first reagent means immobilized against solvent transport and capable of forming a specific binding pair with the labeled specific binding agent; means for transporting solvent from said first end means through said second end means via said solvent transport pathway means; means for transporting with the solvent the specific binding agent in said second zone means by formation of specific binding pairs; and means for transporting with the solvent, label not associated with a labeled specific binding agent capable of forming a specific binding pair, across said second zone means and separate from label associated with the labeled specific binding agent and immobilized in said second zone means.

5. A method for determining the amount of label associated with a labeled specific binding agent in a specific binding agent preparation capable of forming a specific binding pair, which method comprises the steps of: a) providing material comprising capillarity and capacity for solvent transport, the material comprising first end means at which solvent transport begins, second end means at which solvent transport ends, and first zone means proximal to the first end means and distal to the second end means; b) providing first reagent means which, upon application to the material, is immobilized against solvent transport, the application to be made in a subsequent step, and which first reagent means is further capable of forming a specific binding pair with the labeled specific binding agent; c) introducing a quantity of the specific binding agent preparation to the first reagent means, and incubating for a period sufficient to allow formation of specific binding pairs; d) introducing a quantity of the combination of the specific binding agent preparation and first reagent means to the first zone means; e) contacting the material with solvent means, whereby the solvent means traverses at least the first zone means; and f) detecting the amount of label at the first zone means associated with labeled specific binding agent forming the specific binding pair.

6. The method of claim 5 wherein the material further comprises second reagent means immobilized against solvent transport which is not capable of forming a specific binding pair with the labeled specific binding agent, and wherein the second reagent means immobilized against solvent transport substantially blocks non-specific binding of the labeled specific binding agent to the material.

7. A method for determining an amount of label associated with a labeled specific binding agent in a specific binding agent preparation capable of forming a specific binding pair, which method comprises the steps of: a) providing material comprising capillarity and capacity for solvent transport, the material comprising first end means at which solvent transport begins and second end means at which solvent transport ends, and the material comprising first zone means, proximal to the first end means, and second zone means, downstream from the first zone means, with first reagent means immobilized against solvent transport and capable of forming a specific binding pair with the labeled specific binding agent; b) introducing a quantity . of the specific binding agent preparation to the material at a location on or downstream from the first zone means; c) contacting the material with solvent means, whereby the solvent means traverses at least the first zone means and second zone means; and d) detecting the amount of label at the second zone means associated with labeled specific binding agent forming the specific binding pair.

8. The method of claim 7, wherein the first zone means further comprises second reagent means immobilized against solvent transport which is not capable of forming a specific binding pair with the labeled specific binding agent, and wherein the second reagent means immobilized against solvent transport substantially blocks non-specific binding of the labeled specific binding agent to the material.

9. A method for determining the presence of a particulate-associated analyte, the particulate-associated analyte being capable of forming a specific binding pair, in a sample suspected of containing the analyte, which method comprises the steps of: a) providing a labeled specific binding agent capable of forming a specific binding pair with the particulate-associated analyte; b) providing a material comprising capillarity and capacity for solvent transport, and being capable of immobilizing particulate, the material comprising first end means at which solvent transport begins, second end means at which solvent transport ends, and zone means proximal to the first end means and distal to the second end means; c) introducing a quantity of the labeled specific binding agent to the particulate-associated analyte, and incubating for a period sufficient to allow formation of specific binding pairs; d) introducing a quantity of the combination of the labeled specific binding agent and particulate-associated analyte to the zone means, and allowing the particulate to immobilize in the material against solvent transport; e) contacting the first end means of the material with solvent means and allowing the solvent means to traverse the zone means; and f) detecting the presence of the analyte by examining the zone means for the presence of a label.

10. The method of clai m 9 wherein the presence of particulate-associated analyte is quantified, which method further comprises the step of detecting the total quantity of label in the quantity of the combination of the labeled specific binding agent and particulate-associated analyte introduced to the zone means, whereby the percentage of the label detected at the zone means of the total quantity of label introduced to the material is determined.