WO1992020746A1

WO1992020746A1 - Polymeric compositions having bound antibodies

Info

Publication number: WO1992020746A1
Application number: PCT/US1992/004070
Authority: WO
Inventors: Kenneth F. Buechler; Charles P. Lollo
Original assignee: Hybritech, Incorporated
Priority date: 1991-05-14
Filing date: 1992-05-14
Publication date: 1992-11-26
Also published as: AU1929292A

Abstract

A method for assay of analytes and for the syntheses of the corresponding compositions are disclosed. An assay dye compound composed of a polymer substituted by dye moieties and carboxylic and/or sulfonic acid moieties is conjugated to a bispecific antibody or antibody fragment. The antibody or fragment is also immunoreactive with analyte. A second antibody that is immunoreactive with the conjugate analyte of bispecific antibody or fragment is used to immobilize or otherwise distinguish the conjugate. Measurement of the color intensity of the dye of the conjugate indicates the presence and optionally the quantity of analyte.

Description

POLYMERIC COMPOSITIONS HAVING BOUND ANTIBODIES

Background of the Invention

Recently, considerable research has been devoted to the detection and quantitative measurement of various substances in physiological fluids through the use of polymeric, dye-labeled immunoreactive reagents. Typically, the polymers contain multiple pendent groups to which are attached one or more marker substances, such as fluorescent and non-fluorescent dyes, as well as various radioisotopes . In addition, one or more

antibodies, which are immunospecific for the substances to be detected, are covalently bound to the polymer. The particular substance to be detected by these

reagents will be the antigen of the antibody attached to the polymer. The substances sought to be detected by these polymeric compositions can include various

proteins, hormones, enzymes, bacteria, viruses, drugs, peptides and other chemical substances.

In addition to clinical applications, polymeric, dye-labeled immunoreactive reagents can be part of immunoassay systems which can be utilized by a patient at home. For example, such a system may be used by a patient to detect blood or urine levels of

therapeutic drugs, to detect vitamin deficiencies or as pregnancy or fertility tests.

The successful detection and measurement of antigenic substances requires a high degree of

specificity between the antibody and antigen. In addition, the immunological bond between the antibody and antigen should display a low dissociation constant, so that various physical manipulations of the polymeric reagent/antigen complex do not dissociate the reagent from the substance to be detected. Under ideal

conditions, the bond between the immunoreactive, dye-labeled reagent and substance to be detected should be strong and highly specific so that accurate detection and measurement of the substance can be achieved. These conditions are especially important for in-home, patient-controlled assay kits, where controlled

laboratory conditions cannot be maintained. However, the known polymeric, dye-labeled immunoreactive

compositions do not meet these criteria.

Known polymeric, dye-labeled reagents employ covalent binding substrate linking to attach one or more antibodies to the dye-labeled polymers. These binding mechanisms may create such problems as multi-conjugation and cross-linking between the antibody and the dye- polymer which may significantly reduce the specificity of antibody binding to the antigenic

substance to be detected.

Examples of an immunoreactive reagent formed by covalent binding are provided by Azad et al. in U.S.

Patent No. 4,434,150. This patent discloses an

immunoassay reagent formed by coupling an immunoglobulin (antibody) to a water soluble polymer by a covalent bond or a biotin-avidin linkage. In addition, the polymer of this patent is capable of additional substitution with various marker substances, particularly fluorescent dyes. Disadvantageously, the immunological detecting reagents of this patent are capable of attaching

substantially only one immunoglobulin on each polymer.

Similarly, in U.S. Patent No. 4,452,886, Henry et al. disclose utilization of glutaraldehyde or

carbodiimide to link a ligand (antigen or hapten) or its receptor (antibody) to a water soluble polymer of 40 to 600 chromaphoric or fluorescent groups, while in U.S. Patent No. 4,166,105, Hirschfeld et al. disclose similar covalent bonding agents for attaching a single antibody to a polymeric backbone which also is attached to a number of fluorescent dye molecules

Another problem associated with several reported immunoreactive reagents concerns their

utilization of water insoluble dye dispersions When attached to polymeric molecules, such hydrophobic dye groups may inhibit the water solubility of the polymers. In addition, hydrophobic dyes exhibit a low absorption coefficient in aqueous media, thereby decreasing the visual intensity of the dyes, and accordingly the ability to chromagenically detect the dye labeled substance sought to be detected. U.S. Patent No.

4,373,932 (Gribman) describes such an immunoassay reagent system employing hydrophobic dyes or pigments.

Furthermore, published dye-labeled

immunoreactive reagents may only be able to incorporate one antibody per dye-labeled polymer. These polymers often exhibit a coiled, tertiary structure. The single immunoreactive antibody may often be buried in this structure, and be unavailable for complexation with the substance to be detected. The attached antibody may also be in such close physical proximity to several of the dye groups attached to the polymer that its binding specificity and strength are sterically hindered. Azad et al. and Gribman et al, respectively, in U.S. Patent Nos. 4,434,150 and 4,373,932 describe reagents having this character.

In view of the above-noted shortcomings, it is clear that there has existed a need for polymeric, dye-labeled immunoreactive reagents which bind strongly and specifically with the particular substance to be detected in a sample of physiological fluid. The polymers of such reagents should include a multiplicity of antibodies attached by other than covalent means, such that multi-conjugation, cross-linking and steric shielding problems are avoided. The polymers should incorporate water soluble dyes, whose increased dye intensity in aqueous solution enhances the ability to detect and measure the intended substance.

Summary of the Invention

Accordingly, the present invention is directed to polymeric compositions for immunologically detecting analytes and is directed to intermediates of those compositions. The present invention is further directed to methods for making and using these polymeric

compositions to assay a sample for the presence of analyte. As used herein, an analyte is a substance to be analytically detected by means of an antibody-antigen conjugation.

In particular, the polymeric compositions of the present invention can carry dye moieties as well as antibodies, and can accommodate high dye loads without significant reduction in the specificity or binding strength of the bound antibodies. Multi-conjugation and cross-linking problems are avoided by use of a

bispecific antibody conjugate which is immunospecific for both the attached dye moieties and the particular analyte to be detected. Thus, a plurality of antibodies can be bound to. each dye-loaded polymer. In addition, preference for aqueous media allows for the use of more visually intense, water-solvated dyes.

In a first aspect, the present invention provides an assay dye compound composed of a polymer having an all-carbon backbone, a first pendent alkyl amide or sulfonamide group having one or more N- substituted dye moieties, and a second pendent alkyl carboxy or sulfonoxy acid group. Preferably, the ratio of the first pendent amide group to second pendent acid group is from about 100:1 to 1:1. In addition, the polymeric dye compositions may optionally contain a third pendent acidic N-(sulfonoxyalkyl or aryl)alkyl amide or sulfonamide group. Each alkyl substituent on these groups may contain from one to ten carbon atoms . The aromatic group may be benzene or an alkyl , hydroxy, nitro or cyano substituted derivative thereof.

Preferably, the ratio of the first to third pendent substituent is from about 100:1 to 1:1.

The dye moiety or moieties employed in the assay dye compound of the present invention can be any spectrometrically-detectable dye molecule or derivative that contains or is modified to contain a free primary or secondary amine group. Preferably, the dye exhibits minimal hydrophobic binding to proteins and contains functional groups that render it highly water soluble. Examples include a visual dye, phosphorescent dye, fluorescent dye, chemiluminescent dye, a laser dye, an infrared dye, lanthanide chelate or derivatives thereof. In addition, the assay dye compound may advantageously contain two or more distinct dye moieties.

In a second aspect, the invention provides an immunoreactive dye composition which is a combination of the assay dye compound and a bispecific an.tibody. The bispecific antibody chosen is specifically

immunoreactive with both the dye moiety and an analyte to be detected. When reactively combined, the portion of the bispecific antibody which is immunospecific for the dye moiety will bind to the dye moiety, while the portion that is immunospecific for the analyte to be detected will remain uncomplexed. The reactive

combination forms an immunoreactive reagent capable of detecting a corresponding analyte in a physiological or other fluid.

The bispecific antibody to be combined with the assay dye compound of the present invention can be based upon a polyclonal antibody, monoclonal antibody, a recombinant antibody in general such as a chimeric antibody, a synthetic antibody formed by recombination of individual L and H chain pairs or fragments of any of those antibodies, such as Fab, Fab', F(ab)₂, L and H chains, L or H chain fragments and epitopal regions.

When the bispecific antibody is formed of two distinct antibody molecules, i.e., two pairs of coupled

light/heavy chains for the analyte and one for the dye (hereinafter the doublet species), they will be linked together to form the bispecific antibody. The form of each molecule in this instance may be any of those listed above. Alternatively, the bispecific antibody may be two pairs of coupled light/heavy chains with two different binding sites for binding both the analyte and dye (hereinafter the singlet species). When the

bispecific antibody is the singlet species, it will take the form of a monoclonal antibody, a recombinant

antibody in general such as a chimeric antibody, a synthetic antibody of two differing pairs of light and heavy chains, or fragments of any of these antibody forms.

In a third aspect, the invention provides a detectable analyte dye complex of the immunoreactive dye composition, analyte and a second antibody. The second antibody preferably is immobilized on a solid support such as inert particulate material or a membrane but can also function as a precipitating or weight alteration agent for solution detection as discussed below. It is immunospecific for the analyte to be detected, or species specific for the bispecific antibody, or

immunospecific for the complex formed between the analyte and immunoreactive dye composition. The second antibody can be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody or a synthetic antibody formed from recombination of individual pairs of light and heavy antibody chains.

Fragments of these antibodies may also compose the second antibody, these fragments including a Fab, Fab', F(ab)₂, L chains, H chains, L or H chain fragments and epitopal regions. The affinity constants between the bispecific antibody and dye, the bispecific antibody and analyte, and between the complex of analyte,

immunoreactive dye composition and second antibody, should each be sufficient to maintain the corresponding. immun logical conjugations during washing, decanting, separating, exchanging or other physical manipulations of the detectable analyte dye complex. Preferably, these affinity constants will be at least about 10⁸ liters per mole and more preferably 10⁹ liters per mole. In accordance with the present invention, the assay dye compound can be synthesized by several methods which allow for variations in dye solubility in water as a function of pH. In a first method, an amine- containing dye is reacted with an acid polymer having an all-carbon backbone and a plurality of pendent alkyl carboxylic or sulfonic acid groups. The reaction is conducted in aqueous medium, and under conditions for forming amide or sulfonamide bonds between the amine-containing dye and the acid polymer. Preferably, the pH of the medium is maintained between about 6 to 12. The molar ratio of acid polymer to amine-containing dye incorporated is about 1: 1 million to about 1:1,

preferably from about 1:100,000 to about 1:10. The molar ratio of incorporated dye moieties to remaining unreacted pendent alkyl carboxylic or sulfonic acid groups is from about 1,000:1 to about 1:1, preferably about 100:1 to about 1:1, and more preferably about 20:1 to about 1:1.

Alternatively, in a second method, an amine-containing dye and an amino sulfonic (alkyl or aryl) acid compound are reacted with the acid polymer having an all-carbon backbone and a plurality of pendent alkyl carboxylic or sulfonic acid groups. The reaction is conducted in aqueous medium, and under conditions for forming amide or sulfonamide bonds between the acid polymer and the amine groups of (1) the dye, and (2) the amino (alkyl or aryl) sulfonic acid compound.

Preferably, the pH of the medium is maintained between about 4 to 8. The molar ratio of incorporated amine-containing dye moieties to the sum of the incorporated amino sulfonic (alkyl or aryl) acid and unreacted pendent alkyl carboxylic or sulfonic acid groups is from about 1,000:1 to about 1:1, and the molar ratio of acid polymer to amine-containing dye to be incorporated is from^' about 1:1 to about 1:1 million. Preferred molar ratios are as given in the preceding paragraph. In accordance with the present invention, the detectable analyte dye complex can be utilized in methods of detecting and measuring the desired analyte. Under these methods, the assay dye compound, the

bispecific antibody, the second antibody, and a sample containing the analyte to be detected can be combined under conditions for immobilization, or under conditions for solution reaction, and in any order, to yield the detectable analyte dye complex. Thereafter, the

presence and quantity of analyte in the sample can be determined by discrimination of noncomplexed

immunoreactive dye composition from the detectable analyte dye complex and spectrometric measurement of the dye moieties of the detectable analyte dye complex.

Discrimination can be handled for example by

immobilization and washing, by precipitation and

supernatant removal, by variation of dye absorption upon complexation, and by molecular weight change and

centrifugatlon.

While the above-identified components can be combined in any order to form the detectable analyte dye complex, it is preferred that the second antibody and sample containing the analyte to be detected be combined first. Thereafter, the bispecific antibody and assay dye compound are added successively. Alternatively, this same order of combination may be maintained, except that the bispecific antibody and assay dye compound are first mixed together before they are added to the second antibody/sample mixture.

In a preferred embodiment of the above-described methods, the second antibody utilized to form the detectable analyte dye complex can be bound either directly or indirectly to a solid support such as a porous or semiporous membrane. When the second antibody is directly bound to a solid support, the detectable analyte dye complex is formed directly on the support surface. After nonbound components of the testing media have been removed from the membrane, the remaining complex bound to the surface of the membrane can be visually or spectrometrically measured.

Alternatively, the detectable analyte dye complex can be indirectly bound to the solid support. Such means include biotin-avidin, an enzyme-irreversible substrate, an antibody to the analyte complex itself (See El Shami for a description of this technique, A.S. El Shami et al., U.S. Patent No. 4,778,751 published October 8, 1988, the disclosure of which is incorporated herein by reference) and microparticles. For example, the microparticles carry bound second antibody and are combined with the immunoreactive dye composition to form a bound but mobile detectable analyte dye complex. The microparticle-bound detectable analyte dye complex is immobilized by its entanglement within the semiporous membrane or fibrous mat. The microparticles are

composed of water insoluble polymers, such as latex polymer, magnetic particles, glass beads, alumina or polystyrene.

The polymeric compositions and methods of the present invention can be combined into an in-home, analyte assay kit including predetermined amounts of the assay dye compound, bispecific antibody and second antibody in soluble or an insoluble form. The assay kit may also provide a container for containing and mixing therein a fixed amount of a sample to be determined, the assay dye compound, the bispecific antibody and second antibody. As described above, the second antibody may optionally be bound to a microparticle polymer or a membrane support mounted within the container.

Brief Description of Drawings

FIG. 1 shows, a schematic diagram of the immunoreactive dye composition. FIG. 2 shows a schematic diagram of a

detectable analyte dye complex using a solid support or membrane.

FIG. 3 shows a schematic diagram of a detectable analyte dye complex using microparticles and a porous membrane.

Detailed Description of the Invention

In accordance with the present invention, the assay dye compound is an all-carbon backbone polymer with a plurality of pendent dye groups. The pendent dye groups are composed of one or more dye moieties bonded through amide or sulfonamide linkages to alkyl

substituents on the all-carbon backbone. The alkyl substituents contain from 1 to 10, preferably 1 to 4 carbon atoms. Expressed another way, the assay dye compound is composed of a polyacrylic acid or polyvinyl sulfonic acid or a derivative thereof which has been bonded to the amine-containing dye moiety by amide or sulfonamide linkages between the alkylcarboxy or

alkylsulfonoxy substituents and amine substituents.

Preferably, the polymer is derived from a branched or unbranched, olefinically unsaturated carboxylic acid or sulfonic acid monomer of 2 to about 10 carbon atoms.

As used herein, dye refers to any

spectrometrically detectable dye molecule or derivative thereof and amine-containing dye refers to any such dye substituted with a primary or secondary amine, while dye moiety refers to the dye radical covalently bonded to the amine of the amine containing dye. Preferred dye moieties are derived from visual dyes, phosphorescent dyes, fluorescent dyes, lanthanide chelates,

radioisotopes, electron opaque substances and their derivatives. Particularly preferred are the soluble visual dyes, including solvent dyes, pigments, vat dyes, sulphur dyes, mordant dyes, leucovat dyes and species such as fluorescein, sulforhodamine, rhodamine-hydride, rhodamine hydrazide, Texas Red hydrazine, Congo Red, Trypan Blue, Lissamine Blue and the like, as well as oxazine dyes, cyahine dyes, laser dyes and infrared dyes. These and other dyes useful in the present invention are known. See for example "Dyeing and

Chemical Technology of Textile Fibers", Trotman, 45th Ed., C. Griffin & Co., London and "The Chemistry of Synthetic Dyes", Vankataramon (Ed.), Academic Press, New York 1979 the disclosures of which are incorporated herein by reference.

Preferably, the dye is composed of a formula that minimizes non-specific binding of protein to the dye. This property minimizes the effect of non-specific binding on the null control comparison made during the assay. It improves the sensitivity of the assay by minimizing the color or absorbance of the background against which the complex is compared. To maximize this property, it is preferred that the dye be highly water soluble and exhibit minimal hydrophilic binding to protein. Preferably, the first and second N-substituted dye moieties occur in pairs selected from the group consisting of fluorescein with rhodamine and fluorescein with Texas red. The ratio of first to second N-substituted dye moieties may be from about 100:1 to about 1:100.

The assay dye compound of the present invention is derived from an acid polymer having an all-carbon backbone and a plurality of pendent acid groups which form the linkages with the dye moieties. Alkyl carboxy or sulfoxy groups can serve as the pendent acid groups. Particularly preferred polymers include polyacrylic acid, polymethacrylic acid and their derivatives with pendent carboxyl groups. Other pendent groups may also be present, with the sulfoxy, amino and alcohol groups being preferred. The acid polymer employed in the present invention can range in molecular weight from about 10 thousand to 10 million daltons. Preferably the polymer should have a molecular weight over 2 million daltons, most preferably between about 2 million and 8 million daltons. It will be appreciated that the molecular weight of the polymer will be varied in large part according to the number of dye moieties needed to achieve a detectable signal at a designated analyte concentration.

The method for formation of the assay dye compound involves linking the acid polymer and amine- containing dye together through amide or sulfonamide bonds. To form this linkage, a first reactive site (carboxy or sulfoxy) on the polymer is reacted with a second, correlative reactive site (amine) on the amine containing dye to form the assay dye compounds of the present invention. Methods for preparation of the assay dye compound by formation of amide or sulfonamide bonds include techniques to activate the carboxyl or sulfoxyl moiety followed by attack of the amine group of the dye. Such techniques are well-known in the art and include carbodiimide reactions, acid halide or pseudo halide

(e.g., carbonyl diimidazole) reactions under conditions to favor amide or sulfonamide formation, cyanate and isocynate reactions followed by rearrangement of the resulting urea or amidine to form amide, and

substitution reactions to form amides from esters, anhydrides, cyclic anhydrides and specially activated forms such as pivoloyl ester, t-butoxycarboxy ester, p-nitrophenoxy ester and the like. The general conditions for these reactions are known in the art. See for example the "Advanced Organic Chemistry" Jerry March, McGraw-Hill, New York 1968 as well as Organic Synthesis Collective Volume II, 2d ed., H. Gilman and A. H. Blatt, Eds., John Wiley and Sons, (1964), pages 3, 12, 82, 99, 111, 147, 165, 172, 327, 394; Organic Synthesis

Collective Volume I, A. H. Blatt, Editor-in-Chief, John Wiley and Sons, (1943), pages 65, 74, 76, 156, 169, 208, 278, 328, 453, 528, 562, 569; Organic Synthesis Collective Volume III, E. C. Horning, Editor-in-Chief, John Wiley and Sons, (1955), pages 28, 95, 167, 169, 328, 375, 415, 422, 475, 488, 490, 547, 555, 590, 613, 623, 646, 656, 712, 714, 768; and Organic Synthesis Collective Volume IV, N. Rabjohn, Editor-in-Chief, John Wiley and Sons, (1963), pages 6, 34, 62, 88, 154, 263, 285, 339, 348, 411, 513, 521, 554, 608, 616, 620, 715, 739, 780, 900.

Pursuant to the foregoing method, embodiments of the assay dye compound can be made under acidic or basic conditions. In the acidic method for amidation or sulfonamidation of aqueous soluble dyes, the amidation or sulfonamidation reaction between the acid polymer and the amine-containing dye is run in neutral (up to pH 9) or acid aqueous solution, preferably at a pH from about 4.5 to 7.5, most preferably at a pH of about 6-7.

Examples of useful amidation reactions include, without limitation, acid halide or pseudo halide (carbonyl diimidazole) reaction at a pH of from about 7-9 and carbodiimide reaction at a pH from about 4-6.

Although not intended to be a limitation of this invention, it is believed that because of the pKa established by the ionized and un-ionized forms of the acid polymer, this polymer may exist as a partially coiled molecule In acidic aqueous solution. The

uncoiled segment contains the ionized groups while the coiled segment contains the un-ionized groups. As the reaction of the acid polymer and amine containing dye proceeds, the ionized carboxylic acid groups react to form amide groups while the un-ionized groups are believed to be relatively less reactive because they are buried in the hydrophobic domain of the polymer.

Reaction of the exterior pendent groups with dye is believed to cause a shift in the equilibrium to the open form because of the conversion of the ionized pendent groups to amide or sulfonamide groups, particularly because the dyes in use are hydrophilic. Although this uncoiling proceeds slowly, it is believed to result in further reaction of the amine containing dye and the exterior pendent groups until the counterbalance of nonionizable amide groups shuts down further uncoiling. Consequently, factors for time and for solubility of amide or sulfonamide dye pendent groups are

considerations for the reaction parameters of the acidic aqueous solution reaction. These factors can be

accounted for by monitoring the pH to determine where the progress of the reaction plateaus and by observing the reaction medium for cloudiness caused by the

initiation of precipitation of the partially coiled acid polymer.

To enhance the reactability of the pendent groups, especially the interior pendent groups, and to achieve the highest degree of dye substitution, the polymer can be reacted with an additional agent that will promote the solubility and reactability of the intermediate leading to the assay dye compound. The addition of such an agent in conjunction with the aminecontaining dye aids in the unfolding of the polymer and helps to maintain water solubility of the resulting assay dye compound and subsequent compositions

incorporating the assay dye compound. In particular, in addition to the amine-containing dye, the starting reactants for the acidic reaction method preferably include an amino (alkyl or aryl) sulfonic acid

coreactant. According to this practice, the ratio of dye substituted acid groups (i.e., pendent groups with amine-containing dye groups) to remaining unsubstituted acid groups optionally including those with the amine sulfonic acid substitutions (i.e., pendent groups with free carboxy or sulfoxy groups) should be from about 1000:1 to about 1:1, preferably from about 100:1 to about 1:1, and most preferably at about 20:1 to about 1:1. Particularly preferred amine sulfonic acids

include amino alkyl sulfonic acids (NH₂-(CH₂)_n-SO₃H), e.g., taurine (NH₂-CH₂-CH₂-SO₃H) , and aryl sulfonic acids such as orthanilic, metanilic and sulfanilic acid (NH₂- C₆H₄-SO₃H) and the like.

If the reaction to form assay dye compound can be conducted in a medium of higher pH such as greater than 5, preferrably greater than 7, the acid polymer will tend to be in completely ionized form and will be maintained in an uncoiled state. Under such basic conditions, the preference to substitute the starting polymer with sulfonyl groups in order to obtain maximum conversion is lessened. Use of sulfonyl groups as described above is, nevertheless, a preferable option because their presence will help maintain the solubility of the assay dye compound at all pH values especially when the degree of dye substitution is at a maximum.

Optimum basic reaction conditions are between about 8.5 and about 9.0. Those skilled in the art will appreciate though that amide synthetic reactions performed at these high pH values will be affected by competing reactions, one of which is hydroxide ion reaction with the

activated intermediate. Appropriate consideration will be given to the solubility of the dye in basic medium and the ability of the amide or sulfonamide group formation to take place in basic medium.

Typically under both higher pH and acidic conditions, the equivalent percent dye substitution relative to the total equivalents of acid groups

available will be from about 1 to 60 equivalent percent, preferably from about 10 to 40 equivalent percent, more preferably from about 10 to 30 equivalent percent, and most preferably at about 20-25 equivalent percent. If the percent substitution exceeds about 60 equivalent percent, the assay dye compound may develop a tendency to become insoluble and precipitate from solution. The maximum equivalent percent substitution for each species of assay dye compound will vary around the targeted upper value. The maximum substitution can be appropriately and routinely determined for each species and the differing polymer molecular weights by making representative samples and extrapolating to the maximum.

The amine-containing dye and optional amine sulfonic acid are linked to the pendent groups on the polymer through an amide or sulfonamide linkage. For example, in a preferred aspect a polyacrylic acid is reacted with a carboxyl or sulfoxyl activating reagent such as a carbodiimide, carbonyl diimidazole, pivaloyl chloride, thionyl chloride or N-hydroxysuccinimide in an amount sufficient to form the desired proportion of activated acid groups within the acid polymer. The reaction is conducted under appropriate acidic pH conditions to maintain the activated acid in solution. Substantially simultaneous with its formation, the activated acid polymer is reacted with the amine

containing dye to yield an assay dye compound in

accordance with the present invention.

Depending on the particular dye moiety which is attached, the assay dye compound of the present

invention absorbs or emits photons in the visible

spectrum, UV spectrum or near infrared. For example, use of a visual dye such as fluorescein or rhodamine will cause the assay dye compound to absorb photons of specific wavelengths in the visible region (e.g., blue), resulting in the transmission of the complementary wavelength of color (e.g., red) to an observer.

Alternatively, use of a fluorescent dye will cause the assay dye compound to emit photons at a specific

wavelength, which when illuminated will radiate

unpolarized light of a different wavelength due to the return of electrons to the ground state.

In accordance with the present invention, the assay dye compound is combined with a bispecific

antibody to form an immunoreactive dye composition. The bispecific antibody should be immunoreactive with both the dye group of the assay dye compound and with an analyte to be detected. Immunobinding of the bispecific antibody to the dye moiety is superior to other chemical means of linkage, such as covalent bonding or enzyme linkages. Specifically, the immunobinding employed in the present invention prevents multiconjugation and cross-linking problems between the bispecific antibody and other substituents of the polymer chain. The molar equivalent ratio of bispecific antibody to the assay dye compound can range from a saturating amount of antibody (the amount of which varies depending on the assay dye compound molecular weight) to a ratio of about 1:1. The molar equivalent ratio of antibody to dye _'moieties of the assay dye compound. affects the behavior of the complex in the assay; that is, a 1:1 ratio will give the greatest sensitivity but the slowest binding kinetics. Higher molar equivalent ratios of antibody to assay dye compound conversely give faster binding kinetics (i.e., binding of composition to antigen) and lower sensitivity capabilities. For reasons of kinetics it is preferable to have an antibody to assay dye compound molar

equivalent higher than 1:1. Appropriate ratios for each example of immunoreactive dye composition can be

determined experimentally by preparing compositions of several different ratios and extrapolating a plot of the molar equivalent ratio compared with kinetics and sensitivity.

The bispecific antibody may be any

immunoglobulin or fragment thereof which is

immunospecific for two or more distinct antigens. In one version, the bispecific antibody may correspond to a single chimeric antibody, a single recombinant antibody, or, a single synthetic antibody of two differing pairs of light and heavy chains or fragments thereof wherein the single antibody has binding sites for both antigens (the singlet species). In an alternative version

(doublet species), the bispecific antibody may be two or more polyclonal or monoclonal antibodies, recombinant antibodies, chimeric antibodies or two single chain antibodies chemically bound together, or antibody fragments which have been coupled together through the use of known coupling techniques and reagents such as dimaleimide. In addition to complete polyclonal, monoclonal, chimeric, synthetic or recombinant

antibodies, fragments of those antibodies such as Fab₁, Fab₂ and F(ab)₂', L chains, H chains, L or H chain fragments and epitopal regions can be incorporated to form either version of the bispecific antibody according to this invention.

The bispecific antibody and fragment components used in accordance with the present invention are generally known. Methods for their preparation as bifunctional polyclonal, monoclonal and/or recombinant antibodies and all fragments and single chains thereof, as well as methods for developing specific

immunoreactivity, hybridizing with immortal cells and selecting for desired antibody characteristics, are generally known. See "Immunology: An Illustrated

Outline", David Male, C.V. Masby Co., New York (1986); "Molecular Cell Biology", Darnell, Ladish, and

Baltimore, Scientific American (1986), Chapter 24,

Immunity; P.M. Lansdorp, U.S. Patent No. 4,868,109 issued September 19, 1989; M.J. Johnson et al., EPO published application No. 369,566 published May 23, 1990; Hong et al., J. Biol. Chem., 240, 3883 (1965);

Ludtke et al., Biochemistry, 19, 1182 (1980); and

Milstein et al., Immunol. Today, 5, 299 (1984) the entire disclosures of which are incorporated herein by reference.

The anti-dye/anti-analyte bispecific antibody . or fragment can also be formed from two or more

monofunctional antibodies or fragment in accordance with the present invention. Generally, the two antibodies or fragments are derivatized and linked with appropriate coupling agents known in the art. For example, the reagents, procedures and techniques for cross-linking described in such texts as E.A. Rabat, "Structural Concepts in Immunology and Immunochemistry", 2nd Ed., Holt Reinhart and Winston, New York, 1976, or the Pierce Chemical Company Handbook of Reagents, provide an appropriate description for accomplishing such

conjugation. In particular, reagents such as

bismaleimidohexane which reacts with sulfhydryl groups, dimethyl adipimidate dihydrochloride which reacts with amines and photo-coupling groups can be used. A

preferred coupling system is a combination of SPDP and SMCC described below. First, an anti-analyte antibody and anti-dye antibody or the corresponding fragments are separately reacted with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), respectively. Following reduction of SPDP-labeled anti-analyte antibody or fragment with dithiothreitol, the thiol-labeled anti-analyte antibody or fragment is cross-linked with the maleimide-labeled anti-dye

antibody or fragment to yield the desired bispecific antibody conjugate or the corresponding bifunctional fragment conjugate. The conjugate is then further purified by gel permeation chromatography to yield the product shown below.

Either known hybridoma techniques or known recombinant techniques can be used to obtain the

preferred monoclonal antibody starting material for the bispecific antibody. The recombinant techniques for forming antibodies are generally described by Shaw et al. in J. Immun., 138, 4534 (1987); Sun et al. in Proc. Natl. Acad. Sci. USA, 84, 214 (1987); Neuberger et al., Nature, 314, 268 (1985); Boulianne et al., Nature, 312, 643 (1984); and Morrison, Proc. Natl. Acad. Sci. USA, 81, 6851 (1984); M.J. Johnson, et al., EPO published application No. 332,424 published September 13, 1989; M.J. Johnson, EPO published application No. 369,567 published May 23, 1990; A. Pluckthun et al., EPO

published application No. 324,162, published December 24, 1988; Lei et al., PCT application No. WO 89/06283, published July 13, 1989; the disclosures of which are incorporated herein by reference. The preferred

hybridoma technique for forming antibodies is described in Martinis et al., PCT application WO83/03679,

published October 27, 1983; Milstein and Kohler, Nature, 256, 495-497 (1975); Gerhard, Monoclonal Antibodies, 370-371, and Kennett et al., Ed's (Plenum Press, 1980) the disclosures of which are incorporated herein by reference. The resulting monoclonal antibody prepared by either technique can also be used to prepare the preferred F_ab' fragment by pepsin digestion and partial reduction following the procedure of Masuho (see Example 12). Both the monoclonal antibody and the fragment are preferred for preparing the bispecific antibody

conjugate described above.

The hybridoma technique is preferred for preparing this starting material and follows the

procedure of Kohler and Milstein. (See J. Goding,

"Monoclonal Antibodies: Principles and Practice," 2nd Ed., Academic Press, San Diego, CA, 1986, the disclosure of which is incorporated herein by reference.) Briefly, BALB-c mice can be challenged and boosted with antigen composed of the dye in adjuvant with carrier or the analyte in adjuvant with carrier to produce specific immunogenieity. The spleen cells of the mice are

removed and suspended in appropriate adjuvant media.

Cell fusions between the spleen cells and mouse myeloma cells can be conducted with a fusion agent such as polyethylene glycol. Cultures from single cell wells can be grown and tested for production of the desired antibody. The test can be conducted, for example by removing aliquots of cell culturation fluid and

determining the presence of antibody against the dye or against the analyte, by sandwich assay with immobilized antimouse antibody. After washing to remove

nonimmobilized material, detection of the dye by

appropriate spectrometric techniques keyed to the absorption of the dye or detection of the analyte- antibody complex through, for example an enzyme linked or radioimmunoassay will indicate which cultures contain the desired hybridoma. Culturation of the hybridoma following protocols known in the art will yield

production lots of the desired anti-dye monoclonal antibody or the desired anti-analyte monoclonal

antibody .

In accordance with the compositions and methods of the present invention, a novel antibody embodiment which is immunospecific for aminosulforhodamine dye was developed by hybridoma techniques common to those skilled in the art and is for use in forming a

bispecific antibody according to the present invention. The hybridoma secreting this antibody has been deposited at American Type Culture Collection, as Deposit No.

HB10625, dated December 13, 1990, and was prepared according to hybridoma procedures well known in the art. It is assured that the cultures will be maintained in a viable state for at least 30 years and the officials designated by the U.S. Patent Office will be entitled to access. All restrictions placed upon the deposit will be removed at issuance of this application.

An embodiment of an immunoreactive dye composition in accordance with the present invention, formed according to the foregoing technique, is

illustrated schematically in FIG. 1. The composition comprises a polymer having an all-carbon backbone (3), and first (4) and second (5) pendent substituents. The second pendent substituent (5) includes an alkyl carboxy group (6). The first. pendent substituent (4) includes a dye moiety (8), linked to an alkyl group through an amide linkage (9). A bispecific antibody (10), herein shown as two antibodies coupled together, is immunologically bound to the dye moiety (8) at the anti- dye portion (12) of the bispecific antibody. The anti- analyte portion (14) of the bispecific antibody (10) remains uncomplexed until reacted with the analyte to be detected. In practice, a fraction of the multiple number of first pendent substituents present will not actually be bound to bispecific antibody (10).

Moreover, a fraction of the multiple number of

bifunctional antibodies (10) bound to the first pendent substituent (4) will not actually bind to the analyte. Consequently, the actual composition in uncomplexed form will also contain the unbound first, pendent substituent and in complexed form will contain the unbound conjugate of bispecific antibody-first pendent substituent as well.

As noted above, the immunoreactive dye compositions in accordance with the present invention are uniquely suited to detect a wide variety of

analytes, particularly as found in physiological fluids. As used herein, analyte refers to any molecule, such as an antigen or hapten, which can evoke an immunological reaction with an immunoprotein such as an antibody or fragment thereof. For example, analytes may include without limitation, hormones, proteins, vitamins, drugs, viruses, bacteria, enzymes, toxins, carbohydrates, chemicals, and peptides. Particularly preferred in the present invention are analytes which are indicative of a particular physiological state in an individual, such as human chorionic gonadotrophin (HCG), human prolactin (PRL), human placental lactogen (HPL), testosterone, human immune deficiency virus (HIV), hepatitis surface antigen, prostate specific antigen (PSA), CA 125, CA 549, CA 19-9, CA 150-3 (Cancer markers), Human growth hormone (hGH), tissue plasminogen activator (TPA), alpha fetoprotein (AFP), bone alkaline phosphatase (BAP), chlamydia, hepatitis A, hepatitis B (surface antigen), group A streptococcus, group B streptococcus, hepatitis A virus (HAV), hepatitis B core antigen (HBc), blood enzymes and the other similar exogenous or endogenous proteins or polynucleic acids that are indicative of human disease or malconditions.

Generally, the sensitivity of antibodies provides appropriate micro- to nano-molar sensitivity for the method of detection according to the invention. The presence of many dye molecules on the polymer acts as an amplification factor also. Preferably, to detect and measure the analyte sought, the bispecific antibody of the immunoreactive dye composition is sufficiently sensitive to detect the analyte in solution at

concentrations of about less than micro-molar

concentrations. The sensitivity, of course, is

dependent upon the relative concentration of analyte typically found in the unknown sample to be tested.

Under typical circumstances, the molar ratio of

bispecific antibody to analyte should be maintained from about 1:1 to 100:1 preferably from about 1:1 to 40:1, and most preferably at about 1:10.

Conjugation of the immunoreactive dye compositions of the present invention with an analyte and a second antibody yields a detectable analyte dye complex. The second antibody can be immunospecific for the analyte to be detected, or species specific for the bispecific antibody, or immunospecific for the complex formed between the analyte and bispecific antibody.

The detectable analyte dye complex provides a convenient means of detecting and measuring a desired analyte, either by a solution technique or preferably by an immobilized technique. Such complexes can be detected by the spectrometric properties of the dye labeled portion of the detectable analyte dye complex.

The affinity constants between (1) the bispecific antibody and dye, (2) the bispecific antibody and analyte, and (3) the second antibody and analyte should be sufficient to maintain these immunological conjugations during washing, mixing, decanting,

separating, exchanging or other physical manipulations of the detectable analyte complex. In particular, the affinity constants should be at least about 10⁸ liters per mole, preferably at least about 10⁹ liters per mole.

As with the bispecific antibody, the second antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a synthetic antibody, a recombinant antibody, or an antibody fragment such as Fab, Fab', F(ab)₂, and single chains such as the light or heavy chains and fragments thereof carrying the epitopal regions. Its preparation will follow the hybridoma or recombinant techniques outlined above for the bispecific antibody starting material.

The second antibody preferably functions as the immobilizing agent for the detectable analyte dye complex by acting as the moiety that binds the complex to a support such as an inert, solid particulate

material or to a membrane. This immobilization

technique permits noncomplexed immunoreactive dye composition to be separated from the complex, such as by washing, so that spectrometric measurement of the dye concentration will indicate the concentration of analyte present and will not be spoiled by signal from

noncomplexed immunoreactive dye composition.

The second antibody also can enable

discrimination when the detectable analyte dye complex is analyzed by a solution technique. In this

situation, discrimination between complexed and

noncomplexed immunoreactive dye composition can be obtained by any of several options. A first option involves precipitation of the detectable dye complex followed by removal of the supernatant and redissolution of the precipitate. The second antibody can act as a precipitating agent in this situation. A second option involves significantly changing the molecular weight of the detectable analyte dye complex relative to the weight of the immunoreactive dye composition. In this option, the second antibody acts as the weight enhancer such that the complex will centrifugally sediment at a significantly greater rate relative to the sedimentation rate of the immunoreactive dye composition. Separation of these two substances by centrifugation permits discriminatory determination of the concentration of the complex. A third option involves quenching the

absorption of the dye moiety of the immunoreactive dye composition with a second dye moiety attached to the second antibody. This is discussed in detail below. A fourth option involves addition of a known excess amount of immunoreactive dye composition to the unknown

containing the analyte, precipitation of the analyte complex with the second antibody and measurement of the concentration of noncomplexed immunoreactive dye

composition remaining in solution. The difference between the starting concentration and remaining

concentration of immunoreactive dye composition in solution indicates the concentration of analyte present. Generally, the options based upon precipitation or molecular weight variation of the complex will follow procedures known in the art such as addition of

complement and use of centrifugation to sediment

material of differing molecular weights.

According to the foregoing third option for detecting the analyte complex in solution, the

absorption of the dye moiety of the immunoreactive dye composition group is quenched with a second, distinct dye group. Specifically, the analyte dye complex is formed as described except that the second antibody complexed with the analyte is bound to a second dye moiety substituent rather than to a solid support membrane. The second dye moiety is designed to

spectrometrically interact with the dye moiety of the assay dye compound over a short proximity distance. The second dye moiety is positioned on the second antibody such that it does not significantly interfere with immunoreactivity but is within the proximity radius of about 30 to 60 angstroms for quenching the absorbance of the first dye moiety. Upon the formation of this complex using a known amount of immunoreactive dye composition and its irradiation with light specific for the absorptive wavelength of the first dye moiety, a perceptible color change relative to a positive control containing the same concentration of immunoreactive dye composition occurs due to the fluorescence energy transfer. This color change gives a visual and/or a spectrophotometric means of detecting and measuring the detectable analyte dye complex after its formation in solution.

To form the detectable analyte dye complex according to any of the foregoing techniques and

options, the molar equivalent ratio of second antibody to analyte in the sample should be from about 1:1 to about 10 million:1, preferably from about 90,000:1 to about 1 million:1, and most preferably at about 5,000:1 to about 50,000:1. In this regard, it is preferable to maximize the second antibody to analyte ration to obtain the best (i.e., fastest) kinetics of binding. After formation of the detectable analyte dye complex,

detection and measurement of the desired analyte may be performed by a variety of methods.

The order for combining the assay dye compound, the bispecific antibody, the second antibody and a sample containing the analyte to be detected may follow any sequence as long as it is compatible with the

detection of analyte. Preferably, the second antibody should first be combined with the sample containing the analyte. Thereafter, the bispecific antibody and assay dye compound should be added in successive order. In an especially preferred embodiment for the immobilization technique, the second antibody and the sample containing the analyte are combined in association with an immobilizing support to form an immobilized

intermediate. Next, the assay dye compound and

bispecific antibody are combined to form the

immunoreactive dye composition. Finally, the

immobilized intermediate and immunoreactive dye

composition are combined to form the immobilized

detectable analyte dye complex. Washing the immobilized complex will remove noncomplexed immunoreactive dye composition.

Illustrations of a detectable analyte dye complex formed by immobilization of the second antibody upon a solid support is given in FIG's 2 and 3. These illustrations include a polymer having an all-carbon backbone (3), and first (4) and second (5) pendent substituents. The second pendent substituent (5) includes an alkyl carboxy group (6). The first pendent substituent (4) includes a dye moiety (8), linked to an alkyl group through an amide linkage (9). A bispecific antibody (10) is immunologically bound by an anti-dye variable region (12) to the dye moiety (8), and an anti-analyte variable region (14) is bound to the analyte (16) to be detected. The analyte (16) to be detected is further bound to an anti-analyte portion (18) of a second antibody (20) which is immobilized by direct means (Fig. 2) or indirect means (Fig. 3) on a solid support (21) so as to form the detectable analyte dye complex. Alternatively, the anti-analyte portion (18) of the second antibody (20) could be made specifically immunoreactive with the bispecific antibody (10) or the complex formed between the bispecific antibody (10) and the dye moiety (8).

Detection and measurement of the detectable analyte dye complex can be accomplished through various spectrometric or visual means of detection conducted upon the washed immobilized complex or upon a solution of the complex components obtained from the washed immobilized complex by breaking the immunological conjugates with an appropriate ionic reagent such as, for example, 8M ammonium sulfate. The spectral

properties can be observed by the naked eye or through appropriate instrumentation, such as a

spectrophotometer, or colorimetric determination, or measurement of photon absorption or emission in a photometer, fluorometer, fluorescimeter, densitometer, and reflectometer.

According to the immobilization technique for detecting the analyte dye complex according to the present invention, the second antibody (10) may be directly or indirectly bound to a substrate such as the semiporous membrane or fibrous mat (24) as schematically shown, respectively, in FIG's 2 and 3. These techniques follow the practice of Valkirs given in U.S. Patent

No's. 4,632,901 and 4,727,019 issued December 20, 1986 and February 23, 1988 (the '901 and '019 patents, respectively), the disclosures of which are incorporated herein by reference.

Preferred embodiments of the immobilization technique of the present invention produce the

detectable analyte complex bound to a porous or

semiporous membrane. The membrane may be composed of a flexible or rigid matrix made from any of a variety of filtration or chromatographic materials including glass fibers, nylon, nylon 66, polyethylene and polypropylene based materials, cellulosic and nitrocellulosic based materials, polyacetate and polyvinyl based materials, micro-fibers and natural or synthetic materials. Fluids preferably can flow into and pass easily through the membrane. The membrane also preferably will have pore sizes of at least 0.1μ and preferably no more than 2μ. The membrane can be used alone or as part of a more elaborate device. Such devices includes the ICON^® and like devices described in the Valkirs et al. '901 and '019 patents, respectively, herein incorporated by reference. ICON^® is a trademark of Hybritech Incorporated (San Diego, California) for the devices described in the Valkirs et al. patents listed above.

More specifically, Valkirs et al. describe an apparatus for the detection of a target antigen (e.g., analyte) in a liquid sample comprises: (a) a first member which is a porous membrane or filter and to which is bound an antibody against the target antigen

(analyte), which member has upper and lower surface, the sample being applied to the upper surface, and wherein the antibody is bound within an area smaller than the area of the member to which the sample is applied; and (b) a second member which is a body of absorbent

material having a surface over which the first member is placed and having capillaries therethrough which are in communication with the pores on the lower surface of the first member so as to draw liquid added to the upper surface which has permeated the first member into the capillaries of the member, the capillary communication between said first and second members having been established prior to, and maintained during, the

addition of liquids to the apparatus in the immunoassay process.

Still other devices containing porous and semiporous membranes useful in the present invention include the devices of Bauer et al., U.S. Patent No.

3,811,840, issued May 21, 1974; Brown, III et al., U.S. Patent No. 4,916,056, issued April 10, 1990; Cole et al., U.S. Patent No. 4,246,339, issued January 20, 1981; Geigel et al., U.S. Patent No. 4,517,288, issued May 14, 1985; F.S. Intengan, U.S. Patent No. 4,440,301, issued April 3, 1984; M.E. Jolley, U.S. Patent No. 4,704,255, issued November 3, 1987; Katz et al., U.S. Patent No. 4,496,654, issued January 29, 1985; and Tom et al., U.S. Patent No. 4,366,241, issued December 28, 1982, and that described in European Patent Application No. 217,403, published April 8, 1987, all of which are incorporated herein by reference. The present methods also may be accomplished by chromatographic methods such as, for example, those described in Weng et al., U.S. Patent No. 4,740,468, issued April 26, 1988 incorporated herein by reference, and in Yue et al., European Publication No. 186,100, published July 2, 1986. The porous membrane used for immobilization according to the present invention may also be applied to chromatographic assays such as described, for example, in U.S. Patent No. 4,861,711, issued August 29, 1989 to Friesen et al., U.S. Patent No. 4,855,240, issued August 8, 1989 to Rosenstein et al., U.S. Patent No. 4,857,453, issued August 15, 1989 to Ullman et al., in K. May et al., EPO Publication No. 299,428, published January 18, 1989, and S.M. Devereaux et al., EPO Publication No. 323,605, published July 12, 1989, all of which are incorporated herein by reference.

In a first version for binding according to the immobilization technique, the second antibody may be directly bound to the membrane. The direct binding may be covalent or non-covalent and may be accomplished by any method known in the art. Covalent methods include for example, the use of carbodiimide or glutaraldehyde in a solution at a pH from about 4.5 to 10, preferably from about 6 to 8, and most preferably at about 7, and aminosilanes as well as other methods described in

"Immobilized Enzymes", Ichiro Chibata, Halstead Press, New York (1978), Cuatrecasas, J. Biol. Chem. 245: 3059 (1970), and March et al., Anal . Biochem., 60, 149 et seq. (1974). Further methods for covalent binding include amide bond formation as described above to bind the carboxy terminal group or a carboxy side chain group of the antibody to an amine group of the polymer.

Covalent binding may also be accomplished by a cross-linking reaction through linking groups such as the

SPDP-SMCC couple described above or the

bismaleimidohexane group. The non-covalent binding takes advantage of the natural adhesion of antibodies to the non-synthetic and especially the synthetic fibers. Thus, appropriately buffered solutions can be mixed with the membrane then evaporated leaving a coating of the desired antibody on the membrane. Passively coating the polymer membrane with antibody or by absorbing the second antibody onto the surface of the insoluble polymer at a pH near the isoelectric point of the antibody will also accomplish noncovalent binding. The second antibody may also be immunologically bound to the membrane by employing a second bispecific antibody which immunologically binds to the polymer membrane as well as to the analyte, bispecific antibody or complex.

Another version of the immobilization technique according to the present invention involves indirectly binding the second antibody of the detectable analyte dye complex to the membrane through the use of

microparticles of a water insoluble polymer, such as latex. The microparticles are bound to or entrapped by the membrane, such that the microparticles are within the matrix of the membrane, on the surface of the membrane, or bound to other particles which are in turn bound to the membrane. The microparticles may be any shape, preferably spherical. The size of the particles may vary, but in general they may be slightly larger than the minimum pore size of the membrane and smaller than the maximum pore size, and in addition or in the alternative, may be larger than the maximum pore size. Thus, the particles may be bound within the matrix of the membrane, on the surface of the membrane, or to other particles which are in turn bound to the membrane. The particles may be made of a variety of naturally occurring or synthetic materials. Exemplary of such particles are those made from polyethylene,

polyacrylates, polyacrylamide, and preferably

polystyrene or naturally occurring materials such as cross-linked polysaccharides like agarose, dextran, cellulose, starch, latex, particles containing magnetic material and the like. The primary requirement is that the particles do not cause interference, typically light absorption. The second antibody may be covalently or non-covalently bound to the microparticle. Such binding may be accomplished by the methods discussed above for binding the antibody directly to the membrane.

The micro-particles may be applied (or "spotted") to the membrane in a zone within the surface area of the membrane. Thus spotting localizes the antibody-coated microparticles to a discrete area on the membrane to localize the antibody coated microparticles on or within the membrane. Any of the methods known in the art may be employed. One such method employs various mechanical means such as, for example, the Sandy Springs Spotting Machine (Germantown, Maryland) to apply a suspension, frequently aqueous ("latex"), to the membrane.

For example, the microparticle bound detectable analyte dye complex can be poured onto a semiporous membrane or fibrous mat. After the unbound components have passed through the membrane or mat, the remaining microparticle-bound complex adhering to the surface of the membrane or mat can be spectrometrically measured. In addition, the surface of the membrane or mat may be optionally washed with an acceptable buffer solution to remove nonbound components before spectrometric

measurement. Particularly preferred buffered solutions include buffers ranging in pH from 4-12 and containing citrate, phosphate, borate, or carbonate and a nonionic detergent such as Triton X100 or Tween 20. These

techniques follow the practice of Valkirs given in the foregoing '901 and '019 patents, the disclosures of which are incorporated herein by reference.

The methods of preparing and using such microparticles for the instant invention are further discussed in Weng et al., U.S. Patent No. 4,740,468, issued April 26, 1988 (see especially columns 13, 14 and 15), Brown et al., European Patent Application No.

217,403, published April 8, 1987, and A.S. Rubenstein, European Patent Application No. 200,381, published

November 5, 1986, the disclosures of which are

incorporated herein by reference.

The separation steps for the various

immobilized assay techniques may be performed by any of the methods known in the art. For membranes and

filters, additional washing with buffer may often be sufficient, preferably drawing the liquid through the membrane or filter or contacting the opposite side of the filter or membrane with a liquid absorbing member that draws the liquid through, for example, by a

capillary action. The ICON^® device (Hybritech

Incorporated, San Diego, California), which is preferred for use in the present invention, uses the latter method.

Moderate temperatures are normally employed for carrying out the assay. Constant temperatures during the period of the measurement are generally required only if the assay is preformed without comparison with a control sample. The temperatures for the determination will generally range from about 15° to 45°C.

Depending on the nature of the dye, a signal can be detected by irradiating with light and the level of determining absorption and/or fluorescence through the use of visual observation, a fluorometer or a spectrometer. Where the appropriate equipment is not available, it will normally be desirable to have a chromophore produced which results in a visible color. Where sophisticated equipment is involved, any of the techniques is applicable. For a qualitative positive reaction. For a quantitative analysis, the ICON ^® reader and accompanying software (Hybritech Incorporated, San

Diego, California) may be used according to the manufacturer's instructions and are preferred for use in the present invention.

The various compositions and methods of the present invention can be combined into assay test kits for use in various clinical laboratories and at home settings. Thus, in a preferred example of the

microparticles binding technique schematically

illustrated in FIG. 3, a predetermined amount of second anti-HCG antibody (20), which is immunospecific for human chorionic gonadotrophin, bound to a latex polymer (22) is combined with a measured sample of physiologic fluid containing the HCG analyte to be detected (16) to form a first immunological complex of second antibody and analyte. Preferably, about a tenth to 1 ml of a solution containing the second antibody, at a

concentration from about 3×10^-6 to 3×10^-5 mg/ml is provided although this amount will depend upon the assay

configuration. Such a second antibody solution should be sufficiently sensitive to detect HCG at a

concentration between about 0.2mIU/ml to 1000 mIU/ml.

In a second step, after complexation of the second antibody and analyte, a predetermined amount of bispecific antibody (10), which is immunospecific for both the HCG analyte (16)' and an aminosulforhodamine dye moiety (8) is added to the first immunological complex to form the second immunological complex. Preferably, the predetermined amount will be in the range of about 0.100 to 0.200 ml of a solution containing the

bispecific antibody (10) at a concentration from 0.1 to 0.5 mg/ml.

Finally, the second immunological complex formed in the second step is combined with an assay dye compound (28), incorporating an aminosulforhodamine dye . moiety (8), according to the present invention, to yield a latex-bound detectable analyte dye complex.

Preferably, the amount of complex employed will be about 0.100 to 1 ml of a solution containing the assay dye compound (28).

Once formed, the latex-bound detectable analyte dye complex, including the aminosulforhodamine dye moieties, can be visually detected on a membrane (21) by a doctor, medical technician or patient as previously described. The latex-bound complex may be poured onto a semiporous membrane or fibrous mat (21) to aid in the visual detection of the colored complex containing the HCG analyte to be detected. Accordingly, the membrane or mat is a white or off-white coloration to aid in the accurate color-metric determination of the complex. For example, a female patient could utilize the above method in an in-home test kit to determine if she is pregnant. Optionally, if the above test kit components are

utilized in a clinical setting, spectrometric detection may be determined by a device such as an autoanalyzer, photometer or fluorometer.

In addition to the previously discussed embodiments, it will be appreciated that two or more distinct dyes can be attached to the assay dye compounds according to the present invention, such that the combined dye moieties can be attached to the assay dye compounds according to the present invention, such that the color-metric effect of the combined dye moieties can be intensified. Also, it will be further appreciated that two or more assay dye compounds, each being

immunospecific for a different analyte, and each

utilizing distinct dye moieties can be utilized to detect two or more analytes in a sample of physiologic fluid.

The operation and advantages of the present invention will be further described by reference to the following detailed Examples, and it is to be understood that the invention is not limited thereto. EXAMPLE 1

Substitution of Polyacrylic Acid

With Aminofluorescein and Aminosulforhodamine

Polyacrylic Acid (4mg, 2 × 10⁶ daltons,

Polysciences, Inc.) was suspended in water (20ml) and the pH of the mixture was adjusted to 6.5. The solution was cooled to 4°C and ethyl trimethylaminoethyl

carbodiimide chloride (EDC) (10mg) was added while maintaining the pH of the reaction solution at pH 5.5 to 6.0 by the addition of dilute hydrochloric acid. The resultant solution was stirred for 30 minutes at 4°C.

1-amino-2-(amino sulforhodamine) ethane (2mg, available from Molecular Probes, Inc., 4849 Pitchford Avenue,

Eugene, Oregon) then aminofluorescein (2mg) were then added. The resultant mixture was stirred in an ice bath and the pH of the solution was maintained at 7.0.

Additional EDC (2mg) was added and the mixture was stirred overnight. The pH of the reaction solution was taken to 9 by the addition of dilute sodium carbonate and the solution was concentrated and chromatographed on

Sephadex G25/column packed with buffered aqueous

carbonate solution and eluted with neat butyl ethyl ketone. The product portion was determined by

spectrometric detection of the elution fractions

containing the dye bound product (first fraction with absorption at about 500 to 540 nm). The unreacted dye eluted later .

Preparation of 1-amino-2- (aminosulforhodamine) ethane

As an alternative to the purchase of 1-amino-2- (aminosulforhodamine B) ethane, it can be prepared as follows. Ethylene-diamine (2.94ml) was combined with methylene chloride (20ml) and the resultant mixture was purged with Argon. The mixture was stirred in an ice bath for ten minutes and lissamine (0.5g) was added over a 5 minute period. The solution was stirred for 3 hours in an ice bath then stored at -20°C overnight. The solution was stirred for 7 hours in an ice bath, stored again overnight at -20°C, then stirred at room

temperature for 30 minutes. Hexane (130ml) was added, the solution was allowed to stand in the ice bath for 15 minutes, then stirred for 15 minutes at room

temperature. The reaction mixture was filtered, the filter paper was washed with methylene chloride and the filtrate was combined with 1-butanol (4ml) and taken to dryness in vacuo. The residue was suspended in a 3:2 mixture of methylene chloride:chloroform (10ml) and this mixture was centrifuged (10,000 rpm, 10 minutes, 20°C). The supernatant was chromatographed by HPLC on a silica column (Shandon resin 1 × 25cm, 10 μm resin) eluted with methylene chloride and chloroform to give the title product.

EXAMPLE 2

Synthesis of Polyacrylic Acid

Substituted with Rhodamine-hydride, Aminofluorescein, Rhod-amine Hydrazide, l-Amino-2-(aminosulforhodamine)ethane

or Texas Red hydrazine

Polyacrylic Acid (8mg, 2 × 10⁶ daltons,

Polysciences, Inc.) was added to 0.15 M potassium chloride solution (80ml). Upon dissolution, the pH of the solution was adjusted to 6 with KOH, then the solution was cooled in an ice bath to 4°C and EDC (24mg) was added. The reaction solution was stirred and the pH was maintained between 6 and 7 for 20 minutes, then additional EDC (8mg) was added. The resultant solution was stirred for an additional 20 minutes then divided into 5 lots of 10ml each (a concentration of

approximately lmg polymer per aliquot, pH of 6.2). Each of the aliquots was placed in an ice bath and one of the following dyes were added to the aliquots:

1. Rhodamine hydrazide (6.5mg) and

Aminofluorescein (2mg);

2. Rhod-amine hydrazide (8.5mg); 3. 1-Amino-2-(aminosulforhodamine)ethane (8.5mg); and

4. Texas red hydrazide (8.5mg).

The respective dyes were added to each reaction in several portions over a ten minute period. The pH of each solution was maintained between 6 and 7 and the temperature of each solution was controlled with an ice bath. Each solution was stirred for an additional 2 hours then EDC (2mg) was added to each solution.

Each resultant solution was stirred overnight in a cold room. Each aliquot was placed in an ice bath and an additional EDC (2mg) was added then the pH of each was maintained between 6 and 7 and stirred in a cold room for two days. The pH of each aliquot was adjusted to 8.5 by the addition of sodium carbonate at room temperature. Each aliquot was centrifuged at 9000 rpm for 20 minutes. The resultant supernatants were chromatographed on a Sephadex G25 support low pressure liquid chromatography (LPLC) using a mixture of alcohol and water as_ eluant. Aliquot #3 gave a purple product and aliquot #4 gave a blue product.

EXAMPLE 3

Methods of Preparing

1-amino-3,5,7-trioxy-9-(aminosulforhodamine B)

Procedure A

1,9-diamino-3,5,7-trioxynonane

("polyglycolamine", Union Carbide #H-221, 7.7ml) and dry methylene chloride (20ml) were combined, the reaction vessel purged with argon then the flask was cooled to 40°C under a positive argon pressure. Lissamine (0.5) was added to the cooled solution over a 15 minute period and the resultant solution was stirred for an additional 10 minutes and stored under argon overnight at -20°C. The solution was then stirred in an ice bath for 5 hours. The reaction solution was chromatographed over silica gel and eluted with compatible solvent. The dye-containing fractions were combined and taken to dryness. A portion (118mg) of the product collected from the silica gel column was dissolved in a solution of 1:1 acetonitrile/water (2ml) and the solution was

centrifuged. The entire supernatant was chromatographed by LPLC on C₁₈ reverse-phase support (1 × 25 cm column) eluted sequentially with acetonitrile and water to give the above product.

Procedure B

Procedure A was repeated through the point where the reaction solution was stirred overnight at ambient temperature. Hexane (80ml) was added to the reaction solution, the mixture was stirred for several minutes and the liquid was decanted. The precipitate was washed 3 times with a methylene chloride/hexane mixture (60ml each). The washes were combined, filtered with suction, and the filtrate was discarded. The filter was placed over the product containing flash and the filter was washed with chloroform. The resultant suspension was taken to dryness in vacuo. The resultant residue was resuspended in dry methylene chloride (4ml), combined with the oily residue left from the decantation procedure, then chromatographed as in Procedure A. Procedure C

Procedure B was repeated through the hexane wash decantation. The resultant precipitate was washed 3 times by first dissolving the precipitate in methylene chloride (10ml) then adding hexane (40ml) to precipitate the product. With each wash the liquid was decanted and the supernatants thus obtained were combined and

filtered. The filter paper was washed with methylene chloride and this was combined with the earlier obtained precipitate. This product-containing mixture was chromatographed on silica (1cm × 25cm column, 10 μm

Shandon resin) eluted with the following step gradient solvent mixture to yield the title product. time (minutes ) (v/u) chloroform acetone methanol

0.01 100 0 0

5.00 100 0 0

40 20 80 0

90 5 95 0

120 0 50 50

140 0 0 100 EXAMPLE 4

Methods of Preparing Poly (1-N-3,5,7-trioxo-9- (aminosulforhodamine B) amido polyacrylic acid

Condition A

2 × 10⁶ Dalton PAA

Polyacrylic Acid ("PAA", 2 × 10⁶ dalton molecular weight, Polysciences, Inc. 2.4mg) was

dissolved in water (12ml). The pH of the solution was adjusted to 5.5 by the addition of 0.1N potassium hydroxide solution. A 5ml aliquot of this solution was placed in an ice bath. To the aliquot was added EDC

(4mg as an aqueous solution) while maintaining the pH of the solution between about 5.5 and 6.0. The cooled solution was stirred for 30 minutes then 1-amino-3 , 5 , 7- trioxo-9-(-amino sulforhodamine B) (8.25mg), was added as a solution (previously adjusted to pH 7.5 by the

addition of 0.01N potassium hydroxide solution) and the pH was adjusted to 7.5 again after pH of the solution dropped to 7.0. The solution was stirred for 2 hours then additional EDC (2mg) was added and the pH of the resultant solution was adjusted to 7.5 as before. The solution was stirred at 4°C overnight. After

approximately 18 hours, the pH of the solution

stabilized at 7.5 (i.e., without the addition of the aqueous potassium hydroxide solution) and additional EDC (lmg) was added. The pH of the resultant solution was adjusted to 7.5 as before, and when the pH stabilized, the solution was centrifuged (9000 rpm, 15 minutes) and the supernatant was chromatographed by LPLC (Sephadex G25 support, 1 × 25cm column; aqueous eluent to give the title product as determined by spectrometric detection of fractions containing dye bound product (i.e., first fractions absorbing at 500 to 540 nm) . The unreacted dye eluted later.

Condition B

4 X 10⁶ Dalton PAA

Condition A was repeated using a different molecular weight PAA (i.e., 4 × 10⁶ dalton molecular weight) to yield the title product.

EXAMPLE 5

Method of Preparing Poly [1-(N-amido)-2- (aminosulforhodamine-poly-[2-(N-amido') ethylsulforic acid] polyacrylic acid (4 × 10 dalton M.W.) a) Addition of AESA

Polyacrylic acid (4mg, as 0.5mg/ml aqueous solution, 4 × 10⁶ daltons molecular weight) was combined with water (32ml). Ethyl trimethylaminoethyl

carbodiimide ("EDC") and + 2-amino-ethanesulfonic acid ( "AESA" ) were added to the reaction solution in the following fashion, (with periodic adjustments in the pH of the solution by the addition of 0.01N potassium hydroxide solution):

Reagent Amount Water Used PH at addition

EDC 1.25mg 100 microliters 5.87

KOH - - 5.5

KOH - - 6.5

AESA 1.25mg - 7.2

KOH - - 7.3

EDC 1.9mg - 8.3

EDC 2.7mg 100 microliters 6.85

EDC 2.7mg 100 microliters 6.55

After the above series of additions, the solution was stirred overnight at 4°C. b) Addition of Dye

A portion (10ml) of the above solution was placed in an ice bath. EDC and 1-Amino-2- (aminosulforhodamine) ethane ("EDAS" available from Molecular Probes, Inc., Eugene, Oregon) were added in portions to the solution in the following manner, (all while making sure that the addition of the reagents did not take the pH of the reaction solution outside the range of 7.0 - 7.6):

Reagents Amount³

EDC¹ 20

EDAS² 20

EDC 20

EDAS 20

EDC 20

EDAS 20

EDC 20

EDAS 20

EDC 20

EDAS 20

EDC 40

EDA 20

1. EDC was added as an aqueous solution with a concentration of 18mg/20 microliters

2. EDAS was added as an aqueous solution with a concentration of 10mg/400 microliters

3. in microliters

The reaction solution was centrifuged (10,000 rpm for 5 minutes) and the supernatant was

chromatographed on Sephadex G25 with butyl ethyl ketone to give the title product. Product fractions were determined by spectrometric identification of fractions containing the dye bound product (i.e., first fractions absorbing at 500 to 540 nm). The unreacted dye eluted later.

EXAMPLE 6

Basic Method of Preparing

Poly [1-(N-amido)-2-(aminosulforhodaminepoly-[2-(N-amido) ethylsulforic acid]

(4 × 10° daltons M.W.)

To a reaction flask was added a solution of

10ml polyacrylic acid (PAA) as the sodium salt

(Polysciences, Inc., M.W. 1.36 × 10⁶) in aqueous phosphate buffer (0.1 mg of polymer per ml, pH 7).

About 100μl of the dye solution, aminosulforhodamine (ASR) in 50 percent aqueous acetonitrile was next added (Molecular Probes, Inc., 10mg per ml) followed by about 200μl of the coupling agent solution, EDAC in water

(15mg per ml). The reaction mixture at pH 7 was stirred at ambient temperature for about two hours and then allowed to rest for a short time about 15 to 30 minutes. The reaction mixture was then dialyzed with phosphate buffer through 12-14k MECO tubing. The product remained behind while unreacted dye was removed in the dialyzate. High pressure liquid chromatography on a GF250 column (silica gel size exclusion column manufactured by DuPont Chemical Inc., Wilmington, Delaware) with phosphate buffer as an element produced the title product eluting in the void volume as detected by UV absorbance at 560 nm.

Following the foregoing procedure but varying the amount of dye solution added, the results given below were produced. The finding of a precipitate in the reaction mixture indicated that the approximate upper range of dye to polymer binding had been reached for these conditions.

EXAMPLE 7

Preparation of Poly [1-(N-amino)-3,5,7-trioxy- 9-(aminosulforhodamine B)1-poly [2-(N-amino) ethanesulfonic acid! polyacrylic acid (4 × 10 M.W.) a) Addition of AESA

AESA was added to Polyacrylic Acid (4 × 10⁶ daltons molecular weight) in the same manner as

described in Example 5 above. b) Addition of Dye

A portion of the above solution (10ml) was placed in an ice bath. The following reagents were added to the solution in the manner set forth below so as not to drive the pH of the solution beyond the range of 7.10 to 7.60:

Reagents¹ Amount⁴

EDC² 20

PGAS³ 20

EDC 20

PGAS 20

EDC 20

PGAS 20

EDC 20

PGAS 20

EDC 20

PGAS 20

EDC 20

PGAS 20

EDC 60

PGAS 20

EDC 60

PGAS 20

EDC 60

1. In addition each EDC and PGAS were added as a pair, the solution was allowed to stir for a short time, then another addition of the EDC/PGAS pair was made. 2. EDC was added as an aqueous solution with a

concentration of 0.18mg/ 20 microliters.

3. PGAS (1-amino-2,5,7-trioxy-9-(aminosulforhodamine B) was added as an aqueous solution with a

concentration of 10mg/400 microliters.

4. Microliters added. The resultant reaction solution was centrifuged and chromatographed as in Example 5 above to yield the title product. EXAMPLE 8

Preparation of Poly [N-(1-amino)-2- (aminosulforhodamine)ethane] - poly [N(2-amido)

ethanesulfuric acid] polyacrylic acid

(2 × 10º M.W.)

Polyacrylic acid (2 × 10⁶ daltons molecular weight) was dissolved in water to give a solution with a concentration of 0.5mg/ml. A portion of this solution (1ml) was combined with additional water (9ml). The pH of the solution was taken to 7.5 by the addition of 0.001N aqueous -potassium hydroxide solution then DMF

(100 microliters) was added. The resultant solution was cooled to 4°C. The reagents in the chart below were added to the solution, while maintaining the pH of the resultant solution between 6.5 and 7.5 by the addition of the reagents plus occasional additions of 0.01N hydrochloric acid:

Reagents Amount⁵ Concentration⁶

EDC¹ 20 0 .3/20

EDAS² 40 1/40

AESA³ 40 0 .2/40

EDC 20 0 .3/20

EDAS 40 1/40

AESA 40 0 .2/40

(HCL)⁴ to pH 6. 75

(HCL) to pH 6.50

EDC 20 0. .3/20

EDAS 6 1/40

AESA 6 0. .2/40

EDC 40 0, .6/40

EDAS 20 0, .5/40

AESA 20 0, .1/40

(HCI) to pH 6 6.50

EDC 60 0. .9./60

EDAS 20 0. .5/40

AESA 20 0. ,1/40

1. EDC (ethyl trimethylaminoethyl carbodiimide) was

first added to the solution followed closely by an addition by both EDAS and AESA. These portions were allowed to react for a while before the next cycle of EDC/EDAS and AESA was added.

2. 1-amino-2-(aminosulforhodamine)ethane.

3. 2-aminoethanesulfonic acid.

4. sufficient 0.01N(n) HCI was added to bring the pH of the solution to the indicated value.

5. in microliters.

6. expressed as milligram/microliters.

The reaction solution was stored overnight at 4°C. The solution was chromatographed on Sephadex G25 (2.5 cm x 100 cm) (LPLC) with a mixture of 10 millimolar potassium borate, 1% potassium chloride, 1 millimolar EDTA and 10% acetonitrile in water (pH 8.2) to give a solution of 24 ml of the title product. This product was analyzed by UV spectrometry at 572 nm, where its absorbance was 0.272. Assuming an extension coefficient of 50 cm²/μmol, this absorbance indicated a concentration of 0.054 micromols of dye/ml, or 1.3 micromols of dye on the 0.5 mg of unsubstituted PAP used as starting material. This is approximately 7400 dye molecules per molecule of polymer.

EXAMPLE 9

Substitution of Polyacrylic Acid

with 1-Amino-2-(aminosulforhodamine)ethane

Polyacrylic acid (1 mg, 1×10⁶ daltons,

Polysciences, Inc.) was suspended in 10 ml of phosphate buffered saline (PBS), pH 7.0. The solution was kept at room temperature (20° - 22°C). 1-amino-2- (aminosulforhodamine)ethane (10 mg/ml stock solution in

50% aqueous acetonitrile) was added to the reaction vessel in the amount needed to achieve the necessary degree of substitution. 20 ul is approximately maximal to maintain solubility of the final product. To

initiate coupling of the dye moiety and the polyacrylic acid polymer, EDAC was added. From a stock solution of

EDC (15 mg/ml in water) 200 ul is aliquoted and added to the reaction vessel. The reaction is stirred at room temperature for 2 hours at which point the reaction may be considered complete. At this point the net charge of the product may be changed by binding various moieties to the unreacted carboxyls on the polymer backbone.

Ethylene diamine, ammonium hydroxide, ethanolamine and taurine have been used in this respect.

The reaction product was then exhaustively dialyzed in a 12-14,000 MW cutoff dialysis bag against

PBS. The dialysis solutions were changed until no more dye could be detected coming out of the dialysis bag. The free dye in the dialyzate was quantified and used to calculate dye incorporation into the polymer.

The product was deeply colored clear solution.

Analysis by gel filtration indicated a high MW species with strong absorbance at 560 nm. EXAMPLE 10

Preparation of Monoclonal Antibody to Aminosulforhodamine (AS)

A. Cell lines and Media

The enzyme-deficient, nonsecreting myeloma cell line Sp 2.0-Ag 14 is cultured in Dulbecco's modified minimal essential media (DME) containing penicillin, streptomycin (P,S), nonessential amino acids (NEAA), L-glutamine, 10% heat-inactivated horse serum and 5% heat inactivated calf serum (complete media, CM). The 3T6 mouse fibroblast line is used as a feeder cell layer in the cloning of cell hybrids and was cultured in DME containing P,S NEAA, L-glutamine and 10% heat-inactivated calf serum.

B. Preparation of Immunogens and Antigens

The immunogen, aminosulforhodamine(AS) -keyhole limpet hemocyanin (KLH) (AS-KLH) is prepared by the covalent conjugation of the dye to the KLH by amide bond formation with carbonyl diimidazole or of an appropriate activated derivative of the dye, such as the

isothiocyanate for thiourea links to the KLH. The procedure follows the method described by Lopatin & Voss, Biochemistry, 10, 208-213 (1971). The test antigen aminosulforhodamine-bovine serum albumin (AS¬

BSA) is prepared by the same method. Radiolabelled test antigen ([¹²⁵I]AS-BSA) and rabbit antimouse IgG ([¹²⁵I]RAM) are prepared by chloramine-T oxidation Sonoda &

Schlamowitz, Immunochemistry, 7 , 885-898 (1970).

C. Preparation of Immunoadsorbents

Aminosulforhodamine is coupled to Sepharose 6B (Pharmacia) through the introduction of the bisoxirane 1,4-butanedial diglycidyl ether according to the

procedure of Sundberg & Porath, J. Chromatog., 90, 87-98 (1974). D. Cell Fusions and Cloning

Somatic cell hybrids are generated as previously described in Kranz et al., Immun. Commun., 9., 639-651 (1980). On the fourth day following secondary immunization of a BALB-c mouse with 200 μg of AS-KLH, the spleen is removed and approximately 10⁸ washed splenic lymphocytes are added to 10⁷ washed Sp 2.0-Ag 14 myeloma cells and centrifuged. One milliliter of a 50° polyethylene glycol (PEG 1540 Baker Grade) solution (in HEPES-buffered DME at 37°C) is added to the pelleted cells over a period of 1 min. The cells are washed, resuspended in 100 ml of CM and distributed into 48 culture wells (2 ml were COSTAR 24-well cluster plates). The cells are cultured in selection media containing hypoxanthine, aminopterin and thymidine (HAT) for 2-3 weeks. Supernatants from wells are assayed for anti-AS activity using the radioimmunoassay described later.

The cells from anti-AS positive wells are cloned in 0.2% agar (SeaKem) over a 3T6 fibroblast feeder layer as described by Coffino et al. (1972).

After approximately 10 days, individual clones are transferred to a 24-well cluster plate, cultured and assayed for antifluorescein activity. The selected clones are grown in a culture until a sufficient number of cells were available for freezing and for injection into BALB-c mice.

E. Radioimmunoassays

A liquid-phase radioimmunoassay is used to detect anti-AS secreting hybrids. Four hundred

microliters of supernatant, 50μl of IM phosphate buffer, pH 8.0, 50μl of rabbit antimouse gamma globulin and 10μl of [¹²⁵I]AS-BSA are incubated at 37°C for 1 hr. and at 4°C for 2 hr. After the samples are centrifuged and the precipitate washed 3 times with 0.05M phosphate buffer, pH 8.0, the dpm are measured using a gamma counter. F. Purification of Anti-AS Antibodies

Monoclonal murine anti-AS antibodies are purified from the ascites fluid of BALB-c mice, which have been injected with approximately 5×10⁶ of the corresponding hybridoma cells. Antibody purification involves sodium dextran sulfate precipitation,

immunoadsorption and ligand elution with 0.1 M FDS (Watt et al., Molec. Immun., 17, 1237-43 (1980)). Eluted antibodies can be dialyzed against 0.05M phosphate buffer, pH 8.0, and subjected to cation exchange

chromatography to remove free aminosulforhodamine.

Eluted fractions can be monitored for absorbance at 278 and 500 nm. EXAMPLE 11

Preparation of Antihuman Chorionic Gonadotropin Monoclonal Antibody

A. Linkage of Human Chorionic Gonadotropin

(HCG) to Protein Carrier

To prepare the HCG for injection and

immunoassay, 400 umoles of the HCG is dissolved in water. About thirty milliliters of cold ethanol is added and incubated for 30 minutes at 0°C. The reaction mixture is centrifuged at 10,000 g for 30 minutes, and the pellet is washed with 30 ml of cold ethanol. The pellet is dissolved in 200 ml of 40% dimethylformamide pH 4.8, containing 200 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and 1 g of either, bovine serum albumin or keyhole limpet hemocyanin is added to the solution. The reaction mixture is stirred at room temperature overnight. The mixture can be centrifuged as above, resuspended in PBS, and dialyzed overnight at 4°C against 4 liters of PBS. B. Immunization of BALB/c Mice

BALB/c mice receive multiple injections of the HCG-KLH antigen emulsified in Freunds adjuvant (10μg per injection). After the fourth injection, blood from the immunized animal is collected in 0.5 ml of PBS, and each sample is assayed by ELISA for the presence of antigen- specific antibody.

C. Hybridoma Production

The spleen from a mouse testing positive for immunogenicity is removed aseptically and the cells are isolated by dicing the spleen in 5ml of sterile PBS.

The cell suspension is added to a centrifuge tube and tissue fragments are allowed to settle for 1-2 minutes. The cells still in suspension are placed in a similar tube and centrifuged at room temperature. The cells are then washed 3 times by centrifugation in serum-free DMEM (Dulbecco's modified Eagle's medium). Spleen cells are copelleted with P3X63-Ag8.653 myeloma cells at a ratio of 4 spleen cells to 1 myeloma cell. The supernatant fluid is removed, and the pellet is suspended in 1 ml of 35% polyethylene glycol for 1 minute. The polyethylene glycol is gradually diluted by addition of increasing amounts of serum-free DMEM over a period of 15 minutes. The cells were then suspended in HAT medium (Monoclonal Antibodies, Kennett, McKean, Backitt, eds. Plenum press 1981) at a concentration of 2 X 10⁵ myeloma cells per ml, and 4 drops from a 5ml pipet are added to each well of 5 96-well microtiter plates. The plates are incubated in 10% CO₂ at 37°C. Portions of the culture fluid are replenishable from time to time with fresh HT medium (HAT medium without aminopterin), and the plates further incubated. After culturation, approximately 100μl of culture fluid is taken from each well containing

macroscopically visible cell growth, and an RIA

technique described above can be used to identify those culture fluids containing HCG-specific antibodies. EXAMPLE 12

Preparation of Fab' Fragments from AS and HCG

Monoclonal Antibodies

F_ab' fragments of the monoclonal antibodies

(Mab) of Examples 11 and 12 can be prepared by

immobilized pepsin digestion and partial reduction according to the procedures of Masuho and Goding (see Y.

Masuho and T. Hara, Gann, 71, 759 (1980) and J.W.

Goding, J. Immunol. Methods, 13., 215 (1976)). The procedure is as follows.

About 10 mg of the Mab in freshly made buffered aqueous medium (20mM, sodium acetate buffer, pH 4.5) can be placed in a digestion vessel. To this protein solution is added about one half ml of immobilized pepsin on cross-linked agarose beads of about 9000 units of activity per ml of gel (Pierce Chemical Company) to form the F_c and F(_ab')₂ fragments. After about 2 hours incubation, the reaction is treated with about 3 ml Tris HCL buffer (pH 7.5) and centrifuged. The supernatant is applied to an immobilized protein A column (Pierce

Chemical) and the column is washed with appropriate amounts of Tris buffer at pH about 7.5 to elute the F(_ab')₂ fragments. The isolated F(_ab')₂ fragments can be treated with a minimum amount of 2-mercaptoethanol (2mM) in buffered aqueous medium (Tris HCL) to reduce the inner fragment disulfide bond and produce the F_ab' fragments. The F_ab' fragments can be dialyzed against isotonic saline to remove buffers and amino acids.

EXAMPLE 13

Preparation of Bifunctional Di-Fab' Fragment

for HGC and AS

A. Preparation of SPDP-Anti-HGC Fab Fragment

50μl of 12mM SPDP in ethanol is added to 2.0 ml of the anti-HGC F_ab' in 0. IM sodium phosphate buffer, that is 0.1m NaCl, pH 7.5 (5.0 mg protein/ml), and the mixture can be incubated at 26°C for 30 min. The excess SPDP and N-hydroxysuccinimide generated are removed by gel filtration on Sephadex G-25 (0.8×43cm) in

phosphate/EDTA buffer to give anti-HGC F_ab'-SPDP. The average number of 3-(2-pyridyldithio) propionyl groups introduced into one molecule of F_ab' can be determined by reducing the product with 2 mereaptothanol followed by measurement of the absorbance at 343nm due to free pyridine-2-thione generated by reduction. B. Preparation of SMCC Anti-AS Fab' Fragment

The anti-AS F_ab' fragments (2.62 mg dissolved in 1 ml of phosphate-EDTA buffer) can be mixed with a 30- fold molar excess of SMCC (504 μg of SMCC dissolved in 50.4 μl of N,N'-dimethylformamide) for 60 min. at room temperature. The reaction is stopped by gel filtration chromatography on Sephadex G-25 (1.5x30cm) equilibrated with phosphate-EDTA buffer. The anti-AS F_ab' -maleimido fractions are pooled and concentrated to approximately 1 ml.

C. Coupling Reaction

A mixture of 2 ml of anti-HCG F_ab'-SPDP in phosphate-EDTA (3mg protein per ml) can be combined with a stoichiometric amount (relative to protein) of

dithiothreitol and allowed to stir at room temperature for about 30 minutes. To this reaction mixture at about 4°C is then added 2ml of anti-SA F_ab'-SMCC (3mg of protein per ml) in Tris at pH 7.5, 150mmol of NaCI and lmmol of MgCl₂ per L. The resulting mixture is allowed to stir gently at about 4°C overnight to cause coupling and produce anti-HCG F_ab'-SCO-C₆H₁₂-CH₂-N(COCH₂)₂-S(CN₂)₂COS-anti-SA F_ab'. EXAMPLE 14

Demonstration of Binding of Bifunctional Conjugated Anti-HCG/Anti-AS di-Fab'

Polyvinyl chloride microtiter assay plates can be coated with about 100 micromol of antihuman chorionic gonadotropin (anti-HCG) rabbit antibody in aqueous phosphate buffer at pH 7.5 at a concentration of 10 microgram per ml in phosphate buffered saline (PBS).

The plates are incubated overnight to obtain adsorption of the anti-HCG rabbit antibody onto the plate surfaces. The plates are then washed with fresh phosphate buffer to remove nonadsorbed material then blocked with nonfat dry milk to remove all nonspecific protein binding sites on the assay plates.

To half of the plate wells (test wells) is added about 10μg of HCG, about 10μg of the bifunctional conjugate of Example 13 and about 10 mmol of the ASpolyacrylic acid compound of Example 9 all in PBS while to the other half of the wells (control wells) is added the same mixture without HCG. The plate wells are incubated overnight at room temperature, then washed with fresh PBS buffer to remove nonbound material.

Comparison of the test and control wells will show the test well walls to be the distinct color of AS and the control well walls to be colorless. This difference will indicate that immunospecific binding of the

bifunctional conjugate to HGC and the AS assay compound occurs. EXAMPLE 15

ICON ^® Membrane Assay Experiment

100 ul of 1% latex having the second antibody

HCU061 (anti-HCG monoclonal antibody) bound to it, i.e., the HCG ICON ^® Serum II latex prepared according to the procedures given in A.S. Rubenstein EPO application No.

200,381, published November 5, 1986, was incubated with

500 ul of urine that is measured at 40,000 mlU/ml of

Human Chorionic Gonadotrophin (HCG). (This is a typical concentration found in the urine of a pregnant woman who is in the latter part of her first trimester). The mixture was gently rocked at room temperature for five minutes. The mixture was then spun on a centrifuge at 5000 rpm for 5 minutes to pellet the latex. The

supernatant was decanted to remove unbound HCG. To the pellet was added 0.5 ml of 5mM Tris buffer at pH 7.8. The pellet was vigorously vortexed to disperse it once more and then it was recentrifuged.

To this pellet was added 200μl of 0.28 mg/ml of the bifunctional Di-F_ab' conjugate of Example 13 which has affinities for both HCG and aminosulforhodamine.

About 100 ul of dye polymer formed from polyacrylic acid and aminosulforhodamine as described in Example 8 was added to the pellet. This solution was vortexed to disperse the pellet and then incubated for two hours at room temperature.

After incubation 100μl of the solution was spotted onto an ICON^® device. The solution flowed through the membrane leaving a distinct colored spot that is characteristic of the dye polymer. The membrane was washed with 1 ml of Phosphate buffered saline.

A control reaction using a urine sample containing no HCG was prepared in the above manner with the same bifunctional Di-Fab' conjugate and dye assay compound. This sample did not cause any significant colored spot after being passed through the membrane.

The same assay was repeated except that the analyte same of urine was reformulated to contain about 500 mIU/ml of HCG. (This is a typical HCG concentration found in the urine of a pregnant woman who is in her third or fourth week of pregnancy . ) Upon incubation and spotting, a colored spot was observed indicating a positive qualitative test for HCG.

These experimental results demonstrate the feasibility of using bifunctional F(_ab')₂ conjugates or bifunctional monoclonal antibodies with dye polymers for the detection of an analyte in a diagnostic assay.

Claims

WHAT IS CLAIMED IS:

1. An assay dye compound, comprising a polymer having an all-carbon backbone, and first and second pendent substituents, the first pendent substituent being an alkyl amide or sulfonamide having one or more N- substituted dye moieties, and the second pendent substituent being an alkyl carboxy or sulfoxy group, the percent of first pendent substituent relative to the sum of first and second pendent substituents being up to about sixty percent.

2. An assay dye compound according to claim 1 wherein the molar ratio of the polymer backbone to dye moiety is from about 1:10 to about 1:100,000.

3. An assay dye compound according to claim 1 wherein the molar ratio of the first pendent substituent to the second pendent substituent is from about 1,000:1 to about 1:1.

4. An assay dye compound according to claim 1 wherein the first pendent substituent is substituted with first and second N-substituted dye moieties.

5. An immunoreactive dye composition suitable for use in an assay for an analyte, comprising:

(a) a polymer having an all-carbon backbone and

first and second pendent substituents, the first pendent substituent being an alkyl amide or sulfonamide having one or more N-substituted dye moieties, and the second pendent

substituent being an alkyl carboxy or sulfoxy group, the percent of first pendent substituent relative to the sum of the first and second pendent substituents being up to about sixty percent; and (b) a bispecific antibody which is specifically immunoreactive with the polymer and the analyte to be assayed.

6. An immunoreactive dye composition according to claim 5 or an assay dye compound of claim 1 further comprising a third pendent substituent of an N- (sulfonoxyalkyl or aryl) alkyl amide or sulfonamide group for the polymer.

7. An assay dye compound according to claim 6 wherein the ratio of the first to third pendent substituent is from about 100:1 to 1:1.

8. An assay dye compound according to claim 6 wherein the molar ratio of the first pendent substituent to the sum of the second and third pendent substituents is from about 1,000:1 to about 1:1.

9. An immunoreactive dye composition according to claim 5 or an assay dye compound of claim 1 wherein the dye moiety is derived from a spectrometrically- detectable molecule or derivative thereof.

10. An immunoreactive dye composition according to claim 5 wherein the bispecific antibody comprises two or more polyclonal, monoclonal or recombinant

antibodies or single chains or fragments that are covalently linked together.

11. A method for synthesizing an assay dye compound,

comprising combining a dye having amine groups and an acid polymer having an all-carbon backbone and a plurality of pendent alkyl carboxylic or sulfonic groups under conditions for forming amide or

sulfonamide bonds between the dye and the acid polymer thereby forming the assay dye compound.

12. A method of synthesizing an assay dye compound according to claim 11 wherein the pH of the amide or sulfonamide bond forming step is from about 4.5 to about 12.

13. A method for synthesizing an assay dye compound

according to claim 11, further comprising combining of an organic amino sulfonic acid with the acid polymer and the dye and additionally forming amide or sulfonamide linkages between the organic amino sulfonic acid and the acid polymer.

14. A method of synthesizing an assay dye compound

according to claim 13 wherein the pH of the amide or sulfonamide bond forming step is from about 4.5 to about 7.5.

15. A detectable analyte dye complex, comprising:

(a) an assay dye compound of a polymer having an all-carbon backbone and first and second pendent substituents, the first pendent substituent being an alkyl amide or sulfonamide having one or more N-substituted dye moieties, and the second pendent substituent being an alkyl carboxy or sulfoxy group, the percent of first pendent substituent relative to the sum or the first to second pendent substituents being up to about sixty percent;

(b) an analyte to be detected;

(c) a bispecific antibody which is specifically

immunoreactive with the polymer and with the analyte to be detected; and

(d) a second antibody which is species specific for the bispecific antibody, or is immunospecific for the analyte, or is immunospecific for a complex capable of being formed between the analyte and the bispecific antibody.

16. A detectable analyte dye complex according to claim 15 or an immunoreactive dye composition according to claim 5 wherein the bispecific antibody is

immunoreactive with at least one dye moiety.

17. A detectable analyte dye complex according to claim 15 or an immunoreactive dye composition according to claim 5 wherein the bispecific antibody comprises a polyclonal antibody, a monoclonal antibody, a recombinant antibody, a single L or H chain of the polyclonal, monoclonal or recombinant antibody or a fragment of the polyclonal, monoclonal or

recombinant antibody or single or H chain.

18. A detectable analyte dye complex according to claim 15 wherein the second antibody comprises a

polyclonal antibody, a monoclonal antibody, a recombinant antibody, a single L or H chain of the polyclonal, monoclonal or recombinant antibody or a fragment of the polyclonal, monoclonal or

recombinant antibody or single chain.

19. A detectable analyte dye complex according to claim 15 wherein the affinity constants between the bispecific antibody and analyte, and between the second antibody and the analyte, bispecific antibody or complex are sufficient to maintain the

corresponding immunological conjugations during washing, decanting, separating, exchanging or other physical manipulations.

20. A detectable analyte dye complex according to claim 19 wherein the affinity constants are at least about 1 × 10⁸ liters per mole.

21. A method for detecting an analyte in an aqueous mixture comprising:

(a) combining components (i) through (iv) in any order to form a detectable analyte dye complex having dye groups; and

(b) determining the quantity of analyte by

spectrometric measurement or visual

interpretation of the dye groups of the detectable analyte dye complex;

said components (i) through (iv) being

(i) a polymer having an all-carbon backbone and first and second pendent substituents, the first pendent substituent being an alkyl amide or sulfonamide having one or more N-substituted dye moieties, and the second pendent substituent being an alkyl carboxy or sulfoxy group, the percentage of first pendent substituent relative to the sum of the first and second pendent substituents being up to about sixty percent;

(ii) the aqueous mixture containing the analyte to be detected;

(iii) a bispecific antibody which is

specifically immunoreactive with the polymer and the analyte to be detected; and

(iv) a second antibody which is species specific for the bispecific antibody, or is immunospecific for the analyte, or is immunospecific for a complex formable between the bispecific antibody and the analyte.

22. A method according to claim 21, wherein the

detectable analyte dye complex formed in step (a) is prepared by combining components (ii) and (iv) to form a first intermediate, combining components (i) and (iii) to form a second intermediate, and combining the first intermediate with the second intermediate to form the detectable analyte dye complex.

23. A method according to claim 21 wherein component (iv), the second antibody, is immobilized.

24. A method according to claim 23 wherein the

immobilization is accomplished by a solid support.

25. A method according to claim 24 further comprising pouring the detectable analyte dye complex formed in step (a) onto a semi-porous membrane before step (b).

26. A method according to claim 25 further comprising washing the semi-porous membrane after the

detectable analyte dye complex has been poured thereon and before step (b).

27. An analyte assay kit for determining the presence or absence of one or more analytes, comprising:

(a) a predetermined amount an assay dye compound, comprising a polymer having an all-carbon backbone and first and second pendent

substituents, the first pendent substituent being an alkyl amide or sulfonamide having one or more N-substituted dye moieties, and the second pendent substituent being an alkyl carboxy or sulfoxy group, the percentage of first pendent substituent relative to the sum of the first and second pendent substituents being up to about sixty percent; (b) a predetermined amount of a bispecific antibody which is specifically immunoreactive with the polymer and the analyte to be detected; and

(c) a predetermined amount of a second antibody

which is species specific for the bispecific antibody, or is immunospecific for the analyte, or is immunospecific for the complex formable between the analyte and the bispecific antibody.

28. The assay kit of claim 27 further comprising sample containment means for containing and mixing an amount of a sample to be determined, the assay dye compound, the bispecific antibody and the second antibody.

29. An anti-sulforhodamine antibody produced by

hybridoma culture Deposit No. HB10625.