WO1993020448A1

WO1993020448A1 - Non-instrumented enzyme immunoassay: general method utilizing kinetic binding effects

Info

Publication number: WO1993020448A1
Application number: PCT/US1993/003133
Authority: WO
Inventors: William Lasley; Coralie J. Munro
Original assignee: The Regents Of The University Of California
Priority date: 1992-04-01
Filing date: 1993-03-31
Publication date: 1993-10-14
Also published as: AU3972993A

Abstract

A method for detecting the presence or amount of an analyte in a sample by means of an enzyme-labelled immunoassay that utilizes an enzyme-labelled competitive binding compound that competes with the analyte for binding to an anti-analyte antibody specific for the analyte, in which the improvement comprises utilizing as the competitive binding compound a derivative of the analyte comprising an analyte moiety, a bulky immunogenic moiety bound to the analyte moiety at a location on the analyte used to attach an immunogen to the analyte when preparing the anti-analyte antibody, and an enzyme reporting group bound to the bulky linking moiety. The bulky linking group and the enzyme are selected so that together they are of sufficient size to slow diffusion of the competitive binding compound and reduce its ability to bind with the anti-analyte antibody (relative to analyte affinity) by kinetic effects in an amount sufficient to steepen the competitive binding curve for the assay, so that a sharper positive/negative threshold can be distinguished in a non-instrumented enzyme immunoassay without requiring structural manipulations at the antibody binding site.

Description

NON-INSTRUMENTED ENZYME IMMUNOASSAY: GENERAL METHOD UTILIZING KINETIC BINDING EFFECTS

ACKNOWLEDGEMENTS This invention was supported in part by grants from U.S. govenrmental agencies. The U.S. Government has rights in this invention as a result of this support.

INTRODUCTION Technical Field

This invention is related to enzyme-labelled immuno- sorbent assays (ELISA) and is particularly directed to such assays that relay on simple observation to detect results, i.e, non-instrumented enzyme immunoassays (NIEIA) , for small analytes that are normally non-immunogenic.

Background Reagent strips for use in quantitative and qualitative analysis are well known; such strips are widely used for the analysis of low concentrations of various biological compounds, particularly for analysis of clinically signi¬ ficant substances in body fluids, such as the quantitative analysis of glucose in blood.

Two processes are necessary for an analysis in a test strip or on another solid surface: (1) a specific reaction must take place that is indicative of the presence of the component being tested for, and (2) a detectable signal must indicate that the identifying reaction has occurred.- In some cases a single reaction fulfills both functions. In other cases two separate reactions occur.

In both cases, a solid substrate that is capable of adsorbing or absorbing a liquid is impregnated with a reagent capable of reacting with the compound of interest in a test sample. In a typical analysis for a component such as glucose that takes part in a chemical reaction, identification and detection of the compound of interest is achieved by noting, or quantitatively measuring, the appearance of a reaction product or the disappearance of a non-reactant present in the test strip when sample is applied. When the molecule in question cannot be distinguished from other molecules normally present in the test sample by a simple chemical reaction, two reactions are generally used: (1) a binding reaction with a specific binding molecule, such as an antibody, and (2) a chemical reaction which indicates that the binding reaction has taken place.

Many such analyses involve quantitative measurement of color formation using a reflectance photometer. The measured reflectance of the reagent strip after reaction of the sample with the reagent strip containing reactant yields a quantitative colorimetric determination of the concentration of the compound of interest. See, for example, Genshaw, U.S. Patent No. 4,772,561, entitled "Test Device and Method of Determining Concentration of a Sample Component. "

While quantitative results are desirable in some instances, determining the amount of material present in the sample is not required for all analyses. In some cases, the presence of any amount of a material (e.g., lead in the bloodstream) is clinically significant. In other cases, one merely needs to know whether the concentration of a substance is present above (or below) a certain concentration level (e.g. , determination of elevated levels of progesterone as an indication of pregnancy) . The concentration or other properties of the reactant present in a test strip can be adjusted so that color formation or another detectable signal begins when the concentration of the material of interest reaches a certain level, as is well known in the art.

A general technique that is applicable to a variety of analyses on solid surfaces is immunoassay, particularly enzyme immunoassay ("EIA") . In an immunoassay, the presence of an analyte is determine by the binding of that analyte to an antibody. Since antibodies have very specific binding affinities, the reaction is very specific and subject to minimum false negatives. However, the binding reaction itself is not detectable, and a technique must be used to indicate binding between the antibody and the component being analyzed, as described above.

Most assays today use an enzyme label to detect binding of analyte to the antibody. The enzyme is attached either to the antibody itself (direct assays) or to a molecule that competes with the analyte for binding to the antibody (indirect, or competitive, assays) . The enzyme catalyzes reaction of a compound that changes color as a result of the reaction. This color change is indicative of the presence of the enzyme, and, since the enzyme is attached to the a molecule whose presence indicates that binding has

(direct assays) or has not (competitive assays) occurred, the color is in turn indicative of the presence or absence of the analyte.

All competitive immunoassays exhibit a characteristic binding curve for the reaction between the antibody and the analyte/competitor mixture that is bound by the antibody. Figure 1 illustrates the binding curve of a hypothetical competitive antibody binding reaction for a fixed amount of competitor and increasing amounts of analyte, which has a shape commonly referred to as a "S" curve. As shown by the solid line in Figure 1, there is a gradual increase in binding of analyte at the lower end of the curve, a relatively linear central portion, and a tapering off of the curve as maximum binding of analyte is reached. While such a curve is suitable and in fact desirable for quantitative analysis, since there is a relatively broad range over which different amounts of binding can be distinguished, such a binding curve is difficult to use when a qualitative result is desired. For example, if the enzyme reaction produces a blue color, it may be difficult to tell when the very faintest blue color is detectable to the eye, particularly because of the variation in the ability of different persons to detect faint colors. Additionally, since there is no sharp color change, it becomes difficult to determine when the concentration that is indicative of a clinical condition is reached. Accordingly, assays intended for on/off (presence/absence) determinations preferably exhibit a sharper binding curve, as illustrated by the dashed line in Figure 1. Since binding shifts from minimum to maximum over a very narrow range of concentrations, color production will similarly either not occur below that range or fully occur above that range of concentrations. The steeper the binding curve, the more definitive the yes/no decision.

Although NIEIAs have been used for many analytes, NIEIA has not been applied generally to small organic and biologic molecules, such as steroids, since a steep binding curve has been difficult to obtain. In the prior art, the steepness of the curve has been control by controlling the relative affinity of the antibody for the analyte and the competitive binding compound. For example this technology was applied to steroids by using estrone linked to an enzyme label as the competitive binding compound to bind with an antibody that was prepared using estrone glucuronide linked to bovine serum albumin (BSA) as an immunogen. The antibody preparation used in the assay had a higher affinity for an estrone conjugate analyte than it did for the enzyme-labelled estrone competitor as a result of steric effects that reduced the ability of the antibody to bind to the competitor. This was the desired effect, since antibodies with higher affinity for analyte than for competitor produce steep binding curves. However, it is difficult to produce antibodies or manipulate competitive binding compounds to provide such precise affinities, and such antibodies are obtained only by a random antibody production process coupled with manipulation of the structure of the competitor at the region of the competitor bound by the antibody, which is a very unpredictable process. If binding is reduced too much or too little, the desired result will not be obtained. Accordingly, there remains a need for a different technique for producing steep binding curves in competitive binding assays so that NIEIA can be applied to detection of steroids, steroid conjugates, peptides, and other small molecules in a more predictable manner.

SUMMARY OF THE INVENTION The present invention provides an improvement in a method for detecting the presence or amount of a small organic or biologic molecule in a sample using an enzyme- labelled competitive binding assay. In particular, the improvement comprises deriving a competitive binding compound from the analyte/immunogen combination used to produce the antibody used in the assay. The analyte/- immunogen component is bound to a reporter enzyme, in some case by using a bulky linking group to increase the overall bulk of the combination. The immunogen (typically a protein or polysaccharide) is bound to the analyte using conventional techniques (typically through a linking group, usually covalent, that is smaller than the analyte molecule) , and the linking together of other components is also conventional and readily adaptable to a wide variety of assays. The invention provides a general technique for preparing competitive binding compounds for use in non- instrumented enzyme immunoassays without requiring manipulation of the specificity of binding at the location where antibody binds to the competitor. The bulky competitor prepared in this manner diffuses slowly in the reaction medium and thus competes poorly with the actual analyte for binding to the antibody, thus steepening the binding curve without further manipulation.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood by reference to the following detailed description of the invention when considered in combination with the drawing that forms part of the specification, wherein:

Figure 1 is a graph showing hypothetical binding curves for a competitive immunoassay; a normal binding curve is indicated by a solid line ( ) , while a steepened binding curve obtained with a competitor of the invention for use in an NIEIA is indicated by a dashed line ( ) .

DESCRIPTION OF SPECIFIC EMBODIMENTS This invention is directed to an improved enzyme- labelled component for use in competitive binding assays for small organic and biologic molecules and to the assays that use such components to provide present/not-present or "threshold" (+/-) analysis results in non-instrumented enzyme immunoassays. The analyte being detected in the assay (and used as part of the enzyme-labelled component) is a molecule that is small relative to the remaining portions of the enzyme- labelled component used as the competitive binding compound and therefore is not itself generally immunogenic. The improved enzyme-labelled, competitive binding component comprises the analyte molecule (or the portion thereof used to generate the antibody used in the assay) bound to the immunogen that is used to produce the antibody of the assay. An enzyme label is bound to this bulky moiety, preferably through another bulky linker such as an avidin- biotin complex. The use of such an enzyme-labelled competitive binding compound allows antibodies to be used without attempting to manipulate affinity of binding of antibody to competitor while still providing the steep competitive binding curve required for a +/- analysis. As is usual for attachment of im unogens, the analyte is preferably attached to the immunogen through a small linking group so that the bulky protein or other large molecule that forms the immunogen is at a distance from the steroid to improve specific affinity of antibody for the antigen. The assay and new competitive binding component have been demonstrated with steroid analytes, but there are no conceptual limitations on the types of analytes that can be used in the assay, other than the relative bulk/kinetic factors described below in detail.

Although words used herein have their normal meanings when applied generally to the invention, the following definitions apply to preferred embodiments of the invention.

An assay for "detecting the presence or amount" of an analyte is an assay that either (1) gives a yes/no signal (such as a color change) to indicate the presence of an analyte (even though such a signal indicates the presence of a minimum amount of the analyte necessary to produce the signal, the absolute amount of the analyte present is not directly related to a quantitative property of the signal) or (2) gives a quantitative indication of the amount of analyte present as the result of a quantitative property of the signal (such as color development proportional to amount of analyte present) . Preferred embodiments of the invention are directed to on/off (present/not-present) results of assays performed on solid substrates in which no instrument is used, simple observation by the analyst (i.e. , an NIEIA) .

An "analyte" is the material whose presence or amount is being determined by analysis. An "organic" molecule is a molecule that has been synthesized by techniques of organic chemistry (rather than biochemistry) . A "biologic" molecule is a molecule that can be obtained from or is known to occur in a living cell

(which includes procaryotic and eucaryotic cells and viruses that infect cells) . Many molecules, such as pharmaceuticals and steroids, can be both organic and biologic molecules at the same time.

A "steroid" is any organic/biologic compound having a 17-carbon, 4-ring steroidal structure typical of steroids, as well as the usual side chains and functional groups. Steroids typically have functional groups such as hydroxyl groups, keto groups, and unsaturation (either aromatic or non-aromatic) at different locations in the basic steroidal ring system that vary with the class of steroid (e.g., estrogens have an aromatic A ring with a hydroxyl group at the 3 position and an oxygen, either a hydroxyl or a keto group, at position 17) . Exemplary steroid structures can be seen in any edition of the CRC Handbook of Chemistry and Physics, published by the Chemical Rubber Co., such as volume 48, pages C-690 through C-702.

A "steroidal compound" is either a steroid or a steroid conjugate. A "steroidal conjugate" is a compound formed by formation of a covalent linkage of a non-steroidal compound to a steroid. Linkage is typically through a hydroxyl group of the steroidal ring system or of a side group (which is typically aliphatic and can be substituted with hydroxyl and/or other functional groups) . The non- steroidal component can be inorganic (e.g., a sulfate group) or organic (e.g., a glucuronide group) . Examples of preferrednon-steroidal components include glucuronides and sulfates. An "analyte conjugate" is a generic term intended to encompass naturally occurring modifications of analyte molecules exemplified by the steroidal conjugates described in the preceding paragraph. In such cases, the analyte is a biologic molecule that exists in two forms, the analyte molecule itself and the analyte conjugate, which comprises the analyte molecule modified by some biological action

(generally involving the formation of a covalent bond) that results in the binding of another biologic molecule to the original analyte. Analyte conjugates are typically formed in the liver or in secretory organs of an animal that produces or ingests the analyte as part of the process of excreting or secreting an analyte that have limited water solubility. In addition to steroidal conjugates, other typical analyte conjugates include any biologic or organic molecule that is processed by conjugation in the liver or kidneys, such as pharmaceuticals and drugs of abuse.

A "sample" is the material being analyzed and is usually of biological origin, although pre-treatment may have removed some of the normal biological compounds normally associate with the analyte (such as red cells separated from plasma in a whole blood sample) .

An "enzyme-labelled immunoassay" is an assay that uses an enzyme as a detectable reporter group and an antibody as a specific binding compound to detect the presence of an analyte.

A "competitive binding assay" uses competition between an analyte and a different molecule, called the competitive binding compound, for a limited number of binding sites on a specific binding molecule, usually an antibody, to determine whether, or how much of, an analyte is present. An "anti-analyte antibody" is an antibody that binds with an analyte with an association constant of at least o 10 . The antibody is "specific" for the particular analyte of interest if the analyte binds to the antibody in preference to any other compound expected to be present in the same sample.

A compound is a "derivative" of a first compound (as used herein for preferred embodiments) if the derivative compound is formed (or can be form) by reaction of the first compound with another molecule so as to form a new compound either smaller or (usually as used here) larger than the first compound while retaining at least part of the structure of the first compound.

An "immunogen" is a molecule that is capable of inducing an immune response in a vertebrate immune system; i.e, injection of the immunogen into a host animal will result in production of antibodies that recognize and bind with that immunogen. When the immunogen is injected in the presence of another compound bound or otherwise associated with the immunogen, antibodies are also produced that recognize and bind specifically with the other compound, even if that compound is not capable of inducing an immune response if injected by itself. Proteins and carbohydrates from animal or plant species different from the host species in which antibody production is desired are commonly used as immunogens.

A "protein" is a poly(amino acid) produced or producible by a biological process. Some proteins are produced by direct transcription and translation of a gene; others involve post-translational processing, such as glycosylation. Polypeptides are small poly(amino acids) or fragments of larger proteins; many polypeptides are immunogenic in the same manner as proteins.

A "carbohydrate" is simple sugar (i.e., a monosaccharide such as glyceraldehyde or glucose) , a biologic or organic derivative of a simple sugar (e.g., neuraminic acid or 2,3,4,6-tetramethylglucose) , or a molecule formed by linking together more that one simple sugar and/or sugar derivatives (a polysaccharide such as amylose or cellulose and fragments thereof formed by partial hydrolysis) . Generally, simple sugars and derivatives thereof are not immunogenic; polysaccharides are usually quite immunogenic.

Reference to a molecule or part of a molecule as being "linked" or "attached" to another molecule or part usually indicates the presence of a covalent bond. "Bound" includes both covalent and non-covalent associations. Non- covalent associations are preferred to be sufficiently stable so as not to undergo appreciable dissociation during the course of the assay under consideration. When used specifically to refer to types of chemical bonds present, the word "compound" refers to a chemical composition that contains covalent bonds (although protonation, de- protonation, and salt formation of mostly covalent compounds, such as amines or carboxylic acids, are included within this definition) . When used specifically to refer to types of chemical bonds present, the word "complex" refers to a chemical composition that contains non-covalent associations in addition to covalent linkages, such a complex that includes non-covalent binding between avidin and biotin. However, compound is often used herein in its more common meaning; i.e., as a simple descriptor of a chemical composition, such as "competitive binding compound," which, as will be clear from later discussions, can be either a covalently bound compound or a complex containing non-covalent associations.

A "moiety" is a part of a complex derivative molecule that is derived from the indicated original molecule. For example, the "steroid moiety" of a steroid conjugate is the part of the conjugate originally derived from a whole steroid molecule.

"Small," as it is used to describe the analyte moiety of the invention, is a relative term, as is "bulky, " which is used to describe either other components of the competitive binding molecule to which the small analyte moiety is attached or to describe the competitive binding molecule as a whole. These terms are closely related to physical size (volume) of the components, but a more precise comparison relates to the influence of small and bulky components on diffusion rates. Any competitive binding molecule is "bulky" relative to the "small" analyte if the competitive binding molecule as a whole diffuses in the assay medium at a rate less, preferably less than one- half, more preferably less than one-tenth, the diffusion rate of the analyte itself. The bulky overall competitive binding molecule is itself prepared from individual components bound to the analyte as described below; most, of these components are themselves larger than the analyte in order make the competitive binding molecule as a whole large enough to slow its rate of diffusion, although some small linking groups can be used to bind the various components together.

Since diffusion rates are inconvenient to measure (although the measurements are conventional, typically involving diffusion of the material being measured across a porous membrane) , an alternative and equally preferred indication of a bulky competitive binding molecule is one that has a volume at least 400, more preferably at least 600, most preferably at least 1000, times the volume of the analyte. Smaller relative volumes for the competitive binding molecule are satisfactory if the slower diffusion rates indicated above are present for the competitive binding molecule. However, the larger the relative size differences, the steeper the binding curve, so that large relative size differences are preferred. Volume is also related to molecular weight (the relationship is direct for molecules with similar shapes) so that molecular weight comparisons can be used with the same relative comparisons as volume comparisons.

For use in the present invention, an analyte preferably has a molecular weight of less than 6,000, more preferably less than 1,000, and most preferably less than 500. Larger analytes can be used, but the bulk required to achieve the appropriate difference in diffusion rates becomes difficult to achieve for larger analytes without exceeding the solubility of the competitive binding compound. The small linking group that is optionally used to attach the analyte to the immunogen preferably has a molecular weight of from 50 to 200, more preferably from 100 to 500, and most preferably from 300 to 1500. Immunogens can be of any size, since other bulky linking components (such as avidin- biotin linkages) can be added to increase the overall bulk of the competitive binding molecule. However, most commonly used immunogens have molecular weights of from 100,000 to 1,000,000. Furthermore, since the enzyme also contributes to the rate of diffusion, smaller linking groups can be used with larger enzymes to give overall molecules of the indicated sizes. There is no theoretical limit on the upper limit of the size of the competitive binding molecule, since very slowly diffusing molecules can be used with long incubation times. However, the practical upper limits of size for the competitive binding molecules as a whole are determined by solubility. Solubility will vary with the particular combination of components used to make the competitive binding molecule. Typical sizes of the competitive binding molecule as a whole for an analyte having a molecular weight of 1000 would be from 200,000 to

5,000,000, preferably 600,000 to 4,000,000, more preferably

1,000,000 to 3,000,000. Sizes would be proportionally increased or decreased for different sizes of analytes.

The competitive binding compounds of the invention are made by binding the indicated components to each other using standard techniques of synthetic chemistry, which need not be described here in detail. The components can be bound together in a linear or branched configuration, and components can be present merely for the purpose of increasing overall bulk. For example, extra biotin-avidin linkages can be used to attach proteins (in addition to the reporter enzymes) to the immunogen to merely to increase bulk and steepen the binding curve. Thus, a binding curve with practically any desired steepness can be obtained by manipulating the bulk of the competitor at locations far distant from the antibody binding site. No new reactions or synthetic processes are required, merely application of well-known processes for binding together existing organic and/or biologic components in the manner described herein.

Preparation of an enzyme-labeled competitive binding compound of the invention begins by selection of an analyte or analyte derivative that will be the base compound on which other reactions will take place. The specific compound selected will depend on the type of assay being undertaken; for example, if the analyte is a steroid the basic building block will be a member of the same class of steroids. Preferably, the analyte itself is used as the base component of the competitive binding compound. For example, if the analyte is cortisol, cortisol itself is preferably used as the base of the competitive bonding compound. However, a cortisol derivative such as cortisol glucuronide can be used. Likewise, if cortisol glucuronide is the analyte, cortisol can be used as the base compound.

Binding of the remaining portions of the competitive binding compound to the base analyte compound will take place through a functional group or portion of the molecule that is normally used to attach an immunogen when preparing an anti-analyte antibody for use in the immunoassay.

A brief aside here is useful for an understanding of the concept of the immunogen. Although the following discussion of immunogens and antibodies is discussed using steroids as an example, it will be recognized that other analytes can be manipulated in the same manner.

Small molecules such as steroids and pharmaceuticals are generally too small to induce an immune response (antibody formation) in most organisms. Thus, if one wishes to use an immunoassay to analyze for the presence of, for example, a steroid such as estradiol, formation of antibody is induced by injection into an animal (or use of other techniques for antibody production) of a compound formed by attaching the small steroid molecule to a large molecule that is itself capable of including an antibody response. Thus, the antibody response is directed against the entire "foreign object, " which includes the steroid moiety. Some antibodies will be specific for the immunogen while others will be specific for the steroid (or other small molecule) . Those antibodies that are specific for the steroid can easily be isolated by, for example, attaching the steroid to a solid surface (as in an affinity column) in the absence of the immunogen, passing a mixture of antibodies through the column, and then eluting those antibodies which have bound specifically to the steroid on the column surface. Cells capable of producing a monoclonal antibody of the invention can likewise be recognized using standard techniques. Since antibody production is induced in cells by recognition of the surface of molecule that comes in contact with the cell, the "recognition" surface of the analyte molecule is generally that portion of the molecule which is not attached to the immunogen. For example, if an antibody is induced against estradiol attached to an immunogen through one of its two hydroxyl groups, the specificity of the antibody will differ depending on the point of attachment. If attachment is through the hydroxyl group at position 3 (in the A ring) , the antibody will specifically recognize the remainder of the estradiol molecule, notably the structure of the D-ring, which is most removed from the point of attachment. On the other hand, if the hydroxyl group in the D-ring, namely the hydroxyl group at position 17, is attached to an immunogen and used to prepare an antibody, the antibody will most specifically recognize the A-ring, where the hydroxyl group at position 3 is located. Thus, an antibody induced by a steroid attached to an immunogen will recognize a steroid derivative with a higher specific binding constant if the non-steroidal part of the steroid derivative is attached to the steroid at the same position as the immunogen used to generate the antibody. Such attachments are utilized in the compositions of the invention to maximize specificity. While the examples above illustrate the use of naturally occurring functional groups in steroids for attachment of immunogens or the formation of derivatives, natural functional groups need not be used whether in steroids or in other analytes. For example, the B-ring of most estrogens normally does not contain a reactive functional group that will allow formation of derivatives. It is possible, however, to oxidize the carbon atom of the B-ring adjacent to the aromatic A-ring so that a carbonyl group is present. The carbonyl group can be reduced to a hydroxyl group so that attachment can occur through the fi¬ ring. Such attachment allows antibodies to recognize both the A- and D-rings of the estrogens, providing high- specificity. Similar modifications of other steroids at a normally non-reactive location, particularly in the B-ring, are very useful for both induction of antibodies and formative of derivatives for use as competitive bonding compounds, since the A- and D-rings of steroids are the parts of a steroid that are most variable and that therefore account for the principal differences between different steroids. Antibodies that recognize these differences in both the A- and D-rings therefore are more specific than antibodies that recognize differences only in the A-ring or only in the D-ring. Similar modifications are possible for other analyte molecules, such as pharmaceuticals and drugs of abuse.

Returning now to formation of the competitive binding compound of the invention, the analyte is linked to an enzyme through the location on the analyte used to attach the immunogen to the analyte when the anti-analyte antibody was being prepared. In fact, in preferred embodiments the same analyte/immunogen complex is used that was used to induce the antibody. It will be recognized, however, that some variations in the analyte/immunogen complex can exist without departing from the present invention. For example, substitution of one linking group or other component for another having essentially the same physical and chemical properties will not adversely affect the functioning of the resulting compound and can be used interchangeably with the original analyte/immunogen complex; e.g., an adipic acid linker can be used instead of a succinic acid linker, a lupine protein can be used instead of a bovine protein, and the like.

In preferred embodiments the analyte/immunogen complex comprises two parts in addition to the analyte, a bulky moiety with immunogenic properties and a small linking group between the bulky moiety and the analyte. This small linking group is smaller than the analyte and acts as a spacer to allow free access of antibody-producing cells to the analyte moiety so that antibody production can take place. The linking group is generally one of two types of molecules: (1) the non-analyte portion of a naturally occurring analyte conjugate, or (2) a simple organic linking group, typically comprising a linear methylene chain of four to ten carbon atoms with a functional group at each end for attachment 'to the analyte and the protein. These two general classes will be discussed in turn. As before, steroids will be used to illustrate the two possibilities, but the invention is not limited to use with steroids.

Naturally occurring steroidal conjugates are an important biological form of steroids, generally being the form in which a steroid is found upon being excreted, as in urine. Since steroids are lipids, they are not freely soluble in water and therefore require modification before they can be excreted into an aqueous environment. In the bloodstream, they are carried on proteins by non-covalent association, but these proteins are themselves not excreted. Thus, derivatives of these steroids are prepared for excretion by enzymatic reactions in the body. Most steroids are excreted as derivatives of sugar acids, since the polyhydroxyl nature of the sugar increases the water solubility of any compounds to which it is attached. The class of sugar acids typically found in steroid conjugates is referred to as the uronic acids. In uronic acids, only the carbon atom bearing the primary hydroxyl group is oxidized, to a carboxylic acid group, with the secondary hydroxyl groups and (if present) aldehyde carbonyl being unchanged. The uronic acid derived from glucose is known as glucuronic acid. Other important uronic acids are galacturonic acid and mannuronic acid.

Many steroids are excreted in the form of glucosiduron- ides, also known as glucuronides, which are glycosidic excretory products formed from aromatic or aliphatic alcohols by the action of enzymes in the endoplasmic reticulum of the liver. Covalent attachment occurs, not via formation of an ester or amide as might be expected from the presence of the carboxylic acid group at the six position of a normal sugar, but by formation of an acetal between a hydroxyl group of the steroid and the carbonyl carbon of the sugar (carbon 1 for an aldose; carbon 2 for the common ketoses) . This leaves the carboxylic acid group at carbon 6 for further reaction, and either this carboxyl group or the remaining free hydroxyl groups can be used in further derivatizing the steroidal compound to form the competitive binding composition of the invention.

When the steroid analyte is not a steroidal conjugate, the glucuronides can be nonetheless be used to form the base portion of the competitive bonding compound, since the sugar portion of the glucuronide readily acts as a linking group through which attachment can occur to proteins or other bulky molecules. However, it is also possible to use small organic molecules as linkers. These organic molecules are typically bi-functional, having a functional group that is capable of reacting with a hydroxyl group, carboxylic acid group, or amino group at each end of an aliphatic middle portion of the molecule, typically formed by a linear chain of methylene groups, usually four to eight in number. However, the only restrictions on the organic linking group are that it be smaller in size than the analyte molecule and capable of linking the analyte portion of the molecule to the immunogen. Preferred linking groups (after reaction of functional groups that form bonds with the other components) contain no charged functional groups and most preferably comprise carbon, hydrogen, oxygen, and nitrogen atoms (the last two being optional) in the form of aliphatic groups optionally containing carbonyl, hydroxy, carboxylic acid, amine, and amide functional groups.

The protein or other bulky immunogen is bound to the analyte or linker through conventional techniques of complex formation or covalent bond formation. This typically involves an amino group on a protein, although reaction through hydroxyl groups and other functional groups typically found in proteins can occur. In many cases, it is possible to utilize functional groups already present in the small_.linker or analyte moiety to react with functional groups present in the protein. In some cases, activation (in the form of preparation of a reactive intermediate) is useful for rapid reaction. Attachment of proteins to organic molecules is well-known, and there are no limitations that would be relevant to the practice of the present invention.

When the immunogen is not a protein, it will generally be a biologic polymer such as a polysaccharide or other carbohydrate and will contain similar functional groups

(e.g., hydroxyl groups in carbohydrates) that can be used in the same manner. However, strictly organic polymers (e.g., polyesters, polyethers, and polyurethanes) can be used if in the indicated size ranges.

The particular protein or other bulky molecule used as an immunogen/linker in the invention is not limited other than by the desirable size, as previously described. Globular proteins, such as albumins, are preferred but not required when proteins are used. A particularly useful composition, because of its ready availability, is bovine serum albumin (BSA) linked to an analyte as described above. Such compositions are well-known and have previously been used to induce antibody formation. See the examples below. Any protein, including BSA, provides a number of reactive sites that can be used to attach enzymes to the protein. Again, there is no particular limitation on the manner in which the two proteins (i.e., the enzyme and the bulky immunogen/linking protein) are attached to each other) . A useful technique that acts to further increase bulk and which has previously been used in other situations is the formation of avidin-biotin complexes. Biotin is attached to the linking protein and avidin is attached to the enzyme (or vice versa) . Mixing of the two components results in the formation of the desired complex by a non- covalent but extremely high-affinity interaction between the biotin and avidin molecules. Preformed reagents comprising avidin or biotin attached to a reactive moiety for use in attaching. to proteins and other molecules are available commercially and can be used in accordance with their package instructions. Biotin-avidin complexes can also be used to bind the analyte to the immunogen.

The final component of the competitive binding composition of the invention, namely the enzyme, again is not limited but can be any of the enzymes used as reporter groups in enzyme immunoassays. Examples include horseradish peroxidase and alkaline phosphatase. A particularly preferred enzyme for use in non-instrumented- assays is alkaline phosphatase, since the enzyme is very stable and since color formation can be induced to occur with a minimum of manipulation, such as washing steps.

The competitive binding compounds of the invention can be prepared either as complete forms or as incomplete forms. By a complete form is meant a molecule that contains the steroid component at one end, the enzyme reporting group at the other end, and the various linking components described above in between. However, incomplete forms can be prepared and stored for later formation of this complete unit. An example would be two component comprising as the first component a steroid linked to a protein that contains biotin groups and a second component comprising an avidin molecule linked to the enzyme. Mixing of the two components would provide the final complete form. The competitive binding compound of the invention can be used in any immunoassay but is preferred for use in non-instrumented assays in which color formation occurs on a solid substrate. In a typical such assay, antibody prepared against an analyte is attached to a solid surface, such as a microliter plate well, a test tube, or a porous reagent strip (such as cellulose or glass fibers) . The antibody-coated solid surface is then contacted simultaneously with a sample and with the competitive binding compound of the invention. By providing fewer antibody binding sites than are present in the combined total of analyte and competitive binding compound, only a fraction of the molecules in solution will bind to the solid surface. If there are no analyte molecules present, all of the binding sites will be taken up by competitive binding compounds of the invention so that a maximum amount of enzyme is attached to the solid surface. When a substrate for the enzyme is contacted with the solid surface after the sample is washed away, reaction of the enzyme with the substrate provides a detectable signal

(usually formation of a color) that indicates to the user the absence of analyte in the sample (a negative result) .

If analyte is present in the sample, analyte competes for binding sites so that less of the enzyme-labelled competitor can bind. By using a bulky binding composition of the invention, which binds less rapidly to the antibody than does the analyte, and by properly selecting the number of binding sites relative to the amount of sample added (which is a standard technique to one of skill in the art) , analyte present at a concentration above the preselected minimum level will exclude binding of the competitive binding composition and thus binding of the enzyme to the solid substrate. An example of such a selection process to provide different threshold levels is set out in the following examples. Thus, if sufficient analyte is present in the sample, after reaction no enzyme is present to produce a color change and the reaction mixture stays the same (thus a positive reaction using this reaction scheme) . Other reaction schemes can be used in which the formation of color is indicative of the presence of the analyte. The previous example is merely one of many types of competitive binding assays in which the competitive binding compound of the invention can be used.

There are no theoretical differences between using a competitive binding compound of the invention and using previous competitive binding reactions. One merely replaces the competitive binding molecule previously used with the competitor of the invention, replaces the antibody with an antibody specific for the analyte (if the reaction is not already designed for the current analyte) , and carries out the reaction in the normal manner. Some adjustments in concentration and/or amount of reagent may be required because of different solubilities, binding affinities, and the like, but such adjustments of reaction conditions are well within the skill of those who design and carry out competitive binding assays. Thus, the invention can readily be carried out by anyone of ordinary skill following the directions set out in this specification, but provides the advantages described herein.

Antibody production for use in the invention is conventional and is not described here in detail. Techniques for producing antibodies are well known in the literature and are exemplified by the publication Antibodies: A Laboratory Manual (1988) eds. Harlow and Lane, Cold Spring Harbor Laboratories Press, and U.S. Patent Nos. 4,381,292, 4,451,570, and 4,618,577. For an example of production of antibodies specific for estradiol that could be used in the practice of the invention, see Laslev et al.. Fertility and Sterility (1985) 43:861-867, and Munro et al. , Abstract, Society for Gynecologic Investigation, San Diego, March 1989. A brief discussion of general techniques for the production of antibodies specific for steroids is included for those who may be unfamiliar with the process. Again, steroids are used only as an example, and antibodies can be prepared specific for other analytes in the same manner.

An animal is injected with a composition containing the steroid of interest covalently attached to an immunogen, usually a protein, prepared as described above. Multiple injections or the use of an adjuvant will ensure maximum stimulation of the immune system and production of antibodies. If polyclonal antibodies are desired, they can be prepared by simply collecting blood from the immunized animal and separating the antibodies from other blood components by standard techniques. To obtain monoclonal antibodies, the spleen or lymphocytes from the immunized animal are removed and immortalized or used to prepare hybridomas by cell-fusion methods known to those skilled in the art. Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity. For monoclonal anti-steroid antibodies, the antibodies must bind to the steroid of interest. Cells producing antibodies of the desired specificity are selected, cloned, and grown to produce the desired monoclonal antibodies.

Antibody can be attached to a solid surface for use in an assay of the invention using known techniques for attaching protein material to solid support materials. The solid support can include plastic surfaces of test tubes or microtiter plates, polymeric beads, dip sticks, or filter materials. The attachment methods include non-specific adsorption of the protein to the support and covalent attachment of the protein, typically through a free amino group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. The invention now being generally described, the same will be better understood by reference to the following detailed examples, which are provided for illustration only and are not intended to be limiting of the invention unless so stated.

EXAMPLES The method of the invention was demonstrated using estrone glucuronide as the analyte.

A monoclonal antibody (referred to as EG6) that bound specifically to estrone sulfate and to estrone glucuronide

(ElGluc) was used in several of the assay. This antibody had been prepared using estrone-3-glucuronide conjugated to bovine serum albumin by a mixed anhydride reaction

(ElGluc:BSA) as an immunogen. This immunogen was injected subcutaneously into rabbits to induce antibody formation, followed by standard techniques of hybridoma production and antibody selection. Preparation is more fully described in Munro, C.J., and Lasley, B.L., "Nonradiometric methods for immunoassays of steroid hormones," in: Technology and Application in Polypeptide and Steroid Hormone Detection, Eds. Hazeltine and Albertson, Alan R. Liss, Inc., New York, pp. 289-329. In some assays, a polyclonal anti-estrone antiserum, referred to as R522, was used as the antibody preparation. The competitor used in the assay was a complex of estrone glucuronide/bovine serum albumin/biotin- avidin/alkaline phosphatase. This complex was prepared from readily available commercial sources using standard binding reactions for biologic molecules. The first component of the competitor, estrone glucuronide (ElGluc) , which was also the analyte, was purchased commercially from Steroloids, Inc. or Sigma Chemical Co. ElGluc was attached to bovine serum albumin using a mixed anhydride reaction to give ElGluc:BSA. Preparation of ElGluc:BSA is described fully in Munro and Lasley, OJD. cit. ElGluc:BSAwas biotinylated using a technique generally described in Bayer et al., Anal. Biochem., 154:367-370

(1986) and Gretch et al., Anal. Biochem., 163:270-277

(1987) . The technique was varied slightly since the reaction is optimal at pH 8.0 and the pH of the storage buffer (0.10 M PBS; phosphate-bufferred saline) is 7.2. By experiment, the amount of 0.20 M NaOH needed to raise 1.00 ml of 0.10 M PBS (pH 7.2) to pH 8.0 was found to be 90 ml.

The concentration of the complex protein (ElGluc:BSA) being biotinylated in storage buffer was 560 mg/ml; the concentration of biotinamidocaproate-NHS ester in DMF (dimethylformamide) was 230 mg/ml (diluted from a stock of 5.0 mg/ml) . To 1.00 ml (560 mg; 4.7 nmoles) of protein in storage buffer was added, with stirring at room temperature, 90 ml of 0.20 M NaOH. To this solution was added, with stirring at room temperature, 75 ml (17 mg; 37 mnoles) of biotinamidocaproate-NHS ester in DMF; this gives an 8:1 mole ratio of active ester to antibody. The solution was stirred for 1.0 hr, then diluted to 2.5 ml with 0.10M PBS (pH 7.2) and placed into a dialysis bag (12- 14,000 MW cut-off). The solution was dialyzed twice against 2 liters of 0.10M PBS (pH 7.2) at 4°C for 24 hr each time, then divided into 100 ml aliquots and frozen at -70°C. The estimated concentration of biotinylated protein was 220 mg/ml.

After reaction of the competitor and standards with antibody, as described below, streptavidin-alkaline phosphatase was added to supply enzyme for color generation. In some cases, the streptavidin-alkaline phosphatase was added concurrently with the competitor. Commercially available streptavidin-alkaline phosphatase (Pierce Biochemicals) was used in the assay following the instructions provided with the enzyme preparation.

A first assay was carried out in star tubes coated with 500 ml 1:5000 R522 in coating buffer, which is a phosphate buffer at pH 9.4 to allow binding of the antibody to the plastic substrate. Star tubes were obtained from Applied Scientific Co. as Nunc-immunotube polysorb star tubes and were purchased as either "high" or "medium" binding star tubes. High and medium here refer to the relative ability of these star tubes to bind antibody or other proteins coated onto their surfaces. These tubes were used for convenience and illustrate one manner in which the threshold detection level can be set (i.e., by varying the solid surface so that different amounts of antibody will bind) . The tubes were washed (5 times) with wash solution (phosphate buffer at pH 7.0 to keep antibodies bound to the substrate but wash off materials not tightly bound, e.g. excess antibody, steroid or conjugate) . Five hundred ml 1:5000 biotinylated ElGluc:BSA and EIA buffer (0.1M phosphate buffer containing 0.01% BSA; pH 7.0) containing 50-3200 pg/500 ml ElGluc standards were added to each tube. The reaction mixture was incubated at room temperature without agitation in ambient light for 2 hours, decanted, and washed (5 times) by flushing with wash solution at room temperature in ambient light.

Five. hundred ml 1:5000 streptavidin-alkaline phosphatase in EIA buffer were then added to each assay tube. The resulting reaction mixture was then incubated for 2 hours and washed as described above. Five hundred ml of fresh substrate solution (diethanolamine containing 1 mg/ml PNPP; p-nitrophenylphosphate dicyclohexammonium salt) was then added to each tube, and the tubes were incubated for about 20 minutes. The reaction was then stopped with 500 ml 2.5N NaOH. Absorbances of the individual solutions were then read by spectrophotometry at 410 nm. The standard curves obtained in this manner had the steepness required for a non-instrumented enzyme immunoassay.

An additional set of experiments was carried out using the EG6 monoclonal antibody described above. Duplicate assays were carried out in star tubes coated with 500 ml of a 1:30,000 dilution of the EG6 monoclonal antibody. Otherwise, the experiment was carried out exactly as described above. Standard curves obtained in this manner also indicated that the assay was suitable for NIEIA. The assay was modified to use on microtiter plates. Individual wells of the microtiter plates were coated with 50 ml or 100 ml of antibody and incubated in the manner described above for star tubes. In some cases the wells were washed prior to antibody binding, which resulted in less antibody binding to the tubes and thus a lower threshold for a "positive" assay. The assay was carried out using biotinylated ElGluc in the presence of standards or sample as described above. Incubation and washing steps were generally as described above, except that in some instances the reaction mixture was removed without washing to investigate this potential simplification of the assay. Lack of washing at this step did not appear to adversely affect the assay. After incubation, 10 ml of streptavidin- enzyme conjugate were added to each microtiter plate well. The reaction mixture was incubated for two hours and washed as described previously, and 100 ml PNPP was added to each well. After about 20 minutes, the reaction was stopped using sodium hydroxide as previously described and analyzed.

Different sensitivities were achieved with each assay method as shown in following Table 1. The 50% binding levels are indicative of the transition point between a positive reaction and a negative reaction. Thus, it can be seen from Table 1 that different transitions points can readily be obtained by using different techniques of attaching antibody to the reaction surface (i.e. , attaching different amounts of antibody to the reaction surface) .

Table 1

Sensitivity of Different Assay Techniques Using EIA Plates

Sensitivity Assay Conditions (50% Binding)

50 ml antibody, plate washed prior to coating 104.0 pg

50 ml antibody, plate not washed prior to coating 148.0 pg

100 ml antibody, plate not washed prior to coating 245.0 pg

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. In a method for detecting the presence or amount of an analyte in a sample via an enzyme-labelled immunoassay that utilizes an enzyme-labelled competitive binding compound that competes with said analyte for binding to an anti-analyte antibody specific for said analyte, an improvement which comprises: utilizing as said competitive binding compound a derivative of said analyte comprising (1) an analyte moiety bound to an immunogen at a location on said analyte moiety used to bind said immunogen to said analyte when preparing said anti-analyte antibody, and (2) an enzyme reporting group bound to said immunogen.

2. The method of Claim 1, wherein said competitive binding compound diffuses in an assay medium containing said sample at a rate less than one-tenth the diffusion rate of said analyte.

3. The method of Claim 1, wherein said competitive binding molecule has a volume at least 400 times that of said analyte.

4. The method of Claim 1, wherein said competitive binding compound has a molecular weight at least 1000 times that of said analyte.

5. The method of Claim 1, wherein said immunogen comprises a protein that is attached to said analyte through a covalent linking group smaller than said analyte.

6. The method of Claim 1, wherein said immunogen comprises a carbohydrate.

7. The method of Claim 1, wherein said analyte is a steroidal compound.

8. The method of Claim 1, wherein said enzyme is bound to said immunogen via avidin-biotin binding.

9. The method of Claim 1, wherein said enzyme-labelled competitive binding compound comprises an estrone- glucuronide-succinate-(bovine serum albumin) -biotin-avidin- enzyme complex.

10. A competitive binding compound, comprising a derivative of a steroid glucuronide, comprising,: said steroid glucuronide comprising a steroid moiety and a glucuronide moiety, an immunogen moiety comprising a protein attached to said steroid through a hydroxy group of the glucuronic acid moiety, and an enzyme reporting group bound to said protein.

11. The competitive binding compound of Claim 10, wherein said competitive binding compound comprises a steroid-glucuronide- (aliphatic di-acid) -albumin-biotin- avidin-enzyme complex.

12. The competitive binding compound of Claim 10, wherein said competitive binding compound comprises an estrone-glucuronide-succinate- (bovine serum albumin) - biotin-avidin-enzy e complex.

13. The competitive binding compound of Claim 10, wherein said enzyme is alkaline phosphatase.