WO1993020448A1 - Immuno-titrage par enzymes sans instrument d'analyse: procede general recourant a des effets de liaison cinetiques - Google Patents

Immuno-titrage par enzymes sans instrument d'analyse: procede general recourant a des effets de liaison cinetiques Download PDF

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Publication number
WO1993020448A1
WO1993020448A1 PCT/US1993/003133 US9303133W WO9320448A1 WO 1993020448 A1 WO1993020448 A1 WO 1993020448A1 US 9303133 W US9303133 W US 9303133W WO 9320448 A1 WO9320448 A1 WO 9320448A1
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analyte
enzyme
competitive binding
antibody
immunogen
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PCT/US1993/003133
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English (en)
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William Lasley
Coralie J. Munro
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The Regents Of The University Of California
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Publication of WO1993020448A1 publication Critical patent/WO1993020448A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones

Definitions

  • 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.
  • ELISA enzyme-labelled immuno- sorbent assays
  • NEEIA non-instrumented enzyme immunoassays
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • immunoassay particularly enzyme immunoassay ("EIA") .
  • EIA enzyme immunoassay
  • 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.
  • 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
  • the color is in turn indicative of the presence or absence of the analyte.
  • FIG. 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.
  • 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.
  • the steepness of the curve has been control by controlling the relative affinity of the antibody for the analyte and the competitive binding compound.
  • 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.
  • BSA bovine serum albumin
  • 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.
  • 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.
  • 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
  • the immunogen 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.
  • 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 ( ) .
  • 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 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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) .
  • 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.
  • 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.
  • the "steroid moiety" of a steroid conjugate is the part of the conjugate originally derived from a whole steroid molecule.
  • Standard 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the analyte itself is used as the base component of the competitive binding compound.
  • the analyte is cortisol
  • cortisol itself is preferably used as the base of the competitive bonding compound.
  • a cortisol derivative such as cortisol glucuronide can be used.
  • 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.
  • Small molecules such as steroids and pharmaceuticals are generally too small to induce an immune response (antibody formation) in most organisms.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • small 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.
  • linking group (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.
  • the immunogen when it is not a protein, it will generally be a biologic polymer such as a polysaccharide or other carbohydrate and will contain similar functional groups
  • 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) .
  • BSA bovine serum albumin
  • Biotin-biotin complexes 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • analyte competes for binding sites so that less of the enzyme-labelled competitor can bind.
  • 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.
  • reaction mixture stays the same (thus a positive reaction using this reaction scheme) .
  • 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.
  • 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.
  • polyclonal antibodies 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.
  • a monoclonal antibody (referred to as EG6) that bound specifically to estrone sulfate and to estrone glucuronide
  • 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:BSA was biotinylated using a technique generally described in Bayer et al., Anal. Biochem., 154:367-370
  • 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) .
  • 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.
  • 75 ml (17 mg; 37 mnoles) of biotinamidocaproate-NHS ester in DMF this gives an 8:1 mole ratio of active ester
  • 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.
  • streptavidin-alkaline phosphatase 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.
  • 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.
  • 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) .
  • 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
  • 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.
  • 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.

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Abstract

Un procédé permet de détecter la présence ou la quantité d'un analyte présent dans un échantillon grâce à un immuno-titrage par enzymes marquées qui recourt à un composé de liaison concurrent à enzyme marquée qui entre en concurrence avec cet analyte en matière de liaison avec un anticorps anti-analyte spécifique contre ledit analyte. L'amélioration consiste à utiliser en guise de composé de liaison concurrent un dérivé de l'analyte qui contient une fraction, propre à cet analyte, à laquelle est liée une fraction immunogène massive à un site de l'analyte utilisé pour fixer un immunogène à l'analyte lorsqu'on prépare l'anticorps anti-analyte, ainsi qu'un groupe reporter d'enzyme lié à la fraction de liaison massive. On choisit le groupe de liaison massif et l'enzyme de manière qu'ils présentent conjointement une taille suffisante pour ralentir la diffusion du composé de liaison concurrent et pour réduire sa capacité à se lier avec l'anticorps anti-analyte (par rapport à l'affinité avec l'analyte) par des effets cinétiques se manifestant à un degré suffisant pour rendre plus raide la courbe de liaison concurrente de ce titrage, de manière qu'un seuil positif/négatif plus net puisse apparaître au cours d'un immuno-titrage par enzymes sans instrument d'analyse, sans exiger de manipulations structurales au site de liaison de l'anticorps.
PCT/US1993/003133 1992-04-01 1993-03-31 Immuno-titrage par enzymes sans instrument d'analyse: procede general recourant a des effets de liaison cinetiques WO1993020448A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10488408B2 (en) 2006-05-09 2019-11-26 Koninklijke Philips N.V. Detection of target molecules in a sample by using a magnetic field

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0170345A2 (fr) * 1984-03-28 1986-02-05 TECHNICON INSTRUMENTS CORPORATION(a Delaware corporation) Cytométrie à écoulement
US4803170A (en) * 1985-05-09 1989-02-07 Ultra Diagnostics Corporation Competitive immunoassay method, device and test kit
EP0326073A1 (fr) * 1988-01-25 1989-08-02 Roche Diagnostics GmbH Conjugués haptènes-protéines et leur utilisation
EP0378391A2 (fr) * 1989-01-10 1990-07-18 Biosite Diagnostics Inc. Des essais ligands-récepteurs utilisant un seuil
EP0383313A2 (fr) * 1989-02-15 1990-08-22 Mochida Pharmaceutical Co., Ltd. Réactif d'essai immunologique et kit le contenant

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0170345A2 (fr) * 1984-03-28 1986-02-05 TECHNICON INSTRUMENTS CORPORATION(a Delaware corporation) Cytométrie à écoulement
US4803170A (en) * 1985-05-09 1989-02-07 Ultra Diagnostics Corporation Competitive immunoassay method, device and test kit
EP0326073A1 (fr) * 1988-01-25 1989-08-02 Roche Diagnostics GmbH Conjugués haptènes-protéines et leur utilisation
EP0378391A2 (fr) * 1989-01-10 1990-07-18 Biosite Diagnostics Inc. Des essais ligands-récepteurs utilisant un seuil
EP0383313A2 (fr) * 1989-02-15 1990-08-22 Mochida Pharmaceutical Co., Ltd. Réactif d'essai immunologique et kit le contenant

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10488408B2 (en) 2006-05-09 2019-11-26 Koninklijke Philips N.V. Detection of target molecules in a sample by using a magnetic field

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