EP0383860A1 - Methodes de titrage par liaison specifique - Google Patents

Methodes de titrage par liaison specifique

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Publication number
EP0383860A1
EP0383860A1 EP19890907479 EP89907479A EP0383860A1 EP 0383860 A1 EP0383860 A1 EP 0383860A1 EP 19890907479 EP19890907479 EP 19890907479 EP 89907479 A EP89907479 A EP 89907479A EP 0383860 A1 EP0383860 A1 EP 0383860A1
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EP
European Patent Office
Prior art keywords
label
enzyme
analyte
conjugate
solid support
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP19890907479
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German (de)
English (en)
Inventor
James C. Sternberg
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Beckman Coulter Inc
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Beckman Instruments Inc
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Publication of EP0383860A1 publication Critical patent/EP0383860A1/fr
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Classifications

    • 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
    • G01N33/58—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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention is directed to specific binding assays, and, in particular, to improvements in heterogenous specific binding assay methods employing enzym labels.
  • Specific binding assays utilize strong and selective affinities between a pair of species that chemically bind together as a basis for measurement of one member of the pair, using the other as a reagent.
  • the specific binding is generally strong but reversible, and does not normally lead to the formation or breaking of covalent bonds between or within members of the binding pair.
  • Most useful specific binding pairs are of biological origin, such as antigens and antibodies, hormones and hormone receptors, or complementary pairs of nucleic acid molecules.
  • Specific binding assays are used for detecting a variety of analytes, including antibodies, antigens and aptens; receptors and receptor-binding molecules; and specific nucleic acid sequences. Clinical and research use for specific binding assays are numerous, including, but not limited to:
  • Specific binding methods are often classified according to the types of reagents used: as immunoassays when antigen or antibody reagents are used; as hybridization probe assays when nucleic acid probes are used; or as receptor binding assays when other types of specific receptor molecules are used.
  • the detectable label can be the "measured species", or can be an agent which leads to the formation of the measured species.
  • homogeneous specific binding assays In homogeneous specific binding assays, it must be possible to distinguish the bound from the free form of the detectable label through some property of that label while both are simultaneously present. Because of the difficulty in discriminating between the bound and free forms of the detectable label, the sensitivity of homogeneous specific binding assays tends to be limited to the measurement of concentrations down to about 10 " ' to 10" 9 Molar. In order to obtain higher sensitivities, it is generally necessary to separate the bound from the free form of the detectable label molecule, and to make the measurement on one of the separated forms. Such assays, termed heterogeneous specific binding assays, are the subject of this invention, and the more abbreviated term "specific binding assay" will herein be used to identify them.
  • Specific binding assays include both the sandwich- type methods and the competitive binding-type methods.
  • sandwich assay methods a sample that may contain a target analyte to be measured or detected reacts with two analyte- specific binding partners: (i) a first specific binding partner that is attached to, or can subsequently be harvested on, a solid support, and (ii) a second specific binding partner that is linked to a primary label.
  • sandwich assay methods a sample that may contain a target analyte to be measured or detected reacts with two analyte- specific binding partners: (i) a first specific binding partner that is attached to, or can subsequently be harvested on, a solid support, and (ii) a second specific binding partner that is linked to a primary label.
  • binding partners selectively bind to the target analyte rather than to other substances that can be present in the sample; thus these are “analyte-specific binding partners” .
  • the target -analyte binds to both analyte-specific binding partners to form a sandwich in which the target analyte ultimately becomes attached to both the solid support and the label.
  • the bound primary label can itself be directly detectable, or can provide a means for detection, thereby providing a qualitative or quantitative indication of the target analyte.
  • the target analyte being measured competes with a surrogate for, or analogue of, the analyte for binding to an analyte-specific binding partner.
  • the competition can be for binding to a solid support or binding to a label.
  • Either the analogue or its specific binding partner can be attached to, or be able to be harvested on, a solid support; in turn, either the specific binding partner or the analogue can be attached to a primary label, which can itself be directly detectable, or can provide a means for detection.
  • the amount of target analyte present increases, the amount of the labelled species ⁇ the labelled analogue or labelled specific binding partner) that can bind to the solid support decreases.
  • Analyte is generally measured by decrease in detectable label bound to the solid support.
  • Specific binding assays are further classified according to the type of detectable label and/or detection method used.
  • Direct methods are those in which a property of the detectable label can be measured directly; in these cases the primary label is also the detectable label.
  • the primary label is a chromophore
  • its presence can be measured by the absorption of light in a characteristic range of wavelength.
  • the primary label is a fluorophore
  • its presence can be measured by light emitted in a characteristic range of wavelength when stimulated by excitation with light of a specific range (usually shorter) of wavelength.
  • the primary label is a chemilumiphore
  • its presence can also be measured by light emission, but in this case a chemical reaction involving other reagents is required to bring about the emission.
  • the sensitivity of direct methods is limited by the ability to detect the amount of primary label that is directly bound in the specific binding reaction.
  • Indirect methods including especially the use of an enzyme as the detectable label for monitoring the specific binding reaction, provide an advantage in sensitivity through the fact that the enzyme acts as a catalyst. Therefore, each enzyme molecule bound as a result of the specific binding reaction can interact with a substrate reagent to lead to the formation of a multiplicity of molecules of a product which is then measured. This way, enzyme labelled specific binding assays can provide a sensitivity of approximately one to three orders of magnitude greater than direct specific binding assay methods.
  • the detectable labels first used in specific binding methods were radio-isotopes used in radioimmunoassay. Radioactive methods suffer from the drawbacks of low shelf life and problems associated with licensing and disposal, and are progressively losing favor for use in specific binding assays. Thus non-radioactive specific binding assays are becoming increasingly important.
  • Enzyme immunoassays represent a major class of non-radioactive specific binding assays.
  • Sandwich enzyme immunoassays are described in detail in U.S. Patent No. 4,463,090, which is incorporated herein by reference.
  • Competitive binding enzyme immunoassays are described in U.S. Patent Nos. 4,133,873; 4,279,992; and 4,446,233, all of which are incorporated herein by this reference.
  • enzyme specific binding assays can be more sensitive than conventional specific binding assay techniques, there are limitations on the effectiveness of enzyme specific binding assays.
  • the enzyme usually serves as the primary label and catalyzes a reaction in which a substrate species, which is itself undetectable or provides only little response in the detection method used, is converted to a product that is readily detectable.
  • substrates currently used are the chromogenic substrates, which are converted to chromophores (colored, light absorbent product species) by the enzyme, and fluorogenic substrates, which are converted to fluorescent products by the enzyme.
  • enzyme-labelled specific binding assays using fluorogenic substrates that are converted to fluorescent products by a primary enzyme label should be more sensitive than those forming chromophores. Since light emissions of the fluorescent products are measured against an essentially dark background, the percentage change in light intensity associated with the formation of a small amount of a fluorescent product should be great compared with that associated with the formation of a chromophore. Enzyme-labelled specific binding assay methods using fluorogenic substrates are described in Journal of Immunolo ⁇ ical Methods. 56; 341-346 (1983).
  • Sensitivity is limited in the use of fluorogenic substrates, however, because such substrates tend to spontaneously convert to their fluorescent products. I have recognized that because the substrate is non-fluorescent, whereas the product must be fluorescent, this spontaneous conversion is inevitable. In organic chemical species, the phenomenon of fluorescence is generally associated with a high degree of conjugated double bond structure, and the conversion from a non-fluorescent substrate to a fluorescent product requires that the conjugation increases in going from substrate to product. But increasing conjugation also results in greater stability, so that there is an inherent driving force for the substrate to proceed to product, even in the absence of the enzyme.
  • Fluorogenic substrate reagents therefore generally have poor shelf life in aqueous solution and contribute an undesirable amount of background fluorescence to the measurements. The higher background tends to negate the inherent sensitivity advantage of fluorescence methods. Moreover, there are only a few known fluorogenic substrates that satisfy the criterion that a non-fluorescent molecule is converted by an enzyme reaction to a fluorescent product.
  • Enzyme-labelled specific binding assays with chemiluminescent detection are different from enzyme assays used with light absorption or fluorescence detection, and have their own problems.
  • Enzyme-labelled specific binding assays employing chemiluminescent labels can produce chemiluminescence by oxidation of a chemilumiphore substrate with the enzyme peroxidase.
  • the enzyme does not act on the substrate to increase the number of detectable label molecules, but rather produces a transient signal each time it interacts with a substrate molecule. Non-enzymatic oxidation of the chemilumiphore by peroxide interferes in this method.
  • an oxidase such as glucose oxidase as the enzyme label can provide a chemiluminescent response as a measure of the hydrogen peroxide produced by the action of the enzyme on added glucose; the peroxide is measured by using a chemilumiphore substrate plus peroxidase.
  • an oxidase such as glucose oxidase
  • the small amount of the reactive peroxide species formed can be destroyed by side reactions without producing luminescence.
  • Chemiluminescent labels are the most sensitively measured non-radioactive labels, because they are measured against a dark background and do not depend upon a light source for their light emission.
  • Non-specific binding is an additional problem present in both direct or indirect specific binding assays.
  • "Non-specific binding” is defined herein as the binding of labelled material to a solid support, in a heterogeneous specific binding assay system, other than by means of the analyte-specific binding agents.
  • the principal methods currently used to reduce non-specific binding utilize pre- coating of the surface of the solid support with various agents, such as proteins or detergents, and thorough washing of the solid support after the analyte-specific binding reaction has taken place on it. These steps frequently are inadequate to reduce the non-specific binding sufficiently to permit the measurement of low levels of target analyte.
  • the highly sensitive enzyme amplified methods are used, the response associated with non- specifically bound label is amplified. This effect can largely nullify the useful sensitivity gains which can otherwise be achieved through use of the enzyme-amplified methods.
  • Assays of improved sensitivity would have wide applicability. Analytes for which an assay of high sensitivity would be valuable are those usually present in a concentration of below 10 " i x M, and include tumor markers, lymphokines and cytokines such as the growth factors and interferon, and infectious disease agents such as the AIDS-causing virus and papilloma virus. Further, existing assays for thyroid stimulation hormone and parathyroid hormone are very slow because these hormones can be present in a sample in a concentration of 10 " l z molar or less. An assay of improved sensitivity, if available, would allow assays for these hormones to be performed in a timely fashion.
  • the present invention satisfies the need for an improved rapid, accurate, sensitive, and automatable assay method. It is directed to the measurement of an enzyme label that is bound to a solid support through one or more analyte-specific binding agents in the enzyme-labelled heterogeneous specific binding methods of either the sandwich or the competitive binding type. It provides improved fluorescent and chemiluminescent substrates for reaction with the enzyme labels, and provides improved means for reducing errors due to non-specific binding in such assays.
  • a specific binding assay method is useful for measuring the concentration of an analyte An in a sample.
  • the method comprises forming an insoluble complex comprising a first solid support SSI, enzyme means En, and the analyte An or an analogue An * for the analyte.
  • This complex is separated from the sample, and then a mixture is formed by mixing at least the enzyme means En of the separated complex with a substrate/label conjugate.
  • This conjugate has at least one connected non- radioactive label per substrate.
  • This mixture is incubated to cause the enzyme means En to cleave at least some of the conjugate, thereby causing at least some of the label to be released from the conjugate.
  • the number of molecules of the released label in the mixture is greater than the number of molecules of the enzyme means En in the mixture, i.e. the signal produced by the enzyme means is amplified.
  • the released label is separated from the conjugate containing non-released label.
  • the released label is detected for measuring the concentration of the analyte An in the sample. It is important that the released label be separated from the conjugate because the method used for detecting the released label also detects connected labels. In other words, the connected label is directly detectable.
  • directly detectable there is meant that the label, both when connected to the substrate and when released from the substrate, can be detected by the method used for detecting the released label.
  • the directly detectable label can be selected from the group consisting of chemiluminescent, photoluminescent (fluorescent and phosphorescent), chromophoric, electron spin, and enzyme labels.
  • Use of directly detectable labels distinguishes this invention from other enzyme immunoanalysis methods where the enzyme serves to actuate a label that is otherwise not readily detectable, such as a fluorogenic precursor for a fluorescent moiety.
  • the enzyme means is not necessarily an enzyme. It can be an enzyme precursor which in the presence of another reactant acts as an enzyme to release the label from the substrate/label conjugate.
  • a sample containing an analyte An is combined with: (i) the first solid support SSI, (ii) an analyte-specific coupling agent CA for coupling the analyte, directly or indirectly, to the first solid support,
  • the analyte and the two specific binding agents are incubated to an extent dependent upon the concentration of the target analyte in the sample.
  • the incubation step when the analyte is present in the sample, the analyte, the coupling agent, the binding means, and the first solid support combine to form a sandwich complex having the structure:
  • the first solid support is added after the coupling agent, enzyme means, and the binding agents have reacted to substantial completion.
  • the reason for this is that faster reactions can occur in solution before these reactants are bound to the first solid support.
  • the enzyme label means can either contain means for binding to the binding agent BA, or the binding agent and the enzyme label can be pre-connected as a probe BA-En.
  • the sandwich complex is separated from the sample and a mixture is formed by mixing at least the enzyme means of the separated complex with a substrate/label conjugate having at least one connected non-radioactive label per substrate, the connected label being directly detectable.
  • a competitive binding assay method is similar to the sandwich assay method.
  • the analyte in the sample competes with competitive means for binding to the support.
  • the competitive means comprises the analogue An * of the analyte and the enzyme means En prebound together.
  • the coupling agent CA is a specific coupling agent for coupling the analyte and the analogue to the solid support SSI. Upon incubation of these ingredients, two complexes can be formed, a first complex having the structure:
  • a mixture is formed by mixing at least the enzyme means of the first complex with a substrate/label conjugate having at least one connected label per substrate.
  • the mixture is incubated so that the enzyme means En cleaves at least some of the conjugate causing at least some of the label to be released from the conjugate.
  • the number of molecules of the released label in the mixture which is greater than the number of molecules of the enzyme means in the mixture, is detected. It is possible to determine the amount of analyte present in the sample through the measurement of the amount of released label present in the mixture. The more released label present in the mixture, the less analyte originally present in the sample.
  • the analogue bound to or harvestable upon the solid support, competes with the analyte for a limited amount of an analyte-specific binding agent BA, which is attached to the enzyme means En.
  • BA an analyte-specific binding agent
  • the first solid support SSI can include a covalently bound member through which it is attached to the analyte-specific coupling agent CA, and the coupling agent CA, in turn, can include a covalently bound member through which it is attached to the first solid support SSI.
  • the binding agent BA can include a covalently bound member through which it is attached to the enzyme means En, and the enzyme means En, in turn, can include a covalently bound member through which it is attached to the specific binding agent BA.
  • the attachments can be either through direct covalent linkages or through the interaction between complementary members of specific binding pairs which serve to attach the covalently bound members.
  • the preferred labels are- chemiluminescent labels and photoluminescent labels (such as fluorescent and phosphorescent labels).
  • An advantage of these labels over colored labels is that they can be more sensitively measured than colored labels, since the labels providing chemiluminescence and photoluminescence signals provide a much larger .percentage change in signal reaching the light detector than in color measurement.
  • Chemiluminescent and photoluminescent labels are also preferable to radioactive labels (which are not used in this invention) , because radioactive labels have limited shelf life and can require expensive equipment for handling.
  • enzyme immunoassays using fluorescent detectable labels according to this invention it is expected that analytes in concentrations of 10 " 12 M and lower can be measured accurately and rapidly with automated equipment.
  • enzyme immunoassays with chemiluminescent detectable labels it is expected that analytes in concentrations of 10- 13 M and lower can be measured accurately and rapidly with automated equipment.
  • cleavable link between the analyte or analogue and the solid support SSI.
  • the coupling agent CA used for coupling the analyte or analogue to the first solid support SSI is (or becomes) attached to the solid support by means of a cleavable link.
  • cleavable link or "cleavable bond” as used herein means a chemical link. including covalent and/or non-covalent bonds, that is cleavable and which when cleaved releases enzyme means En bound to the solid support SSI through the specific binding reactions. Cleaving the cleavable bond does not release non-specifically bound material from the solid support.
  • Use of a cleavable link reduces background noise due to non ⁇ specific binding to the solid support SSI, thus greatly improving the signal-to-noise ratio in the measurement of the label.
  • the cleavable link is severed after the complex is separated from the sample to form a soluble enzyme-containing compound.
  • the soluble enzyme-containing compound is washed from insoluble compounds, including compounds containing SSI, to remove any compounds containing the enzyme where the enzyme was not originally bound to SSI through either An or An * .
  • the coupling agent can be preattached to the solid support.
  • the substrate/label conjugate can be either soluble or insoluble in the mixture. If it is insoluble, to detect released labels, it is only necessary to wash the released labels from the insoluble conjugate before measuring the amount of free label. However, if the conjugate is soluble, it is necessary to insolubilize the conjugate containing unseparated label, such as with a harvesting solid support SS2, so that the released label can be distinguished from unreleased label in the detection step.
  • the present invention is for an improved enzyme- labelled specific binding assay method that is (1) rapid, (2) can be used with automated equipment, (3) improves the sensitivity of enzyme-labelled specific binding assays;
  • the invention provides an enzyme-labelled specific binding assay method that can improve the sensitivity of other existing (non- radioactive) specific-binding assay techniques.
  • a method according to the present invention can be applied to either a sandwich assay or a competitive binding assay.
  • a sandwich assay method for detecting an analyte in a sample
  • the principal steps in the method are (a) forming a sandwich complex containing the analyte and an enzyme means, bound through the analyte to a solid phase, (b) mixing at least the enzyme means of the complex with a substrate/conjugate containing at least one label so that the enzyme means releases label from the conjugate, and (c) detecting the released label.
  • the method can comprise the following steps:
  • Step 4 Separate cleaved enzyme
  • Step 5 ' 3 ' Release label a[CA-An-BA-En] + n[H-Sl-S2-L] a[CA-An-BA-En] + x[S2-L] + (n-x) [H-S1-S2-L]
  • Step 6 Insolubilize unreleased label: add SS2 to give a[CA-An-BA-En] + X(S2-L) + (n-x) [SS2-H-S1-S2-L]' ⁇
  • Step 8 Detect released label
  • a sandwich containing at least the analyte An, the analyte-specific coupling agent CA, and the analyte-specific binding agent BA is formed by combining a sample containing An in solution with (i) the analyte-specific coupling agent CA, which is bound to or capable of selectively binding to a first solid support SSI; and (ii) the analyte-specific binding agent BA, which is bound to or capable of selectively binding to an enzyme means En.
  • These ingredients when combined, are allowed to incubate so that when the analyte is present in the sample, a sandwich complex is formed having.the structure:
  • the first solid support SSI can be precombined with CA or can be provided with a group CA' that can subsequently serve as a specific bridge between the coupling agent CA and the solid support SSI.
  • the solid support is added after sandwich complex (1) is formed so that the complex can form quickly in solution.
  • the binding agent BA can be either precombined with the enzyme means En or provided with a group BA' that can subsequently serve to specifically bind the enzyme means En to the binding agent BA.
  • the resulting sandwich can have many structures including:
  • the complex is separated from the solution such as by washing the soluble ingredients from the formed complex.
  • the enzyme means is cleaved from the solid support. This can be effected by using a cleavable bond between the enzyme means and the solid support.
  • the bond between CA and CA' can be cleaved by means such as an enzyme, pH adjustment, temperature adjustment, or the like. This forms the solubl complex:
  • the immunochemical bond between CA and An, or between An and BA, or both can be cleaved to release into the solution a fragment or fragments containin En. Cleavage can be effected through the use of a reduced pH, the addition of a chaotropic salt (such as sodium thiocyanate), the use of a detergent (such as sodium dodecyl sulfate) , the addition of an organic species known to have such an effect (such as dimethyl sulfoxide), or a combination of the these.
  • a chaotropic salt such as sodium thiocyanate
  • a detergent such as sodium dodecyl sulfate
  • organic species known to have such an effect such as dimethyl sulfoxide
  • the released complex containing En is then separated from insoluble complexes present, including complexes containing non-specifically bound enzyme means, such as by washing.
  • the substrate/label conjugate has the general formula H-Sl- S2-L where:
  • H is a harvestable moiety, which can either be pre-connected to a solid support, SS2, or can subsequently be selectively harvested on a solid support SS2;
  • SI and S2 are separate portions of the substrate
  • L is a detectable label
  • the mixture of the enzyme means and the conjugate is incubated to cause the enzyme means to cleave at least some of the conjugate, causing at least some of the label to be released from the conjugate.
  • the label can be released by itself, or can be released with a portion S2 of the substrate as shown in Table I. Generally not all of the label is released. Preferably the amount of label released is such that the number of label molecules in the mixture is greater than the number of En molecules in the mixture.
  • a harvesting agent such as solid support SS2 having a binding factor capable of combining with the harvestable moiety H is added for insolubilizing conjugate containing non-released label.
  • the soluble fragments containing label are then separated from the insolubilized conjugate, such as by washing.
  • the released label fragments of the conjugate remaining in solution are detected.
  • detect there is meant that either the presence of a label is determined, or the concentration of the label in solution is determined as a measure of the concentration of analyte originally present in this sample.
  • detect refers to both detecting the label itself directly, or detecting the presence of the label indirectly such as where the label is a chemilumiphore, which is detected by means of light released when an additional reagent is added to the solution containing the chemilumiphore, or the label is an enzyme that acts on other compounds to form a product which is then detected.
  • the detection method used for the label is the same (i.e., the same chemical reaction is used to detect an enzyme label), whether or not it had been released from the label conjugate.
  • a fluorophore or chromophore label would be detectable whether or not they had been cleaved from the conjugate.
  • the conjugate, which is the substrate for the label already contains an entity which is measurable by the same means, both prior to and subsequent to action of En, as used for measuring released label.
  • this invention requires separation an measurement of a fraction which becomes, or is enabled to remain, free as a result of the action of the En.
  • Variations of the method shown in Table I are available. For example, it is not necessary to include steps 3 and 4 of the method. In other words, it is not necessary to separate En from the insoluble complex, particularly whe non-specific binding is not a' problem. Thus, the entire insoluble complex can be used in step 5 for releasing the label.
  • the substrate/label conjugate can be insoluble by having H already attached to SS2. This eliminates the need for step 6. However, if the substrate/label conjugate is insoluble, it is necessary that steps 3 and 4 be employed to render th enzyme means soluble, because it is extremely difficult to obtain reasonable reaction rates between two insoluble complexes.
  • a competitive binding assay method according to the present invention is very similar to the sandwich assay method.
  • the competitive binding assay method can be structured in either of two principal versions. The first version has the following steps:
  • Steps 2 - 8 See Table I .
  • the remaining steps are the same as steps 2-8 in Table I except SSl-CA-An * -En replaces SSl-CA-An-BA-En
  • Steps 2-8 See Table I. The remaining steps are the same as steps 2-8 in Table I except SSl-An * - BA-En replaces SSl-CA-An-BA-En
  • a sample containing an analyte An in solution is combined with (i) competitive means An * -En for the analyte An, the competitive means comprising the enzyme means En and an analogue An * of the analyte An that is capable of being coupled to, and competes with the analyte for coupling to, a coupling agent CA; and (ii) a coupling agent CA capable of coupling either the analyte or its analogue to a solid support, SSI. Subsequently, the solid support SSI is added. This results in the formation of two insoluble complexes:
  • Complex (5a) is formed only if the analyte is present in the sample. The more analyte present in the sample, the less complex (4a) that is formed.
  • the analyte analogue An* can either be prebonded to En or can be coupled to an agent which provides for its rapid subsequent coupling to the enzyme means.
  • the sample containing the analyte An competes with the analogue An * , which is preattached to or harvestable on the solid phase SSI, for binding to the specific binding agent BA.
  • the binding agent BA can be prebonded to the enzyme means En, or can be coupled to an agent which provides for its rapid subsequent coupling to the enzyme means. This leads to the formation of the insoluble complexes: SSI - An * - BA - En (4b) and, when An is present in the sample, the soluble complex:
  • the addition of the harvesting solid support SSI can take place at the time the principal reaction occurs. However, it is preferred that after the specific binding reaction occurs, the solid support SSI is added to harvest and insolubilize formed complexes containing ' , including any En present in the formed complexes.
  • Steps 2-8 for the competitive binding assay method are substantially identical to steps 2-8 of the sandwich assay method, respectively, except that the enzyme- containing complex used is that of structure (4a) or (4b) rather than that of structure (2a), (2b), (2c), or (2d).
  • the enzyme- containing complex used is that of structure (4a) or (4b) rather than that of structure (2a), (2b), (2c), or (2d).
  • the amount of label detected is inversely proportional to the amount of analyte originally present in the sample.
  • the more analyte that was originally present in the sample the lower the amount of analogue and enzyme means that are bound to the first solid support, and thus the less enzyme available for releasing label from the substrate/label conjugate.
  • Antigens, antibodies, and haptens can be measured, either quantitatively or qualitatively.
  • Antigens include agents of infectious disease, such as viruses, fungi, bacteria, and toxins, allergens, tumor markers, lymphokines and cytokines, and cell surface markers.
  • Haptens can include drugs and steroids, among others.
  • DNA and RNA sequences for which a complementary sequence is available can be measured, and a receptor substance or its corresponding ligand can be measured.
  • receptor substances include steroid hormone receptors (such as estrogen receptors), lymphokine receptors (such as interleukin-2 and epidermal growth factor receptors), catecholamine hormone receptors (such as epinephrine and dopamine receptors), peptide hormone receptors (such as insulin, growth hormone, and thyroid stimulating hormone receptors), receptors for therapeutic drugs and drugs of abuse, and histamine receptors.
  • steroid hormone receptors such as estrogen receptors
  • lymphokine receptors such as interleukin-2 and epidermal growth factor receptors
  • catecholamine hormone receptors such as epinephrine and dopamine receptors
  • peptide hormone receptors such as insulin, growth hormone, and thyroid stimulating hormone receptors
  • receptors for therapeutic drugs and drugs of abuse and histamine receptors.
  • ligands corresponding to these receptor substances are, respectively, estrogen, IL-2, EGF, epinephrine, dopamine, insulin, HGH, TSH, morphine, and histamine.
  • Solid supports SSI and SS2 for the method of this invention can be those solid supports conventionally used in specific binding assays, including carbohydrate-based polymers (such as celluloses, agaroses, dextran and their derivatives that have hydroxyl groups amino groups or aldehyde groups available for binding) ; silica-based materials (including glass, quartz, and solid gels); polyamide polymers (such " as Nylon (Trademark) and polyacrylamides) including such materials as Pel 102 (Trademark); polystyrene and derivatized polystyrenes; acrylates; polyethylene; and polypropylene.
  • carbohydrate-based polymers such as celluloses, agaroses, dextran and their derivatives that have hydroxyl groups amino groups or aldehyde groups available for binding
  • silica-based materials including glass, quartz, and solid gels
  • polyamide polymers such " as Nylon (Trademark) and polyacrylamides) including such materials as Pel 102 (Trademark
  • Preferred solid supports have- a high ratio of specific to non-specific binding - i.e., they provide a large number of sites for the specific binding reagents without displaying high non ⁇ specific binding.
  • Cellulose and cellulose derivatives are among the preferred supports.
  • Cleavable bonds used for binding a coupling agent CA to a solid support SSI or to an analyte An or its analogue An * can be based on either covalent or affinity binding. It is desirable that such cleavable bonds be cleaved readily under sufficiently mild conditions that sensitive biological reagents, such as enzymes, are not destroyed.
  • Covalent linkages that can be readily cleaved include disulfide, ester, diazo, and thioester linkages.
  • Useful affinity bonds include antigen/antibody, hapten/antibody, nucleic acid hybridization, and ligand/receptor bonds.
  • affinity bond between iminobiotin and avidin satisfies these requirements, with very tight binding at a pH of 9 or above, and rapid release when the pH is reduced to 4; the range from 4 to 9 is generally well tolerated by most biological reagents.
  • Table III present representative cleavable bonds and corresponding cleaving agents.
  • Cleavable-Bond Cleaved Bv Avidin-iminobiotin Reducing the pH to 4 or lower with acid or acetate buffer. Can be aided by displacement with biotin.
  • Disulfide Reagent containing sulfhydryl group such as 2-mercapto-ethanol and dithiothreitol.
  • Oxidant such as performi ' C acid.
  • a preferred cleavable bond is avidin-iminobiotin.
  • An advantage of the avidin-iminobiotin cleavable bond is that it can be cleaved by merely adjusting the pH of the solution to 4 or lower.
  • Iminobiotin is commercially available attached to agarose from Calbiochem of La Jolla, California.
  • Another preferred cleavable bond is a disulfide bond as discussed in Schwarzberg, U.S. Patent No. 4,272,506, which is incorporated herein by reference.
  • a disulfide bond can be cleaved with reagent containing a sulfhydryl group, for example dithiothreitol.
  • binding agents BA can be used for binding an enzyme to an analyte or its analogue.
  • the linking of enzymes to antibodies and antigens is generally discussed in Scand. J. Immunol.. Vol. 8, Suppl. 7, 7-23, 1978.
  • the choice of binding agent depends upon the specific functional groups that are to be coupled.
  • analyte and analogue pairs are known for use in competitive binding assay methods.
  • Analogues of analytes are most commonly derivatives of the analyte molecules themselves, in which another functionality is attached to provide either a label or a means for attaching a label molecule.
  • an analogue of an analyte can be an analyte conjugated to a hapten, a ligand, an enzyme, a fluorescent species, or a chemiluminescent species.
  • an analogue of an analyte can be analyte-biotin conjugate, which can be used to attach a label that is conjugated to avidin.
  • the analogues can be conjugates of the analyte to various haptens, so that the label can be attached by subsequent reaction with a conjugate of the anti-hapten antibody and the label " species (e.g., enzyme, fluor, etc. )
  • species e.g., enzyme, fluor, etc.
  • label moieties can be used, including chemiluminescent, fluorescent, electron spin, and colored labels. Pyrogallol, acridinium-ester and luminol are useful chemiluminescent labels. Chemiluminescent labels can be detected with a luminescence instrument commercially available from Berthold of West Germany.
  • fluorescent labels can be used with this invention, including fluorescein, dansyl, rhodamine, fluorescamine, pyrene, acridine, methoxy-2, 4-diphenyl- 3(2H)- furanone (MDPF) , pyranine, eosin, acriflavine, safranine, derivatives of porphyrins and phthalocyanines, and anthocyanins and fluorescein mercuric acetate.
  • MDPF 4-diphenyl- 3(2H)- furanone
  • Fluorescent labels can be detected with the use of a commercially available Amin ⁇ o- Bowman fluorometer, as described in U.S. Patent 4,576,912, or with a flash fluorescence device of the type described by Noller in U.S. Patent 4,133,873.
  • Exemplary electron spin labels include nitroxide spin labels such as piperidinyl, pyrollidinyl, oxazolidinyl, maleimide, iodoacetamide, and isothiocyanate. Electron spin intensity can be measured with an electron spin response spectrometer.
  • any colored label such as a formazan dye can be used, with detection measured as light absorption in a spectrophotometer.
  • Enzyme means/substrate pairs include those listed in Table IV.
  • Proteases pepsin, papain, Proteins or oligopeptides bromelain, trypsin, chymotrypsin, pronase
  • Carbohydrases amylase starch or oligosaccharide lysozyme chitin or oligo amino sugars such as a chain of N-acetylgluccellulase groups cellulase cellulose dextranase dextran
  • the enzyme means of Table IV cleaves bonds of the corresponding substrate of Table IV, thereby releasing one or more labels pre-attached to the substrate.
  • the enzyme means can be proenzymes which are activated during the assay. Examples of suitable proenzymes, activators and substrates are described in U.S. Patent No. 4,463,090, which is incorporated by this reference.
  • the harvested enzyme- containing complex and the insolubilized substrate/label conjugate are separated from solution. This can be accomplished with such techniques as centrifugation, decanting, liquid chromatography, and filtration. A preferred method of separation is by filtration.
  • Two preferable materials commercially available for filters are tetrafluoroethylene (TFE) fluorocarbon polymer, commercially available as Teflon (Trademark) from DuPont of Delaware and tetra-fluoroethylene matrix membrane material available under the trade name "Zytex" from Chemplast Inc. of Wayne, New Jersey.
  • the washing or separation step preferably comprises washing the material on the filter with deionized water containing a buffer such as TRIS-HC1 buffer.
  • Harvesting can be accomplished by having an avidin group on a substrate as the harvestable agent and combining with a solid support such as Sepharose (Trademark) which has attached iminobiotin, as described in Hoffmann, et al. "Iminobiotin/Af inity Columns and Their Application to Retrieval of Streptavidin” , Proc. Natl. Acad. Sci. USA, Vol. '77, No. 8, pp 4666-4668-1980 and in Journal of Biochemical and Biophysical Methods. B (1986) 103-112. Biotin and other biotin derivatives can be used in place of iminobiotin for harvesting. The use of iminobiotin is preferred where it is desired subsequently to cleave the bond under mild conditions.
  • a Sepharose (Trademark) solid support can have avidin attached to harvest a substrate with attached iminobiotin.
  • Harvesting can be also be accomplished by using other specific binding pairs, such antibody/hapten or antibody/antigen, or by using ion exchangers to harvest specifically charged species or by using lectins to harvest carbohydrates (such as organic galactosides or organic glucosides or starches) .
  • the harvestable agent can be a galactose harvestable by a solid support containing lectin as the receptor; or the harvestable agent can be a hapten such as a drug, and the receptor species on the solid support can be a corresponding antibody.
  • parathyroid hormone PTH
  • Step 1 - A 50 microliter sample containing the analyte parathyroid hormone (present in a concentration of about 10 " - ⁇ M) is combined with 200 microliters of a solution containing (i) 50 mM tris-glycine, (ii) the coupling agent CA, and (iii) the binding agent BA.
  • the pH of the combination is 7.4.
  • the coupling agent CA is anti-parathyroid hormone attached to iminobiotin, and is present at a concentration of about 10 " l ° molar.
  • the coupling agent can be prepared according to the method described in "Enzyme Immunoassay for the detection of Retrovirus Antigen and Antibody” , Yolken et al. , in Manual of Clinical Laboratory Immunology. 3rd Ed (1986) - American Society for Microbiology.
  • the iminobiotin is attached to the anti-PTH by reacting the anti- PTH with an excess of the N-hydroxysuccinimide ester of iminobiotin, and subsequently removing the unreacted ester by dialysis.
  • the binding agent BA is another antibody also present at a concentration of 10" - ° molar connected in a 1:1 molar ratio to the enzyme DNAse (with staph. nuclease A) .
  • the mixture is stirred for one minute and then is incubated at 37°C for about one and one-half hours while stirring continues.
  • the formed specific binding reaction complex is then harvested by adding 200 microliters of a solution containing the solid support SSI in a buffer containing 250 mM tris and 400 mM sodium chloride at pH 9.6. The harvesting occurs in approximately ten minutes at 37°C.
  • the solid support SSI comprises cellulosic particles of about 25 to 100 micron diameter which are derivatized to avidin.
  • the particles are prepared by coupling aldehyde-containing cellulosic particles (commercially available as Acti.gel A-Superflow from Sterogene Biochemicals of San Gabriel, California), to avidin using the recommended Sterogene coupling procedure (described in Appendix A attached hereto and incorporated herein by reference) .
  • Step 2 The harvested complex of the primary specific binding reaction of step 1 is then separated from the solution by filtering on a sintered tetrafluororoethylene matrix membrane material approximately 0.005 inch thick, having a functional pore size of about 10- 20 microns.
  • a sintered tetrafluororoethylene matrix membrane material approximately 0.005 inch thick, having a functional pore size of about 10- 20 microns.
  • Such filter material is available from Chempla ⁇ t, Inc. of Wayne, New Jersey under the tradename "Zytex", catalog number A-145, E249-122 as set forth in Che plast publication number C-100-10M680N.
  • the filter is then washed three times with one milliliter portions of a buffer containing 20 mM TRIS and 400 mM NaCl at pH 9.6.
  • Steps 3 and 4 One milliliter of an acetate buffer containing 125 mM sodium acetate and acetic acid, and having a pH of 4, is then added to the filter to cleave the avidin - iminobiotin link and to wash the enzyme through the filter. Pressure is applied to assure that a maximum amount of the released enzyme passes through the filter.
  • the pH of the solution which passes through the filter is increased to above 9 by adding 1/2 ml of 750 mM phosphate buffer at pH 9.6.
  • Step 5 Cellulosic solid particles containing a substrate label conjugate are added to the solution.
  • the particles consist of avidin derivatized particles of the type described in Step 1, to which a biotinylated synthetic oligonucleotide has been added.
  • Biotin is attached to the terminal adenosine, and each oligonucleotide contains at least one fluorescein label, L, attached to one of the cytosines.
  • step 6 to insolubilize unreleased label is not required.
  • Steps 7 and 8 - The solution is then pressure forced through a Zytex (Trademark) filter of pore size 10 to 20 microns into a flow cell of a fluorimeter.
  • a flash source is applied to induce fluorescence of the fluorescein.
  • the fluorescence is detected by a photo-multiplier detector in the fluorimeter.
  • Another advantage of the present invention is that substantially none of the substrate of the conjugate/label substrate is present when measurement is made on the released label. Thus, possible interference of the unreacted substrate on the measurement is eliminated.
  • Another advantage of the present invention is that it can provide rapid assays of analytes present in low quantities. For example, it is believed that an automated assay method according to the present invention can accurately detect an analyte present in a concentration as low as 10 " l 3 molar in about 1 to 2 hours.
  • the present invention also helps overcome problems associated with non-specific binding. Further, it enhances the sensitivity of fluorometric enzyme immunoassay methods. The technique also enhances the sensitivity of chemiluminescent methods.
  • the enzyme probe be able to convert a non-detectable label to a detectable one; for example, a fluorogenic compound to a fluorescent one or a non-colored compound to a colored one.
  • the labels can be already colored, fluorescent, chemiluminescent, or have electron spin. Detection does not require the conversion of a non-detectable label to a detectable label, such as conversion of a fluorogenic substance into a fluorescent one or of a non-colored label into a colored one.
  • the enzyme means acts to release the label or labels from the backbone, thereby making them accessible to measurement.
  • the label can be coupled to the substrate by extremely stable bonds because the technique is not dependent on breaking the bond coupling the label to the rest of the substrate, but rather cleaving one portion of the substrate containing the label from the remainder of the substrate as a result of specific enzymatic reaction. This assists in overcoming high background signals that result from prior art techniques where a label had to be undetectable, even though present, before becoming converted into a detectable form.
  • the present invention has been described in considerable detail with reference to certain versions thereof, other versions are possible.
  • the present invention can improve the sensitivity of all enzyme specific binding assays, including those using chromophoric and electron spin detectable labels.
  • the invention can also improve enzyme specific binding assays in which a second enzyme means is the secondary label.
  • the cleavable bond can be a bond other than those specific embodiments described herein.
  • the cleavable bond can also be used in a variety of locations in the primary specific binding reaction complex other than the locations described above.
  • analytes which can be detected by the method of this invention are not limited to the examples discussed herein, but can include any analyte which can be detected by a specific binding assay. Therefore the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained herein.

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Abstract

Des méthodes de titrages par liaison spécifique, permettant de mesurer la concentration d'un analyte dans un échantillon, consistent à former un complexe comprenant un support solide, l'analyte ou un analogue de l'analyte, et une enzyme. On utilise l'enzyme pour cliver des marques non isotropiques directement détectables à partir d'un conjugué substrat/marque, afin de produire une pluralité de marques libérées dans l'état libre, que l'on sépare ensuite du conjugué contenant la marque non libérée. On détecte les marques libérées pour mesurer la concentration de l'analyte dans l'échantillon. L'analyte ou analogue est de préférence fixé au support solide par une liaison clivable, de sorte que l'on peut séparer l'enzyme du support solide pour l'utiliser dans le clivage enzymatique de marques à partir du conjugué. Cette version préférée réduit l'effet désavantageux d'une liaison non spécifique sur la sensibilité et la précision du titrage.
EP19890907479 1988-06-27 1989-05-31 Methodes de titrage par liaison specifique Withdrawn EP0383860A1 (fr)

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DE69132567D1 (de) * 1990-10-15 2001-05-03 Tecnogen Scpa Verfahren zur Feststellung von proteolitischen Wirkungen und/oder deren Inhibitoren
DE4217474A1 (de) * 1992-05-27 1993-12-02 Boehringer Mannheim Gmbh Verfahren und Mittel zur Bestimmung eines Analyts
GB9622524D0 (en) 1996-10-29 1997-01-08 London Biotechnology Ltd Enzyme labels for assays
EP1437594B1 (fr) 2001-09-19 2011-01-12 Sekisui Medical Co., Ltd. Polymere luminescent utilisable dans des bioessais
WO2019213583A1 (fr) * 2018-05-04 2019-11-07 Abbott Laboratories Procédé d'échantillonnage séquentiel pour améliorer la sensibilité et la cinétique de dosage immunologique d'échantillons de faible volume

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US4331590A (en) * 1978-03-13 1982-05-25 Miles Laboratories, Inc. β-Galactosyl-umbelliferone-labeled protein and polypeptide conjugates
US4289748A (en) * 1979-05-31 1981-09-15 United States Of America Ultrasensitive enzymatic radioimmunoassay method
US4272506A (en) * 1979-08-31 1981-06-09 Syva Company Purification of reagents by disulfide immobilization
EP0040728A1 (fr) * 1980-05-19 1981-12-02 Pharmacia Diagnostics Ab Méthodes d'essai comportant des réactions d'affinité biospécifique

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