EP1977253A2 - Dosage immunologique de l'homocysteine - Google Patents

Dosage immunologique de l'homocysteine

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Publication number
EP1977253A2
EP1977253A2 EP06848972A EP06848972A EP1977253A2 EP 1977253 A2 EP1977253 A2 EP 1977253A2 EP 06848972 A EP06848972 A EP 06848972A EP 06848972 A EP06848972 A EP 06848972A EP 1977253 A2 EP1977253 A2 EP 1977253A2
Authority
EP
European Patent Office
Prior art keywords
homocysteine
antibody
polyanion
immunoassay
sample
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.)
Withdrawn
Application number
EP06848972A
Other languages
German (de)
English (en)
Inventor
Toru Yoshimura
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of EP1977253A2 publication Critical patent/EP1977253A2/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine

Definitions

  • the present invention relates generally among other things to a system for the detection of homocysteine using an immunoassay.
  • the invention relates among other things to diagnostic tests, methods of use, and kits related to the assessment of homocysteine levels in a biological sample.
  • diagnostic tests, methods of use, and kits related to the assessment of homocysteine levels in a biological sample.
  • kits related to the assessment of homocysteine levels in a biological sample.
  • the influence of inhibiting substances in the biological sample is eliminated by inclusion in the assay of a polyanion.
  • the homocysteine concentration is measured after a pretreatment step comprising a first stage where homocysteine bound to components in blood by a disulfide bond is dissociated, and a second stage where the dissociated homocysteine is reacted with an enzyme (S- adenosylhomocysteine hydrolase) together with an auxiliary substrate (adenosine) so as to convert the homocysteine into a form capable of being measured by immunoassay.
  • an enzyme S- adenosylhomocysteine hydrolase
  • auxiliary substrate adenosine
  • HPLC high performance liquid chromatography
  • MS mass spectral analysis
  • a treatment method for use in conventional and automated immunoassays.
  • the biological sample is directly diluted or the volume of the reaction solution is increased.
  • Such a treatment method is disadvantageous in that it requires increased labor for the measurement of homocysteine and results in an unnecessary decrease in sensitivity due to the dilution.
  • the dilution step causes a decrease in the speed of sample processing as well as an inability of some of the measuring apparatuses to handle the increased volume of the reaction solution.
  • the present description provides an improvement of a immunoassay of a biological sample (e.g., an assay of a factor for cardiovascular disease, including a homocysteine immunoassay), characterized in that the sample is reacted with antibody in the presence of an extrinsic polyanion.
  • the polyanion is selected from the group consisting of heparin, polyacrylic acid, and dextran sulfate.
  • the polyanion concentration ranges from about 1 ⁇ g/mL to about 100 mg/mL.
  • the method of the invention can be employed for an immunoassay (e.g., an assay of a factor for cardiovascular disease, including a homocysteine immunoassay) using any sort of appropriate immunoreaction carried out on any appropriate instrument.
  • the immunoassay comprises a competitive immunoassay.
  • the immunoassay is carried out using an automated measuring apparatus.
  • the immunoassay comprises a sandwich assay.
  • the invention thus provides a method for assaying a biological sample for an analyte of interest (e.g., a cardiovascular antigen such as homocysteine), optionally wherein the method comprises:
  • an analyte of interest e.g., a cardiovascular antigen such as homocysteine
  • obtaining a biological sample from a subject e.g., from a human subject
  • the reaction of the antibody with analyte in the presence of polyanion is done where the polyanion is either added before, during, or after the reaction of the antibody with analyte. In one embodiment, polyanion is added either before or during the reaction of the antibody with analyte.
  • kits to be used for the immunoassay according to by the invention e.g., an assay of a factor for cardiovascular disease, including a homocysteine immunoassay
  • the kit comprises a polyanion.
  • FIG. 1 is a graph of changes in signal intensity caused by addition of the polyanion heparin in a measuring system for homocysteine, as described in Example 1.
  • the ordinate shows the measured signal whereas the abscissa shows the concentration of the added heparin in the reaction ( ⁇ g/mL).
  • FIG. 2 is a graph of the ratio of the concentration of homocysteine in the absence of the polyanion heparin as compared to the known value measured using a chemiluminescence automated measuring apparatus, as described in Example 2.
  • the ordinate shows the measured values for known values (%) whereas the abscissa shows known homocysteine concentrations ( ⁇ M).
  • FIG. 3 is a graph of the ratio of the concentration of homocysteine in the presence of the polyanion heparin as compared to the known value measured using a chemiluminescence automated measuring apparatus, as described in Example 2.
  • the ordinate shows measured values for known values (%) whereas the abscissa shows known homocysteine concentrations ( ⁇ M).
  • FIG. 4 is a bar chart showing the influence of polyanion on the dissociation rate where the apparent homocysteine concentration is from a "high" concentration sample group and a "normal” concentration sample group, as described in Example 3.
  • the ordinate shows dissociation rate whereas the abscissa shows the amount of polyanion added.
  • Bars (left to right): (a) no polyanion added; (b) 4.2 ⁇ g/mL of heparin; (c) 14 ⁇ g/mL of heparin; (d) 42 ⁇ g/mL of heparin; (e) 84 ⁇ g/mL of heparin; (f) 420 ⁇ g/mL of heparin; (g) 420 ⁇ g/mL of dextran sulfate; (h) 42 ⁇ g/mL of polyacrylic acid; (i) 420 ⁇ g/mL of polyacrylic acid; (j) 2.8 rag/raL of gelatin; and (k) 281 ⁇ g/mL of bovine ⁇ -globulin.
  • the present description relates to a method for improving an immunoassay for by the addition of a polyanion.
  • a substance which is the same as or similar to polyanion in terms of either structure of function appears to be causing inhibition of the immunoreaction in a homocysteine immunoassay, and that variations in the amount of this polyanion-like substance contained in a sample result in dispersion in immunoassayed values of homocysteine.
  • the description herein provides a method and means for substantially reducing or eliminating the influence of the immunoreaction inhibiting substance by adding to the immunoassay a sufficient amount of a polyanion.
  • the present invention thus provides, among other things, diagnostic tests, methods of use, and kits for the assessment of a cardiovascular factor such as homocysteine.
  • samples or “biological samples” that can be assayed using the methods of the present invention include biological fluids, such as whole blood, serum, plasma, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural effusion, urine, as well as tumor tissue or any other bodily constituent or any tissue culture supernatant that could contain the analyte of interest.
  • biological fluids such as whole blood, serum, plasma, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural effusion, urine, as well as tumor tissue or any other bodily constituent or any tissue culture supernatant that could contain the analyte of interest.
  • Preferred biological samples in an immunoassay of homocysteine are further described below.
  • analyte refers to the substance to be detected, which may be present in the sample (i.e., the biological sample).
  • the analyte can be any substance for which there exists a naturally occurring specific binding partner or for which a specific binding partner can be prepared.
  • an analyte is a substance that can bind to one or more specific binding partners in an immunoassay.
  • an analyte as described herein is an endogenous antigen, including but not limited to homocysteine.
  • binding partner is a member of a binding pair, i.e., a pair of molecules wherein one of the molecules binds to the second molecule. Binding partners that bind specifically are termed "specific binding partners.” In addition to the antigen and antibody binding partners commonly used in immunoassays, other specific binding partners can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding partners can include partner(s) that is/are analog(s) of the original specific binding partner, for example, an analyte-analog. Immunoreactive specific binding partners include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal, and complexes thereof, including those formed by recombinant DNA methods.
  • epitope refers to a site(s) on any molecule that is recognized and is capable of binding to a complementary site(s) on its specific binding partner.
  • the molecule and specific binding partner are part of a specific binding pair.
  • an epitope can be a polypeptide, protein, hapten, carbohydrate antigen (such as, but not limited to, glycolipids, glycoproteins or lipopolysaccharides) or polysaccharide and its specific binding partner, can be, but is not limited to, an antibody, e.g., an autoantibody.
  • an epitope is contained within a larger antigenic fragment (i.e., region or fragment capable of binding an antibody) and refers to the precise residues known to contact the specific binding partner. It is possible for an antigenic fragment to contain more than one epitope.
  • telomere binding pair e.g., an antigen and antibody
  • telomere binding pair e.g., an antigen and antibody
  • analyte e.g., an endogeneous antigen such as homocysteine
  • Antibodies or antibody fragments that specifically bind to an analyte can be identified, for example, by diagnostic immunoassays (e.g., radioimmunoassays ("RIA") and enzyme-linked immunosorbent assays ("ELISAs") (See, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989)), BIAcore® (Sweden), KinExA® (Kinetic Exclusion Assay, available from Sapidyne Instruments (Boise, Idaho)) or other techniques known to those of skill in the art.
  • diagnostic immunoassays e.g., radioimmunoassays ("RIA") and enzyme-linked immunosorbent assays ("ELISAs”)
  • BIAcore® Sweden
  • KinExA® KinExA® (Kinetic Exclusion Assay, available from Sapidyne Instruments (Boise, Idaho)
  • binding preference e.g., affinity
  • the binding preference for the target molecule/sequence is at least 2-fold, more preferably at least 5-fold, and most preferably at least 10- or 20-fold over a non-specific target molecule (e.g. a randomly generated molecule lacking the specifically recognized site(s)).
  • a “solid phase,” as used herein, refers to any material that is insoluble, or can be made insoluble by a subsequent reaction.
  • the solid phase can be chosen for its intrinsic ability to attract and immobilize a capture agent.
  • the solid phase can have affixed thereto a linking agent that has the ability to attract and immobilize the capture agent.
  • the linking agent can, for example, include a charged substance that is oppositely charged with respect to the capture agent itself or to a charged substance conjugated to the capture agent.
  • the linking agent can be any binding partner (preferably specific) that is immobilized on (attached to) the solid phase and that has the ability to immobilize the capture agent through a binding reaction.
  • the linking agent enables the indirect binding of the capture agent to a solid phase material before the performance of the assay or during the performance of the assay.
  • the solid phase can, for example, be plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon, including, for example, a test tube, microtiter well, sheet, bead, microparticle, chip, and other configurations known to those of ordinary skill in the art.
  • microparticle refers to a small particle that is recoverable by ultracentrifugation. Microparticles typically have an average diameter on the order of about 1 micron or less.
  • Capture agent is used herein to refer to a binding partner that binds to analyte, preferably specifically. Capture agents can be attached to a solid phase. As used herein, the binding of a solid phase-affixed capture agent to analyte forms a "solid phase-affixed complex.”
  • labeled detection agent is used herein to refer to a binding partner that binds to analyte, preferably specifically, and is labeled with a detectable label or becomes labeled with a detectable label during use in an assay.
  • a “detectable label” includes a moiety that is detectable or that can be rendered detectable.
  • a "direct label” is a detectable label that is attached, by any means, to the detection agent.
  • an "indirect label” is a detectable label that specifically binds the detection agent.
  • an indirect label includes a moiety that is the specific binding partner of a moiety of the detection agent.
  • Biotin and avidin are examples of such moieties that are employed, for example, by contacting a biotinylated antibody with labeled avidin to produce an indirectly labeled antibody.
  • the term "indicator reagent” refers to any agent that is contacted with a label to produce a detectable signal.
  • an antibody labeled with an enzyme can be contacted with a substrate (the indicator reagent) to produce a detectable signal, such as a colored reaction product.
  • an "antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. This term encompasses polyclonal antibodies, monoclonal antibodies, and fragments thereof, as well as molecules engineered from immunoglobulin gene sequences.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50 - 70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • Antibodies exist as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab')2, a dimer of Fab which itself is a light chain joined to VH-CHl by a disulfide bond. The F(ab')2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into a Fab' monomer.
  • the Fab' monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N. Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
  • Preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (sFv or scFv), in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • the single chain Fv antibody is a covalently linked VH- VL heterodimer which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker. Huston, et al.
  • VH and VL are connected to each as a single polypeptide chain, the VH and VL domains associate non-covalently.
  • the scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three dimensional structure substantially similar to the structure of an antigen- binding site are known to those of skill in the art (see, e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778).
  • the term "about” refers to approximately a +/-10% variation from the stated value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
  • the present invention provides among other things an immunoassay of a biological sample, including an immunoassay of a cardiovascular factor, e.g., homocysteine, present in the sample.
  • the method comprises inclusion of a polyanion in the immunoassay (e.g., a homocysteine immunoassay) in the reaction of the biological sample with an antibody.
  • a polyanion in the immunoassay e.g., a homocysteine immunoassay
  • it is possible to reduce or eliminate the influence of inhibiting substances existing in a sample without substantial dilution of the sample, or without using a large amount of an assay buffer solution, at the same time giving a highly sensitive and highly reliable measured value of homocysteine in a simple and convenient manner. This is particularly advantageous where the immunoassay (e.g., the homocysteine immunoassay) is fully automated and a large amount of sample is to be treated within a short time.
  • the immunoassay employed in the method of the present invention can be any immunoassay so long as it is a detection (e.g., quantitative) method that utilizes an antigen-antibody reaction. Accordingly, it can be any of the methods routinely employed for immunoassay including but not limited to a competitive method and a non-competitive method such as a sandwich method.
  • the assay methods of the invention are generally carried out on samples derived from an animal, preferably a mammal, and more preferably a human. These methods can be carried out on samples from asymptomatic subjects or subjects with one or more symptoms of disease.
  • the methods of the invention can be carried out using any sample that may contain analyte of interest, e.g., that may contain homocysteine.
  • Convenient samples include, for example, blood, serum, and plasma.
  • the biological sample which is an object for the measurement of homocysteine in the immunoassay of the present invention can be any biological sample so long as it is a liquid sample derived from a living organism such as a body fluid or a tissue extract and, usually, it is preferred to use serum, plasma or urine.
  • the sample may be pretreated, as necessary or desired, by dilution in an appropriate buffer solution or other solution, or optionally may be concentrated.
  • an appropriate buffer solution or other solution or optionally may be concentrated.
  • homocysteine immunoassay since many homocysteines bind to other thiol or protein such as albumin by means of a disulfide bond in a biological sample, it is typical for the measurement of total homocysteine in plasma, urine, and other samples to subject the sample to pretreatment with a reducing agent such as dithiothreitol (DTT).
  • DTT dithiothreitol
  • homocysteine is subjected to an enzymatic treatment or the like to be converted into a molecule which can be recognized by antibody.
  • the enzyme and any auxiliary substrate used in such a pretreatment step can be any appropriate reagents so long as they are enzyme and auxiliary substrate which are able to convert homocysteine into a molecule which is can be measured immunologically.
  • S-adenosylhomocysteine hydrolase as an enzyme and adenosine as an auxiliary substrate. In that case, homocysteine is converted to S-adenosylhomocysteine and is subjected to an immunoassay.
  • An auxiliary substrate such as adenosine preferably is charged in a solution of a reducing agent, but optionally ca be added to any reagent so long as it is a reagent which can be added to a pretreated solution or during the first reaction without deleteriously impacting the reaction.
  • a polyanion is "exogenous" in the sense that it typically is added to an immunoreaction, as described herein.
  • the optimum amount of the polyanion used according to the present invention varies depending upon the type of the polyanion employed, optionally not less than about 1 ⁇ g/mL of polyanion is added during the reaction with a sample.
  • the serum amount in the reaction solution is from about 6% to about 20%. optionally about 10%.
  • Use of not less than about 1 ⁇ g/mL of polyanion typically provides that the influence of the inhibiting substances is substantially reduced, if not completely eliminated.
  • the optimum polyanion concentration is, therefore, within a range of from about 1 ⁇ g/mL to about 100 mg/mL (e.g., from about 1 ⁇ g/mL to about 100 ⁇ g/mL, from about 100 ⁇ g/mL to about 100 mg/mL, or from about 50 ⁇ g/mL to about 50 mg/mL) during the reaction of the antibody with the biological sample, in an immunoassay in which the serum or plasma amount in the reaction solution is about 10%.
  • polyanion coexists during the reaction of homocysteine in the sample with the antibody.
  • Any route may be employed for addition of the polyanion.
  • polyanion may be added to any reagent so long as it is a reagent which participates and does not interfere with the reaction of homocysteine with antibody.
  • exogenous polyanion may be added to any solution such as solid-phase antibody solution, labeled solution, assay buffer solution, pretreatment solution, and the like.
  • Exemplary solutions include but are not limited to: Tris buffer; phosphate buffer; borate buffer; Good's buffer; SSC buffer; TBE buffer; TAE buffer; and any buffer that is routinely employed in an immunoassay.
  • a "polyanion” is a molecule in which the anion is present in multivalent form, i.e., is in more in one molecule such that there is a valence of three or more, and optionally is tetravalent. Where the anion existing in one molecule is more than decavalent (i.e., has a valence greater than ten) and the molecular weight is several hundred or more, the molecule shows a significant property as polyanion.
  • the upper limit of molecular weight of a polyanion employed as described herein is set within limits such that the viscosity does not deleteriously impact the measuring system (i.e., immunoassay).
  • a molecular weight of several hundred thousand to several million is the upper limit for the polyanion.
  • any type may be used so long as it meets the aforementioned definition.
  • Suitable r polyanions include but are not limited to: polyacrylic acid where the carboxyl group is present in a multivalent state in a polymer chain of carbon, and substances similar thereto; dextran sulfate where the polysaccharide is substituted with a sulfate group; heparin; heparin sulfate; poly(methyl methacrylate) (PMMA); poly(vinylsulfonic acid) (PVSA); poly-L-aspartic acid; and carboxymethyl cellulose (CMC).
  • Other polyanions that optionally can be employed include but are not limited to chondroitan sulfate, hyaluronic acid, dermatan sulfate, and dextran sulfate.
  • Such a polyanion optionally can be used in the form of a salt, e.g., sodium salt, lithium salt, or other similar salt.
  • the polyanion used can be a sole polyanion, or can be a mixture of different types of polyanions (e.g., a so-called "plurality" wherein the plurality optionally comprises between two and five, added either simultaneously or sequentially).
  • the antibody used in the present invention preferably is an antibody that is able to recognize analyte of interest (e.g., homocysteine).
  • analyte of interest e.g., homocysteine
  • the homocysteine is first subjected to a conversion treatment so as to render it capable of being recognized by an antibody.
  • an anti-S-adenosylhomocysteine antibody is used.
  • Such an antibody can be any polyclonal antibody or monoclonal antibody.
  • the antibody can be not only a complete antibody but also any type of an antibody fragment including Fab, Fab', and F(ab') 2> or antibody fragment where only the active site is taken out by means of genetic recombination, so long the antibody provides a specific activity (i.e., a reactivity). Scoring
  • the immunoassays according to the invention optimally are scored in accordance with standard practice and, optionally include the use of positive and/or negative controls and/or or standards (calibrators) containing known concentrations of antibodies to the analyte of interest.
  • the level of analyte e.g., homocysteine
  • a control level or control range which can be determined when the assay is carried out or, more conveniently, can be predetermined.
  • reagents used in the immunoassay of the present invention e.g., reagents such as antibody, labeled substance, reducing sugar and enzyme
  • substances which are routinely used in homocysteine immunoassays can likewise be employed in the assay as described herein according to their ordinary and customary conditions for use. This is further expanded upon below.
  • the immunoassay methods of the invention can be carried out in any of a wide variety of formats. These formats merely are modified as described above to include polyanion in the reacting step of analyte antigen (e.g., homocysteine) with antibody.
  • analyte antigen e.g., homocysteine
  • an immunoassay method of the invention can be performed by contacting a biological sample suspected of containing an analyte of interest, with an antibody reactive therewith in the presence of polyanion, and under conditions sufficient for binding of the antibody to any analyte present in the biological sample.
  • Analyte is detected/quantitated by detecting complex(es) comprising the analyte antigen bound to the reactive antibody.
  • Such assays can be homogeneous or heterogeneous (i.e., employing a solid phase).
  • a capture agent that binds to the analyte is typically affixed to a solid phase.
  • Analytes such as homocysteine can be measured in a non-competitive immunoassay, wherein the amount of analyte bound to antibody is positively correlated with the concentration of analyte present in the biological sample.
  • the biological sample is contacted with the antibody reactive with analyte (and which may, but need not, be affixed to a solid phase) and also contacted with another antibody that reacts with analyte so as to form a "sandwich" where the analyte is bound between two antibody reagents.
  • Analyte is detected/quantitated by detecting complex(es) comprising the antigen bound to the reactive antibodies.
  • the first antibody is affixed to a solid phase
  • binding of analyte antigen present in the biological sample to the antibody forms a solid phase-affixed complex
  • detecting comprises detecting a signal from the solid phase-affixed complex.
  • the solid phase-affixed complex is detected using a second antibody also reactive with analyte antigen and that is directly or indirectly labeled.
  • the bound entities are separated, if necessary, from free labeled antibody, typically by washing, and the signal from the bound label is detected.
  • Analyte e.g., antigen such as homocysteine
  • analyte antigen such as homocysteine
  • the biological sample is contacted with an antibody (which may, but need not, be affixed to a solid phase) and also is contacted with competing labeled (directly or indirectly) antigen.
  • This step is carried out under conditions sufficient for specific binding of the labeled antigen and analyte antigen to the antibody.
  • the labeled antigen and analyte antigen compete with each other for binding to the antibody. Accordingly, the higher the level of analyte antigen (such as homocysteine)
  • ⁇ as homocysteine in a biological sample, the lower is the binding of labeled antigen to the antibody.
  • the biological sample may be contacted with the labeled antigen and the antibody either simultaneously or sequentially, in any order.
  • binding of the analyte antigen present in the biological sample to antibody forms a solid phase-affixed complex
  • detection entails detecting a signal from the solid phase-affixed complex.
  • the bound entities are separated, if necessary, from free labeled antigen, typically by washing, and the signal from any bound label (displacing analyte antigen) is detected.
  • Capture agents useful in the immunoassay methods of the invention include those that bind to analyte antigen (e.g., homocysteine) and can be affixed to a solid phase.
  • Convenient capture agents include antibodies specific for the analyte antigen.
  • Any endogenous antigen can be used (e.g., assessed as the analyte antigen or included in a kit as a calibrator or control) in the immunoassay methods of the invention.
  • the endogenous antigen is an endogenous antigen amino acid sequence that can be derived from any organism.
  • Endogenous antigen amino acid sequences useful in the invention are generally those derived from vertebrates, preferably from birds or mammals, more preferably from animals having research or commercial value or value as pets, such as mice, rats, guinea pigs, rabbits, cats, dogs, chickens, pigs, sheep, goats, cows, horses, as well as monkeys and other primates.
  • the endogenous antigen amino acid sequence is derived from a human polypeptide.
  • the methods of the invention can employ full-length endogenous antigens or one or more fragments thereof. Fragments will generally have at least one epitope to which an antibody can bind.
  • Such fragments can have a length, e.g., of about 125, 100, 75, 50, 25, or 15 amino acids or a length that falls within a range with endpoints defined by any of these values (e.g., 15-125, 25-100, 50-75, 15-100, etc.).
  • the endogenous antigen amino acid sequence can be a wild-type amino acid sequence or an amino acid sequence variant of the corresponding region of a wild-type polypeptide.
  • endogenous antigens include a wild-type endogenous antigen amino acid sequence or an endogenous antigen amino acid sequence containing conservative amino acid substitutions, as defined above.
  • Endogenous antigens useful in the invention can include other amino acid sequences, including those from heterologous proteins. Accordingly, the invention encompasses fusion polypeptides in which an endogenous antigen amino acid sequence is fused, at either or both ends, to amino acid sequence(s) from one or more heterologous proteins. Examples of additional amino acid sequences often incorporated into proteins of interest include a signal sequence, which facilitates purification of the protein, and an epitope tag, which can be used for immunological detection or affinity purification.
  • Endogenous antigen polypeptides according to the invention can be synthesized (e.g., for use as calibrators or controls in the kits according to the invention) using methods known in the art, such as for example exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, and classical solution synthesis. See, e.g., Merrifield, J. Am. Chem. Soc, 85:2149 (1963). For a description of solid phase peptide synthesis procedures, see John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
  • Endogenous antigen polypeptides can also produced using recombinant techniques. Tn certain embodiments, the sequence of an endogenous antigen coding region is used as a guide to design a synthetic nucleic acid molecule encoding the endogenous antigen polypeptide that can be incorporated an expression vector. Methods for constructing synthetic genes are well-known to those of skill in the art. See, e.g., Dennis, M. S., Carter, P. and Lazarus, R. A., Proteins: Struct. Funct. Genet., 15:312-321 (1993).
  • the expression vector includes one or more control sequences capable of effecting and/or enhancing the expression of an operably linked polypeptide coding sequence.
  • Control sequences that are suitable for expression in prokaryotes include a promoter sequence, an operator sequence, and a ribosome binding site.
  • Control sequences for expression in eukaryotic cells include a promoter, an enhancer, and a transcription termination sequence (i.e., a polyadenylation signal).
  • An expression vector according to the invention can also include other sequences, such as, for example, nucleic acid sequences encoding a signal sequence or an amplifiable gene.
  • a signal sequence can direct the secretion of a polypeptide fused thereto from a cell expressing the protein.
  • nucleic acid encoding a signal sequence is linked to a polypeptide coding sequence so as to preserve the reading frame of the polypeptide coding sequence.
  • a wide variety of host cells are available for propagation and/or expression of vectors. Examples include prokaryotic cells (such as E. coli and strains of Bacillus, Pseudomonas, and other bacteria), yeast or other fungal cells (including S. cerevesiae and P. pastoris), insect cells, plant cells, and phage, as well as higher eukaryotic cells (such as human embryonic kidney cells and other mammalian cells).
  • Vectors expressing endogenous cardiovascular antigen can be introduced into a host cell by any convenient method, which will vary depending on the vector- host system employed. Generally, a vector is introduced into a host cell by transformation or infection (also known as "transfection") with a virus (e.g., phage) bearing the vector.
  • the host cell is a prokaryotic cell (or other cell having a cell wall)
  • convenient transformation methods include the calcium treatment method described by Cohen, et al. (1972) Proc. Natl. Acad. Sci., USA, 69:2110-14.
  • the vector can be introduced into the host cell by transfection.
  • Yeast cells can be transformed using polyethylene glycol, for example, as taught by Hinnen (1978) Proc. Natl. Acad. Sci, USA, 75:1929-33.
  • Mammalian cells are conveniently transformed using the calcium phosphate precipitation method described by Graham, et al.
  • the culture conditions should allow transcription, translation, and protein transport between cellular compartments.
  • Factors that affect these processes are well-known and include, for example, DNA/RNA copy number; factors that stabilize DNA; nutrients, supplements, and transcriptional inducers or repressors present in the culture medium; temperature, pH and osmolality of the culture; and cell density.
  • the adjustment of these factors to promote expression in a particular vector-host cell system is within the level of skill in the art. Principles and practical techniques for maximizing the productivity of in vitro mammalian cell cultures, for example, can be found in Mammalian Cell Biotechnology: a Practical Approach (Butler ed., IRL Press (1991).
  • any of a number of well-known techniques for large- or small-scale production of proteins can be employed in producing the polypeptides of the invention. These include, but are not limited to, the use of a shaken flask, a fiuidized bed bioreactor, a roller bottle culture system, and a stirred tank biorcactor system. Cell culture can be carried out in a batch, fed-batch, or continuous mode.
  • a polypeptide including a signal sequence can be recovered from the culture medium or the periplasm. Polypeptides can also be expressed intracellularly and recovered from cell lysates.
  • the expressed polypeptides can be purified from culture medium or a cell lysate by any method capable of separating the polypeptide from one or more components of the host cell or culture medium. Typically, the polypeptide is separated from host cell and/or culture medium components that would interfere with the intended use of the polypeptide.
  • the culture medium or cell lysate is usually centrifuged or filtered to remove cellular debris. The supernatant is then typically concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification.
  • polypeptide can then be further purified using well-known techniques. The technique chosen will vary depending on the properties of the expressed polypeptide. If, for example, the polypeptide is expressed as a fusion protein containing an epitope tag or other affinity domain, purification typically includes the use of an affinity column containing the cognate binding partner. For instance, polypeptides fused with green fluorescent protein, hemagglutinin, or FLAG epitope tags or with hexahistidinc or similar metal affinity tags can be purified by fractionation on an affinity column.
  • affinity column containing the cognate binding partner. For instance, polypeptides fused with green fluorescent protein, hemagglutinin, or FLAG epitope tags or with hexahistidinc or similar metal affinity tags can be purified by fractionation on an affinity column.
  • Antibodies useful in the immunoassay methods of the invention include polyclonal and monoclonal antibodies.
  • Polyclonal antibodies are raised by injecting (e.g., subcutaneous or intramuscular injection) an immunogen into a suitable non-human mammal (e.g., a mouse or a rabbit).
  • a suitable non-human mammal e.g., a mouse or a rabbit.
  • the immunogen should induce production of high titers of antibody with relatively high affinity for the target antigen.
  • the endogenous antigen i.e., analyte of interest
  • a carrier protein by conjugation techniques that are well known in the art.
  • Commonly used carriers include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The conjugate is then used to immunize the animal.
  • the antibodies are then obtained from blood samples taken from the animal.
  • the techniques used to produce polyclonal antibodies are extensively described in the literature (see, e.g., Methods of Enzymology, "Production of Antisera With Small Doses of Immunogen: Multiple Intradermal Injections," Langone, et al. eds. (Acad. Press, 1981)).
  • Polyclonal antibodies produced by the animals can be further purified, for example, by binding to and elution from a matrix to which the target antigen is bound.
  • Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal, as well as monoclonal, antibodies see, for example, Coligan, et al. (1991) Unit 9, Current Protocols in Immunology, Wiley Interscience.
  • mAbs monoclonal antibodies
  • the general method used for production of hybridomas secreting mAbs is well known (Kohler and Milstein (1975) Nature, 256:495). Briefly, as described by Kohler and Milstein, the technique entailed isolating lymphocytes from regional draining lymph nodes of five separate cancer patients with either melanoma, teratocarcinoma or cancer of the cervix, glioma or lung, (where samples were obtained from surgical specimens), pooling the cells, and fusing the cells with SHFP-I . Hybridomas were screened for production of antibody that bound to cancer cell lines. Confirmation of specificity among mAbs can be accomplished using routine screening techniques (such as the enzyme-linked immunosorbent assay, or "ELISA") to determine the elementary reaction pattern of the mAb of interest.
  • ELISA enzyme-linked immunosorbent assay
  • antibody encompasses antigen-binding antibody fragments, e.g., single chain antibodies (scFv or others), which can be produced/selected using phage display or yeast display technology.
  • scFv single chain antibodies
  • phage display or yeast display technology The ability to express antibody fragments on the surface of viruses that infect bacteria (bacteriophage or phage) makes it possible to isolate a single binding antibody fragment, e.g., from a library of greater than 1010 nonbinding clones.
  • an antibody fragment gene is inserted into the gene encoding a phage surface protein (e.g., pill) and the antibody fragment-pill fusion protein is displayed on the phage surface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137).
  • a phage surface protein e.g., pill
  • phage-bearing antigen-binding antibody fragments can be separated from non- binding phage by antigen affinity chromatography (McCafferty et al. (1990) Nature, 348: 552-554). Depending on the affinity of the antibody fragment, enrichment factors of 20-fold - 1,000,000-fold are obtained for a single round of affinity selection. By infecting bacteria with the eluted phage, however, more phage can be grown and subjected to another round of selection. In this way, an enrichment of 1000-fold in one round can become 1 ,000,000-fold in two rounds of selection (McCafferty et al.
  • Human antibodies can be produced without prior immunization by displaying very large and diverse V-gene repertoires on phage (Marks et al. (1991) J. MoI. Biol. 222: 581-597).
  • natural VH and VL repertoires present in human peripheral blood lymphocytes are isolated from unimmunized donors by PCR.
  • the V-gene repertoires can be spliced together at random using
  • PCR to create a scFv gene repertoire which can be cloned into a phage vector to create a library of 30 million phage antibodies (Id.).
  • binding antibody fragments have been isolated against more than 17 different antigens, including haptens, polysaccharides, and proteins (Marks et al. (1991) J. MoI. Biol. 222: 581-597; Marks et al. (1993). Bio/Technology. 10: 779- 783; Griffiths et al. (1993) EMBO J. 12: 725-734; Clackson et al. (1991) Nature. 352: 624-628).
  • Antibodies have been produced against self proteins, including human thyroglobulin, immunoglobulin, tumor necrosis factor, and CEA (Griffiths et al. (1993) EMBO J. 12: 725-734).
  • the antibody fragments are highly specific for the antigen used for selection and have affinities in the 1 nM to 100 nM range (Marks et al. (1991) J. MoI. Biol. 222: 581-597; Griffiths et al. (1993) EMBO J. 12: 725-734). Larger phage antibody libraries result in the isolation of more antibodies of higher binding affinity to a greater proportion of antigens.
  • antibodies can be prepared by any of a number of commercial services (e.g., Berkeley Antibody Laboratories, Bethyl Laboratories, Anawa, Eurogenetec, etc.).
  • Solid Phase For embodiments of the invention that employ a solid phase as a support for the capture agent, the solid phase can be any suitable material with sufficient surface affinity to bind a capture agent.
  • Useful solid supports include: natural polymeric carbohydrates and their synthetically modified, crosslinked, or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross- linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; inorganic materials such as sulfates or carbonates of alkaline earth metals and
  • Nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents including monoclonal antibodies. Nylon also possesses similar characteristics and also is suitable.
  • Preferred solid phase materials for flow-through assay devices include filter paper such as a porous fiberglass material or other fiber matrix materials.
  • the thickness of such material is not critical and will be a matter of choice, largely based upon the properties of the biological sample or analyte being assayed, such as the fluidity of the biological sample.
  • the solid phase can constitute microparticles.
  • Microparticles useful in the invention can be selected by one skilled in the art from any suitable type of particulate material and include those composed of polystyrene, polymethylacrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar materials. Further, the microparticles can be magnetic or paramagnetic microparticles, so as to facilitate manipulation of the microparticle within a magnetic field.
  • Microparticles can be suspended in the mixture of soluble reagents and biological sample or can be retained and immobilized by a support material. In the latter case, the microparticles on or in the support material are not capable of substantial movement to positions elsewhere within the support material.
  • the microparticles can be separated from suspension in the mixture of soluble reagents and biological sample by sedimentation or centrifugation.
  • the microparticles are magnetic or paramagnetic the microparticles can be separated from suspension in the mixture of soluble reagents and biological sample by a magnetic field.
  • the methods of the present invention can be adapted for use in systems that utilize microparticle technology including automated and semi-automated systems wherein the solid phase comprises a microparticle.
  • Such systems include those described in pending U.S. App. No. 425,651 and U.S. Patent No. 5,089,424, which correspond to published EPO App. Nos. EP 0 425 633 and EP 0 424 634, respectively, and U.S. Patent No. 5,006,309.
  • the solid phase includes one or more electrodes.
  • Capture agent(s) can be affixed, directly or indirectly, to the electrode(s).
  • capture agents can be affixed to magnetic or paramagnetic microparticles, which are then positioned in the vicinity of the electrode surface using a magnet.
  • Systems in which one or more electrodes serve as the solid phase are useful where detection is based on electrochemical interactions. Exemplary systems of this type are described, for example, in U.S. Patent No. 6,887,714 (issued May 3, 2005). The basic method is described further below with respect to electrochemical detection.
  • the capture agent can be attached to the solid phase by adsorption, where it is retained by hydrophobic forces.
  • the surface of the solid phase can be activated by chemical processes that cause covalent linkage of the capture agent to the support.
  • a charged substance can be coated directly onto the solid phase.
  • Ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer described in U.S. App. No. 150,278, corresponding to EP Publication No. 0326100, and U.S.App. No. 375,029 (EP Publication No. 0406473), can be employed according to the present invention to affect a fast solution-phase immunochemical reaction.
  • an immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged polyanion/immune complex and the previously treated, positively charged matrix and detected by using any of a number of signal-generating systems, including, e.g., chemiluminescent systems, as described in U.S. App. No. 921,979, corresponding to EPO Publication No. 0 273,115.
  • the surface must generally be activated prior to attaching the specific binding partner.
  • Activated silane compounds such as triethoxy amino propyl silane (available from Sigma Chemical Co., St. Louis, Mo.), triethoxy vinyl silane (Aldrich Chemical Co., Milwaukee, Wis.), and (3-mercapto- propyl)-trimethoxy silane (Sigma Chemical Co., St. Louis, Mo.) can be used to introduce reactive groups such as amino-, vinyl, and thiol, respectively.
  • Such activated surfaces can be used to link the capture directly (in the cases of amino or thiol), or the activated surface can be further reacted with linkers such as glutaraldehyde, bis (succinimidyl) suberate, SPPD 9 succinimidyl 3-[2- pyridyldithio] propionate), SMCC (succinimidyl-4-[Nmaleimidomethyl] cyclohexane-1-carboxylate), SIAB (succinimidyl [4iodoacetyl] aminobenzoate), and SMPB (succinimidyl 4-[lmaleimidophenyl] butyrate) to separate the capture agent from the surface.
  • linkers such as glutaraldehyde, bis (succinimidyl) suberate, SPPD 9 succinimidyl 3-[2- pyridyldithio] propionate), SMCC (succinimidyl-4-[N
  • Vinyl groups can be oxidized to provide a means for covalent attachment. Vinyl groups can also be used as an anchor for the polymerization of various polymers such as poly-acrylic acid, which can provide multiple attachment points for specific capture agents. Amino groups can be reacted with oxidized dextrans of various molecular weights to provide hydrophilic linkers of different size and capacity.
  • oxidizable dextrans examples include Dextran T-40 (molecular weight 40,000 daltons), Dextran T-110 (molecular weight 110,000 daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight 2,000,000 daltons) (all of which are available from Pharmacia, Piscataway, N.J.), or Ficoll (molecular weight 70,000 daltons; available from Sigma Chemical Co., St. Louis, Mo.). Additionally, polyelectrolyte interactions can be used to immobilize a specific capture agent on a solid phase using techniques and chemistries described U.S. App. No. 150,278, filed Jan. 29, 1988, and U.S. App. No.
  • solid phase Other considerations affecting the choice of solid phase include the ability to minimize non-specific binding of labeled entities and compatibility with the labeling system employed. For, example, solid phases used with fluorescent labels should have sufficiently low background fluorescence to allow signal detection. Following attachment of a specific capture agent, the surface of the solid support may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding.
  • Detectable labels suitable for use in the detection agents of the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oregon, USA), chemiluminescent compounds such as acridinium (e.g., acridinium-9-carboxamide), phenanthridinium, dioxetanes, luminol and the like, radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), catalysts such as enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta- galactosidasc and others commonly used in an ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40-80 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polys
  • Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • the label can be attached to the detection agent prior to, or during, or after contact with the biological sample.
  • so-called "direct labels” are detectable labels that are directly attached to or incorporated into detection agents prior to use in the assay. Direct labels can be attached to or incorporated into detection agents by any of a number of means well known to those of skill in the art. In contrast, so-called "indirect labels” typically bind to the detection agent at some point during the assay.
  • the indirect label binds to a moiety that is attached to or incorporated into the detection agent prior to use.
  • an antibody used as a detection agent can be biotinylated before use in an assay.
  • an avidin-cpnjugated fluorophore can bind the biotin-bearing detection agent, to provide a label that is easily detected.
  • polypeptides capable of specifically binding immunoglobulin constant regions can also be used as labels for detection antibodies.
  • These polypeptides are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non- immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally Kronval, et al. (1973) J. Immunol., I ll : 1401-1406, and Akerstrom (1985) J. Immunol., 135: 2589-2542).
  • polypeptides can thus be labeled and added to the assay mixture, where they will bind to the detection antibody, as well as to the species-specific antibody, labeling both and providing a composite signal attributable to analyte and autoantibody present in the biological sample.
  • Some labels useful in the invention may require the use of an indicator reagent to produce a detectable signal.
  • an enzyme label e.g., beta-galactosidase
  • a substrate e.g., X-gal
  • the present invention is for example applicable (e.g., adaptable) to the jointly owned commercial Abbott Point of Care (i-STAT®) electrochemical immunoassay system which performs sandwich immunoassays for several cardiac markers, including TnI, CKMB and BNP. Immunosensors and ways of operating them in single-use test devices are described in jointly owned Publication Nos. US 20140060600A1
  • i-STAT® Abbott Point of Care electrochemical immunoassay system which performs sandwich immunoassays for several cardiac markers, including TnI, CKMB and BNP.
  • Immunosensors and ways of operating them in single-use test devices are described in jointly owned Publication Nos. US
  • the solid phase can include a plurality of different capture agents, including one that captures endogenous antigen or analyte of interest (e.g., homocysteine).
  • the solid phase can have affixed thereon a plurality of antibodies, wherein each is intended to test for the presence of different analytes (e.g., homocysteine and endogenous analytes) in the biological sample.
  • the solid phase can consist of a plurality of different regions on a surface, wherein each region has a particular antibody affixed therein.
  • Multiplex formats can, but need not, employ a plurality of labels, wherein each label is used for the detection of a particular antigen.
  • each label is used for the detection of a particular antigen.
  • multiple, different analytes can be detected without using a plurality of labels where a plurality of capture agents, such as antibodies having different specificites, are affixed to the solid phase at different known locations. Because the specificity of the capture agent at each location is known, the detection of a signal at a particular location can be associated with the presence of antigen bound at that location.
  • Examples of this format include microfluidic devices and capillary arrays, containing different capture agents at different locations along a channel or capillary, respectively, and microarrays, which typically contain different capture agents arranged in a matrix of spots ("target elements") on a surface of a solid support.
  • each different capture agent can be affixed to a different electrode, which can, for example, be formed on a surface of a solid support, in a channel of a microfluidic device, or in a capillary.
  • the immunoassays as described herein can be used in kits for commercial platform immunoassays (e.g., homocysteine blood screening assays on Abbott's Prism®, AxS YM®, ARCHITECT® and/or EIA (Bead) platforms, as well as in other commercial and/or in vitro diagnostic assays.
  • commercial platform immunoassays e.g., homocysteine blood screening assays on Abbott's Prism®, AxS YM®, ARCHITECT® and/or EIA (Bead) platforms, as well as in other commercial and/or in vitro diagnostic assays.
  • test kits for assaying biological samples for analytes such as homocysteine and other endogenous antigens.
  • Test kits according to the invention include one or more reagents useful for practicing one or more immunoassays according to the invention.
  • a test kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as admixture where the compatibility of the reagents will allow.
  • the test kit can also include other materials), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or any other material useful in biological sample processing, washing, or conducting any other step of the assay.
  • a test kit includes a polyanion, wherein the polyanion is employed in the reaction of analyte antigen with antibody. If desired, this component can be included in the test kit in multiple concentrations, and/or by provision of a variety of different types of polyanions (including mixtures).
  • Kits according to the invention can include a solid phase and a capture agent affixed to the solid phase, wherein the capture agent is an antibody specific for the analyte being assessed in the biological sample.
  • the kits can additionally include a labeled detection agent.
  • the test kit includes at least one direct label, such as acridinium-9-carboxamide.
  • Test kits according to the invention can also include at least one indirect label. Tf the label employed generally requires an indicator reagent to produce a detectable signal, the test kit preferably includes one or more suitable indicator reagents.
  • the solid phase includes one or more microparticles or electrodes.
  • Test kits designed for multiplex assays conveniently contain one or more solid phases including a plurality of antibodies that are specific for a plurality of different analytes of interest (e.g., homocysteine or endogenous antigens).
  • a test kit designed for multiplex electrochemical immunoassays can contain a solid phase including a plurality of electrodes, with each electrode bearing a different antibody.
  • Test kits according to the invention preferably include instructions for carrying out one or more of the immunoassays of the invention. Instructions included in kits of the invention can be affixed to packaging material or can be included as a package insert.
  • instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions.
  • An anti-S-adenosylhomocysteine mouse monoclonal antibody (procured from Abbott Laboratories, U.S.A.) was bonded onto a magnetic fine particles modified by the addition of carboxyl group (procured from Abbott Laboratories, U.S.A.) using EDC (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Sigma Aldrich)) to give fine particles where the antibody was made into a solid phase.
  • EDC N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • the antibody in a solid phase was added to a BisTris buffer solution containing Tween 20 (manufactured by Kanto Kagaku), EDTA (sodium ethylenediaminetetraacetate) and sodium chloride to prepare a solution of fine particles of the antibody in a solid phase.
  • S-adenosylcysteine labeled with acridinium derivative was added to an MES buffer solution containing Triton X 100 (manufactured by Sigma Aldrich) to prepare a tracer solution.
  • S-adenosylhomocysteine hydrolase (procured from Axis Shield, United Kingdom) was added to a buffer solution containing 30% (by volume) of glycerol to prepare an enzyme solution.
  • DTT and adenosine were added to an aqueous solution of citric acid to prepare a reducing agent solution.
  • a sample (18 ⁇ L) was mixed with 79 ⁇ L of the enzyme solution, 50 ⁇ L of the fine particles solution of antibody in a solid phase, and 10 ⁇ L of the reducing agent solution, and the first reaction was started.
  • the following reactions were generated: (1) a bonded product of homocysteine in the sample was liberated to a free homocysteine; (2) the liberated homocysteine was converted to S-adenosylhomocysteine; and (3) the converted S- adenosylhomocysteine was bonded to the particles of antibody in a solid phase.
  • the tracer solution 50 ⁇ L was further mixed therewith and the reaction was continued for 4 minutes.
  • an emission signal was observed using an emission trigger reagent (also was exclusive for this instrument).
  • a standard curve was prepared by a logistic 4 para method using an Abbott AxSYM® homocysteine calibrator (manufactured by Abbott Japan Co., Ltd.) as a reference solution, and the homocysteine concentration was calculated based on the signal obtained from the sample, whereupon the concentration of the homocysteine in the sample was determined.
  • Example 1 The concentration of homocysteine contained in the sample was determined by prior art methods including a diluting operation using a commercially-available AxSYM® Homocysteine Assay Reagent and AxSYM® Analyzer (both manufactured by Abbott Japan Co., Ltd.).
  • This measuring method is a fluorescence polarization immunoassay where a fluorescent substance is utilized in a labeled substance and, in order to eliminate the influence of an inhibiting substance, the sample was diluted to an extent of about 300-fold upon a competitive reaction.
  • Plasma (A) in blood collected with heparin was similarly investigated and experimental results were obtained similar to those for the serum where the homocysteine concentration was apparently highly measured (•). Because of that, it is also strongly suggested that a heparin-like polyanionic substance derived from a living organism was contained in the serum where homocysteine concentration was apparently highly measured.
  • a rheumatoid factor participates as an inhibiting substance in a measuring system for homocysteine
  • concentration of rheumatoid factor was quantified for the sample used in the Examples.
  • only one among the samples where homocysteine was able to be precisely measured contained the rheumatoid factor in an amount of more than the standard value while all other samples were within a range of normal value. This suggests that a rheumatoid factor does not participate as a reaction inhibitor in an immunoassay of homocysteine.
  • FIG. 4 shows rates of dissociation in the higher sample group and the normal sample group at each of the concentrations of dextran sulfate, polyacrylic acid, gelatin and ⁇ -globulin. Each 7 and 8 samples were used as the higher and the normal sample groups, respectively.
  • Typical heparin has one anion per a molecular weight of 150 whereas in some polyanions, there are molecules in which the anions are more densely present.
  • polyacrylic acid has one anion per a molecular weight of 71.

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Abstract

Il est connu que, dans les dosages immunologiques d'homocystéine, les valeurs mesurées sont dispersées en raison de l'influence des substances d'inhibition présentes dans un échantillon biologique sur l'immunoréaction. L'invention concerne notamment un procédé selon lequel l'influence des substances d'inhibition de l'immunoréaction est supprimée ou éliminée de manière aisée et efficace ; l'invention porte également sur un kit utilisé pour la mise en oeuvre dudit procédé. L'influence des substances d'inhibition de l'immunoréaction est supprimée ou éliminée quand l'homocystéine contenue dans les échantillons biologiques est mise en réaction avec un anticorps en présence de polyanion extrinsèque.
EP06848972A 2005-12-22 2006-12-21 Dosage immunologique de l'homocysteine Withdrawn EP1977253A2 (fr)

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