EP0248038A1 - Idiotypisches antigen-konjugierungs-bindetestverfahren - Google Patents

Idiotypisches antigen-konjugierungs-bindetestverfahren

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Publication number
EP0248038A1
EP0248038A1 EP19860906648 EP86906648A EP0248038A1 EP 0248038 A1 EP0248038 A1 EP 0248038A1 EP 19860906648 EP19860906648 EP 19860906648 EP 86906648 A EP86906648 A EP 86906648A EP 0248038 A1 EP0248038 A1 EP 0248038A1
Authority
EP
European Patent Office
Prior art keywords
antibody
analyte
complex
antigen
specimen
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Withdrawn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
EP19860906648
Other languages
English (en)
French (fr)
Inventor
Miles Gerald Hossom
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.)
Murex Corp
Original Assignee
Murex Corp
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 Murex Corp filed Critical Murex Corp
Publication of EP0248038A1 publication Critical patent/EP0248038A1/de
Withdrawn legal-status Critical Current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
    • G01N33/53—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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype

Definitions

  • the method of the present invention relates to the performance of an assay for the determination of the presence or quantity of a biological substance.
  • the substance of interest is an antigen but can also be an antibody or other substance.
  • Numerous techniques have been developed to measure the presence and/or amount of antigen present in a sample of biological fluid. Certain of these involve the use of a solid phase system whereby a substance which will form an immunocompiex with the analyte is bound to an insolublized carrier or material, such as latex, cellulose, and the like. In this manner the immunocompiex when formed can be separated in its solid phase from the rest of the solution containing extraneous uncomplexed material.
  • a material which has been labeled or "tagged" with a signal generating molecule is added to the immunocompiex so that the material binds to the analyte.
  • the sample is read and the amount of signal measured is related to the amount of analyte present to which it is bound.
  • binding material assays have been developed so that less analyte is required to produce a reliably measurable signal.
  • an antigen is the analyte of interest
  • two assays have been developed wherein two different antibodies are used: one is bound to the solid support and the other is labelled. The two antibodies bind to two different anti genie determinants on the antigen. More recently, monoclonal antibodies developed for this type of use can provide much greater selectivity and specificity for a given antigen.
  • US Patent No. 4,376,110 issued to David et al., discloses a sandwich immunoassay using two monoclonal antibodies which are site specific, yet may be a product of the same or different cell lines.
  • a "sandwich,” or "two-site,” immunoassay relies upon the formation of an insolublized anti body: anti gen: labelled antibody complex.
  • the two monoclonal antibodies are designed to minimize cross-reactivity and interference with each other when binding to the antigen. However, there still remains the problem of nonspecific binding and incomplete washing away of unbound materials.
  • Some small amount of labelled antibody can remain in the reaction area by binding nonspecifically to either the reaction support surface or extraneous material in the area other than the insolublized antibody.
  • labelled compounds Since there are few methods currently available for conveniently measuring the amount of antigen or antibody directly, labelled compounds have been developed which, when attached to the analyte, emit a signal of some kind which can be measured,thereby giving an indirect quantitation of analyte.
  • labels such as radioisotopes, such as 132I; enzymes, such as alkaline phosphatase; fluorescent, such as flourescein; and luminescent, such as luciferin have been developed for use in immunoassays.
  • the primary concern with these labeled compound assay systems is the interference of background signal noise. Background noise occurs, in part, because of nonspecific binding of labeled compound to material in the sample other than the analyte.
  • US Patent No. 4,446,233 issued May 1, 1984 to Auditore-Hargreaves et al., discloses a homogeneous immunoassay using covalent hybrid antibodies.
  • the antibody contains one site specific for an analyte and one binding site specific for an indicator.
  • the unique feature of that invention is the use of a single antibody which has been designed so as to contain two distict binding sites on the same antibody, joined through inter-heavy-chain disulfide bonding.
  • the antibody is composed of two different heavy-chain light-chain half-molecules or two idiotypic determinants. The method is limited, however, by the difficulty and expense of designing and producing an antibody with two different chains.
  • the present invention remedies the deficiencies of the prior art to provide a highly sensitive and reliable method for detection of analytes.
  • the present invention relates to a method for detection or quantitation of an analyte of interest suspected of being in a specimen, comprising (a) contacting a first antibody with said specimen in such a manner that said first antibody reacts with any of the analyte present in said specimen to form a first complex; (b) contacting with any of the first complex a second antibody which recognizes and binds preferentially to the juncture of the idiotypic determinant of the first antibody and the antigenic determinant of the analyte of the first complex to form a second complex; and (c) determining the presence or amount of the second complex as an indication of the presence or amount of the analyte of interest in the specimen. DESCRIPTION OF THE INVENTION
  • Immune response reactions have been identified as an area of extreme interest by scientists searching for tools to detect and monitor disease states. The ability of an organism to react to exposure to a foreign entity provides the basis for the exploration of the relationships and interactions between host and pathogen. Two of the myriad of components involved are antibodies and antigens, and much effort has been expended in developing assays using antibodies to detect and quantify a given antigen.
  • an antigen as an analyte is merely by way of illustration but not limitation; many other kinds of analytes are detectable utilizing the method of this invention. Many types of materials can be assayed for using the method of this invention, including but not limited to bacteria (e.g.
  • Chlamydia trachomatis Chlamydia trachomatis
  • fungi e.g. Candida albicans
  • parasites e.g. Trichomonas vaginails
  • mammalian cells e.g. tumor cells
  • macromolecules e.g. OMP MC gonorrhea protein
  • enzyme complexes e.g. creatinine phosphokinase
  • IgM viruses
  • viruses e.g. hepatitis B
  • Antigens are substances that are able to provoke a specific immune response by reacting with the components of the specific immune response, e.g., antibodies or specifically sensitized T-lymphocytes. There are exposed reactive areas on the surface of antigens containing surface groupings of a molecular nature, such as ami no acids or carbohydrate side chains. These chemical structures when reactive with antibodies are called antigenic determinants, or epitopes, and can be of two kinds: (1) sequential determinants, e.g., primary amino acid sequences; or (2) conformational determinants, i.e., determined by secondary, tertiary or quaternary structure. It is these antigenic determinants that enable a particular antigen to be recognized by a specific antibody.
  • sequential determinants e.g., primary amino acid sequences
  • conformational determinants i.e., determined by secondary, tertiary or quaternary structure. It is these antigenic determinants that enable a particular antigen to be recognized by
  • Antibodies are proteins comprised of two pairs of polypeptide chains. These polypeptide chains consist of constant regions and variable regions. The constant regions vary little from one antibody to the next; however, the variable regions of every monoclone of an antibody has a unique amino acid sequence. These sequences are able to recognize a complementary sequence on an antigen against which the antibody is directed. The areas on the variable region are called idiotypes and specifically bind to the antigenic determinant present on the surface of the antigen. In a manner similar to the enzyme-substrate lock and key phenomenon, an antibody's variable region matches with a particular antigenic determinant, either via a sequential determinant or a conformational determinant.
  • Monoclonal antibodies are prepared generally according to the process described by Milstein and Kohler in Nature 256:495-497, 1975. The process is well known in the art and need not be repeated here. Let it suffice to say that a monoclonal antibody can be designed to recognize and bind to the juncture created between the antibody:antigen immunocompiex described above. The procedures are set out in the Examples contained hereinbelow.
  • a system with a plurality of antibodies can be used to detect or quantify an analyte, such as an antigen, present in a patient specimen, such as urine, feces, sputum, semen, mucus, blood, and the like.
  • an analyte such as an antigen
  • a patient specimen such as urine, feces, sputum, semen, mucus, blood, and the like.
  • a signal generating substance the emitted signal of which can be read and measured.
  • a homogeneous assay is carried out in a single phase, commonly a liquid medium.
  • a heterogeneous assay uses two phases: solid and liquid; whereby one antibody is insolublized by adsorbing it onto a solid support surface, such a plastic beads, cellulose, glass fiber filter paper, glass, polymers, and the like. Usually the antibody that is not insolublized is conjugated with a labelling compound.
  • a first antibody is insolublized on a solid support surface.
  • An analyte of interest such as an antigen, is then added, and, an immunocomplex of first antibody:antigen forms. At this point a new molecular entity has formed which can be recognized by a second antibody.
  • This second antibody has been labelled with a signal-generating substance and is added to the immunocompiex. As previously discussed, this novel second antibody will recognize the juncture at the first-antibody:antigen binding site and bind to it, creating a new immunocompiex of first anti body: anti gen: second antibody.
  • the second antibody is designed so as to distinguish and bind specifically to the first antibody: anti gen complex rather than to the idiotypic determinant of the first antibody alone or the antigenic determinant on the antigen alone.
  • antigen immunocompiex a wash solution, typically a buffer, is added to separate bound (insolublized) material from unbound material.
  • the reaction area is then read and the amount of signal produced by the label is measured. .pa
  • the amount of signal is correlative with the concentraton of antigen originally present in the patient sample.
  • anti gen binding junture By utilizing the distinctive sequential and/or comformational characteristics of a specific anti body: anti gen binding junture a highly specific assay can be obtained.
  • the novel use of a labelled antibody that recognizes only the complex formed when antigen is present in the sample and binds to the insoluble antibody permits a specific and unique method for measurement of said antigen.
  • a triple antibody system can be used, wherein an insolublized first antibody "captures" the analyte, a second antibody binds to an antigenic determinant different and distinct from that of the first antibody, and a labelled third antibody which recognizes only the junction of the analyte and second antibody.
  • an assay would be useful in the analysis of large analytes such as bacteria, fungi, parasites, mammalian cells (e.g., tumor cells), macromolecules (e.g., OMP MC gonorrhea protein), enzyme complexes, IgM, etc.
  • analytes are sufficiently large or bulky that when an insolublized antibody binds to it, that access to the junction between the two could be sterically hindered and the second antibody that binds to the juncture would be blocked.
  • a second antibody is designed to bind to an antigenic determinant different and distinct from that of the insolublized first antibody.
  • a third antibody can bind to the junction between the second antibody and the analyte because it would not be sterically hindered by the presence of a large antigen- bearing molecule or cell.
  • the capture antibody can be either a monoclonal or a polyclonal anti body , whi l e the second and thi rd anti bodi es are preferably monoclonals.
  • second antibody and third antibody are in close proximity to each other when complexed with an analyte, their physical juxtaposition can serve as a basis for an assay based on the i nteracti on of two l abel l i ng components , wherei n the second antibody is conjugated with one constituent of a labelling scheme and the third antibody, which recognizes the juncture between second antibody and analyte, labelled with a second labelling constituent.
  • An illustration of such a method would employ a second antibody conjugated with a coenzyme, and third antibody conjugated with a corresponding enzyme.
  • the enzyme and coenzyme When the enzyme and coenzyme are brought in proper physical and geometric proximity (i.e., when in a complexed form as described above), they will react with an added substrate to produce a detectable signal.
  • the method is highly specific because substrate will be acted upon only when second antibody and third antibody are very close to each other and in a particular geometric alignment, which can be created by designing the antibody- conjugate constituents properly. In this way, there is a selectivity and proximity interaction requirement which effectively minimizes nonspecific reaction between unbound second antibody or third antibody and their labelled conjugates.
  • Alternative labelling systems embodying this triple antibody concept include, but are not limited to, a fluorescent label and a quencher molecule; an enzyme and an inhibitor molecule; a primary enzyme that acts on a substrate, which, in turn produces a product that is the substrate for a secondary enzyme that will act on the product-substrate to produce a signal.
  • a fluorescence quenching assay can be performed utilizing the selective binding of an antibody to the antigen:first antibody complex.
  • antigen is labelled with a quencher molecule and second antibody is labelled with a chemi luminescent substance.
  • second antibody recognizes the bound antigen:first antibody complex, it binds to the antibody:antigen junction.
  • Quencher is now in proximity to chemi luminescer and by its interaction will reduce the amount of signal emmitted by the chemiluminescer. The reduction of signal is indirectly related to the amount of analyte present.
  • An alternative procedure is to add a labelled first antibody to an antigen to form an immunocompiex. Subsequently, an insolublized second antibody is added which binds to the juncture of the labelled first antibody and antigen. This method would be advantageous where steric problems between an insolublized first antibody and antigen might occur.
  • This antibody is prepared according to the general procedure disclosed by Milstein & Kohler in NATURE 256:495-497, 1975.
  • a complex is formed of human chorionic gonadotropin ("hCG”) and an antibody directed against it. This complex serves as the new antigen to which the monoclonal antibodies are designed.
  • hCG human chorionic gonadotropin
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the hCG:antibody complex antigen, with an appropriate myeloma cell line. The resultant product is then cultured in a standard HAT (hypoxanthine, aminopterin, and thymidine) medium. Screening tests for the specific monoclonal antibodies are employed utilizing immunoassay techniques which will be described below.
  • the immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present invention will be described in connection with mice.
  • the mouse is first immunized by injection of the antigen chosen generally for a period of eleven weeks. When the mouse shows sufficient antibody production against the antigen, as determined by conventional assay, it is given a booster injection of the antigen, and then killed so that the immunized spleen may be removed. The fusion can then be carried out utilizing immunized spleen cells and an appropriate myeloma cell line.
  • the fused cells yielding an antibody which give a positive response to the presence of the antigen are removed and cloned utilizing any of the standard methods.
  • the monoclonal antibodies from the clones are then tested against standard antigens to determine their specificty for the particular antigen.
  • the monoclonal antibody selected, which is specific for the antigen or species, is then bound to an appropriate label.
  • Amounts of antibody sufficient for labelling and subsequent commercial production are produced by the known techniques, such as by batch or continuous tissue culture or culture in vivo in mammals, such as mice.
  • the monoclonal antibodies may be labelled with a multitude of different labels, such enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like.
  • Some of the enzymes utilized as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase, urease, and the like.
  • Such linkage with enzymes can be accomplished by any one of the conventional and known methods, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.
  • Staphylococcal Protein A method the glutaraldehyde method
  • benzoquinone method the benzoquinone method
  • periodate method the periodate method.
  • first antibody directed against hCG polyclonal or monoclonal
  • urine suspected of containing hCG is added one to three drops of urine suspected of containing hCG.
  • the mixture is incubated for one minute to permit formation of a first complex of the first antibody with any hCG present.
  • alkaline phosphatase labelled second antibody prepared in Step A above, is added.
  • the mixture is allowed to incubate for two minutes to permit formation of a second complex of the first complex and second labelled antibody.
  • the mixture is filtered. Any second complex will remain within or on the filter and is washed with Tris buffered saline to remove unbound reactants.
  • To the insolublized second complex is then added indoxyl phosphate substrate to develop color. After one to three minutes a blue color will appear if a second complex has been formed indicating that hCG is present in the specimen.
  • Streptococcus is prepared according to the procedure described in Example 1 part A above, except that group A Streptococcus antigen is substituted for hCG.
  • EXAMPLE 1 STEP 1 The procedure for idiotypic-anti genie conjunction binding antibody preparation of EXAMPLE 1 STEP 1 above is followed except that the hCG used is substituted each in turn with Chlamydia trachomitis, Candida albicans, Trichomonas vaginalis, OMP MC gonorrhea protein, creatinine phosphokinase, hepatitis B, and the like.
  • Step B EXAMPLE 1 or Step B, EXAMPLE 2
  • an antigen such as one in the list described above.
  • An appropriate first antibody is selected as being directed against the analyte suspected of being in a given specimen.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
EP19860906648 1985-10-22 1986-10-22 Idiotypisches antigen-konjugierungs-bindetestverfahren Withdrawn EP0248038A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US79007785A 1985-10-22 1985-10-22
US790077 1985-10-22

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EP0248038A1 true EP0248038A1 (de) 1987-12-09

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EP (1) EP0248038A1 (de)
AU (1) AU6479986A (de)
WO (1) WO1987002779A1 (de)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0250119A3 (de) * 1986-06-20 1990-04-18 Vasocor Atherosclerotischer anti-idiotypischer Antikörper, Immunotest und Reagenzien
AU599639B2 (en) * 1987-03-24 1990-07-26 Diagnostic Technologies Ltd. Method for the detection of pregnancy disorders
SE8902043L (sv) * 1988-11-10 1990-05-11 Pharmacia Ab Foerfarande foer karakterisering av makromolekyler
US5876985A (en) * 1991-04-25 1999-03-02 Board Of Regents, The University Of Texas System Methods and compositions for the preparation of recombinant Trichomonas vaginalis proteins and peptides
GB0129776D0 (en) * 2001-12-13 2002-01-30 Sec Dep For Environment Food & Assay device and method
WO2011060015A1 (en) * 2009-11-11 2011-05-19 Genentech, Inc. Methods and compositions for detecting target proteins

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1587193A (en) * 1976-08-25 1981-04-01 Univ Birmingham Process for preparation of antisera
DE3364347D1 (en) * 1982-04-09 1986-08-07 Fujirebio Kk Anti immune complex antibody and preparation thereof
US4514508A (en) * 1982-07-06 1985-04-30 Biond Inc. Assaying for a multiplicity of antigens or antibodies with a detection compound

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8702779A1 *

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AU6479986A (en) 1987-05-19
WO1987002779A1 (en) 1987-05-07

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