WO1991010907A1 - Anticorps de selection de composes pharmacologiquement actifs et analyse hierarchique de conglomerats - Google Patents

Anticorps de selection de composes pharmacologiquement actifs et analyse hierarchique de conglomerats Download PDF

Info

Publication number
WO1991010907A1
WO1991010907A1 PCT/US1991/000447 US9100447W WO9110907A1 WO 1991010907 A1 WO1991010907 A1 WO 1991010907A1 US 9100447 W US9100447 W US 9100447W WO 9110907 A1 WO9110907 A1 WO 9110907A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibodies
drug
compounds
theophylline
receptor
Prior art date
Application number
PCT/US1991/000447
Other languages
English (en)
Inventor
Edward T. Maggio
Manfred E. Wolff
David B. Waller
Original Assignee
Synbiotics Corporation
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 Synbiotics Corporation filed Critical Synbiotics Corporation
Publication of WO1991010907A1 publication Critical patent/WO1991010907A1/fr

Links

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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • 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
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • 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
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • the invention relates generally to methods for screening for ligands which bind to receptors. More particularly, the invention relates to the use of monoclonal antibodies which are specially produced or selected to mimic critical binding elements of the three dimensional surface structure of drug or hormone receptors.
  • Monoclonal antibodies are immunoglobulins produced by hybridoma cells.
  • a monoclonal antibody reacts with a single antigenic determinant and provides greater specificity than conventional, serum-derived polyclonal antibodies. Furthermore, screening a large number of monoclonal antibodies makes it possible to select an individual antibody with desired specificity,, avidity and isotype.
  • Hybridoma cell lines provide a constant, inexpensive source of chemically identical antibodies and preparations .of such antibodies can be easily standardized. Methods for producing monoclonal antibodies are well known to those of ordinary skill in the art, e.g., U.S. Patent Nos. 4,198,265 and 4,792,528; Kohler and Milstein, Nature 2.56:495 (1975) and Schook, ed.
  • Antigen recognition by a monoclonal antibody is attributable to a specific combining site in the N-terminal region of the immunoglobulin (Ig) molecule.
  • Ig molecules are thought to react with antigens via the same types of short range forces characteristic of all protein-protein interactions.
  • Antigen-antibody interactions are highly specific because of the complementary three-dimensional shapes of the antibody combining site and of the corresponding antigenic * determinant or epitope. Such complementary shapes permit the molecules to approach each other closely and to interact through a variety of intramolecular attractive forces over a substantial surface area.
  • the specificity of antibody-antigen interactions is evidenced by the fact that even slight changes in the configuration of the antigenic determinant result in marked decreases in specificity and/or binding constant of the antigen to the antibody.
  • the biological and pharmacological properties of drugs, hormones, and other biological modifiers are determined to a large degree by the way these ligands bind specific receptors in cells and tissues.
  • purified receptors or cells expressing specific receptor populations are being adapted as bioassays to screen chemical libraries for pharmaceutical leads.
  • Computer-generated models, based on physical data, are also being used to predict ligand-receptor binding.
  • receptor technology is useful, but limited: Only a fraction of important biological receptors are known and available as screening reagents. Furthermore, bioassays using natural receptors are often labile, subject to artifacts, and may contain only low concentrations of a desired receptor. Computer simulations, too, are hampered by the lack of structural data for many receptors. Now, however, the antigen binding sites of antibodies can be designed to mimic the structure of a variety of biological receptors. The resulting receptor "models" can then be used as chemical screens in the discovery and tailoring of new drugs and other bioactive compounds. It is known that the effects of a drug often result from complex interactions that may involve an initial ligand, secondary metabolic products, and a heterogeneous population of receptors operating under variable physiological conditions. Still, for many bioactive compounds, the ability to specifically bind a given population of receptors correlates well with biological activity.
  • ligands as antigens from which to derive antibodies that mimic receptors are extremely valuable. Not all anti-ligand antibodies, however, mimic the ligand , s receptor(s) . Where the ligand contains multiple epitopes, and where only some of- these correspond to the ligand , s receptor-binding region, only subpopulations of antibody would be expected to be receptor mimics. Likewise, only antibodies which bind to drug in the same spatial orientation which the natural receptor binds to drug would be expected to function as receptor mimics.
  • Antibodies may be generated to virtually any drug or other low molecular weight substance (referred to as haptenic substances) by attachment of such low molecular weight substances to a carrier molecule such as a protein or other polymeric material.
  • haptenic substances a drug or other low molecular weight substance
  • the site of attachment and the length of the linker arm between the haptenic substance and the macromolecular carrier molecule determine how the hapten is presented to the immune system. This in turn determines the nature of the immune response. If, for example, a drug has a number of epitopes to which antibodies can be generated, and this is typically the case, attachment of the drug to a acromolecule by epitope A will result in the generation of antibodies against epitopes B, C, D, etc. , and a lack of response to epitope A.
  • Linthicu et. al., Bio Essays, 3_ : 213-217 (1985) disclose that antibodies directed against pharmacologically active ligands have a three-dimensional binding site which is somewhat analogous to the natural receptor and that anti-idiotypic antibodies produced against these anti-ligand antibodies can have a three-dimensional shape which mimips the original ligand.
  • the anti-idiotypic antibodies generated in this fashion are able to.bind to cell surface receptors.
  • U.S. Patent No. 4,262,089 to Singh, et. al. discloses theophylline derivatives useful in competitive protein binding assays.
  • Theophylline is substituted at the 3-position and conjugated to antigens for production of antibodies which specifically recognize theophylline as distinct from structurally similar analogues such as caffeine.
  • This invention includes the discovery that a certain class of monoclonal antibody may be of substantial commercial value in the screening for, or design of, drugs or hormone analogues or in the production of low molecular weight replacements of naturally occurring polypeptide drugs such as insulin, growth hormone, erythropoietin, interleukin, tnf, tpa, interferon and the like.
  • the invention therefore relates to a method of screening for low molecular weight ligands which are capable of binding to a naturally occurring receptor which comprises producing a set of one or more monoclonal antibodies capable of reacting with a specific receptor agonist or antagonist and screening for a low molecular weight structural mimic of the agonist or antagonist.
  • the mimic is a binding ligand to a receptor, it may be either an agonist or an antagonist.
  • the invention further relates to a method of producing or screening for a low molecular ligand capable of specific binding to a naturally occurring receptor comprising the steps of (1) attaching a acromolecular carrier at one or more different points on a haptenic substance, (_) sensitizing B-cell lymphocytes in vivo or in vitro to produce an array of monoclonal antibodies to the haptenic substance, and (3) using a set of one or more of the resulting antibodies to screen chemical compounds for their ability to bind to said antibodies.
  • An additional step may include comparing the binding profiles of these compounds by hierarchical cluster analysis.
  • Hierarchial cluster analysis is a non-parametric method of multivariate statistics whose purpose is to place objects into groups or clusters not defined a priori, objects in a given cluster tending to be similar and objects in different clusters dissimilar.
  • One or more of the final classes of hierarchical subclusters contain the compound(s) of interest as drugs.
  • receptor a molecule or molecular complex that will combine specifically with its particular ligand molecule(s) . It is those receptors which on binding with their particular ligand(s) mediate a biological function that are of most interest.
  • Examples of receptors include, but are not restricted to, the common class of receptors associated with the surface membrane of cells and include, for instance, the immunologically important receptors of B-cells, T-cells, macrophages and the like.
  • Other examples are the receptors for acetylcholine on nerve cells. These receptors cause a nerve impulse to be transmitted down the length of the neuron when the receptor molecule reacts with its ligand, acetylcholine.
  • epitope the specific surface of an antigen molecule that is delineated by the area of interaction with the subclass of receptors known as antibodies.
  • complementary refers to the matching together of the reacting surfaces of an ligand molecule and its receptor.
  • the receptor and its ligand can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.
  • paratope the combining site of an antibody which is complementary to a particular epitope.
  • ligand a molecule which binds to a particular receptor and when bound to it mediates the biological function associated with that particular receptor.
  • Drug Receptor - Drug Interaction Some drugs exhibit biological activity in minute concentrations. For this reason they are described as structurally specific. The effect produced by such drugs is attributed to interaction with a specific cellular component which is the drug receptor. As a result of this interaction the drug forms a complex with its receptor.
  • receptors are integral parts of certain macromolecules of living organisms. In most instances, they are segments of proteins. Often, they are the catalytic sites or the allosteric sites of enzymes. In some cases, drug receptors are parts of nonenzyme proteins.
  • Receptors for some chemotherapeutic agents are nucleic acids (DNA or RNA) , which can react chemically with these drugs through covalent bond formation or lodge them between their base pairs and the phosphate groups, by complexing with them by means of relatively weak interactions. Receptors can also be parts of lipoprotein complexes, principally of cellular membranes.
  • DNA or RNA nucleic acids
  • Receptors can also be parts of lipoprotein complexes, principally of cellular membranes.
  • Acetazolamide carbonic anhydrase actinomycins DNA adrenergies adenylate cyclase cimetidine H2 receptor allopurinol xanthine oxidase amodiaquine DNA amphotericin B cell membrane antifolates dihydrofolate reductase aspirin eye1ooxygenase atro ine muscarinic receptor butaclamol DA2 receptor captopril dipeptidyl carboxypeptidase I carbidopa aromatic
  • MAO inhibitors amine oxidase mepacrine DNA methotrexate dihydrofolate reductase methylsergide 5-HT1 receptor methylxanthines adenosine receptor mitomycin DNA nalidixic acid DNA topoisomerase
  • Adenosine Receptors The methylxanthines most widely used in medicine are caffeine, theophylline, and theobromine. In small doses these drugs are taken especially as coffee, tea, cocoa, and chocolate drinks, in order to enhance mental alertness and wakefulness, to lessen fatigue and to produce diuresis. Excessive amounts, however, cause insomnia and restlessness in some people.
  • caffeine is the most potent cerebral stimulant; theophylline follows, whereas theobromine is almost devoid of stimulant properties. Theophylline produces more diuresis than theobromine and the latter more than caffeine.
  • Xanthine derivatives because they relax smooth muscle contraction of bronchioles, are useful as adjuncts in the symptomatic treatment of asthma, bronchitis, emphysema and other obstructive pulmonary disorders.
  • the negative inotrophic and vasodilatory actions of adenosine have been known for some time and the purine has also been shown to cause sedation when given centrally or peripherally.
  • the major breakthrough in defining a physiological role for adenosine was the observation that the xanthines, caffeine and theophylline, might function as adenosine antagonists. See Molecular Pharmacology, ⁇ 5, 13-23, (1970).
  • xanthine derivatives such as 8-phenyl theophylline (8-PT) , l,3-diethyl-8-phenylxanthine (DPX) and 1,3 dipropyl-8(2-amino-4-chloro)phenylxanthine (PACPX) have been developed which are selective adenosine antagonists.
  • 8-PT 8-phenyl theophylline
  • DPX l,3-diethyl-8-phenylxanthine
  • PPPX 1,3 dipropyl-8(2-amino-4-chloro)phenylxanthine
  • the pharmacological characteristics of the cell surface adenosine receptor sites differ markedly.
  • the potent xanthine adenosine antagonist PACPX has marked differences in its interaction with Al and A2 receptor subtypes and in its effects at Al receptors from brain and cardiac tissue.
  • the alkylxanthine, 3- propylxanthine appears to have little affinity for CNS adenosine receptors, although it retains its activity for bronchial receptors.
  • PIA Ns-phenylisopropyladenosine
  • CADO chloroadenosine
  • NECA 5'-ethyl- carboxa ide adenosine.
  • Agonists or antagonists whether they be of high or low molecular weight contain multiple epitopes capable of recognition by antibodies having different paratopes.
  • the particular structure of a given paratope is determined in part by the orientation of the agonist or antagonist (when used as an immunogen) as it interacts with an enormous repertoire of B cells many of which have the capability to be stimulated by interaction with the agonist or antagonist.
  • the orientation of binding can be quite varied, limited only by the underlying genetic variability of the immune system of the animal from which the B lymphocytes were.obtained.
  • a large number of antibodies containing different paratopes and in effect, different structural perspectives of the agonist or antagonist are obtainable.
  • the nature of the specific paratopes generated can be manipulated.
  • all of the epitopes on a given drug structure can be exposed to B cells generating antibodies having paratopes which likewise have the capability to bind to the drug molecule from all spatial perspectives within the genetic capability of the animal supplying the B lymphocytes.
  • B cells By stimulating a large number of lymphocytes for example 10 8 cells, using drug conjugates attached by a variety of sites on the molecular structure of the drug, a large library of monoclonal antibodies may be generated which contains nearly all three dimensional molecular perspectives of the original drug.
  • the detection of antibody binding to drug may be conducted through a variety of means including enzyme immunoassay and the like.
  • enzyme immunoassay for example, if the monoclonal antibodies were prepared in mice, an enzyme labeled antimurine antibody could be used to detect the presence of monoclonal antibody bound to drug. If a solution containing free drug were first placed in the microtiter well containing the bound drug conjugate, the free drug competes with anti-drug antibody, reducing or preventing binding of anti-drug antibody to the drug conjugate, thus resulting in a diminished enzyme immunoassay signal. Likewise, the effect of structurally dissimilar drugs, active metabolites, and inactive metabolites may be tested for their ability to diminish antibody binding to conjugate. As each antibody in the antibody array is examined in this way, a number of possible outcomes are observed. These are shown in Table 2.
  • This technique composes the final classes by hierarchically grouping sub-clusters; hierarchical clusters are organized so that one cluster may fall entirely within another cluster but no partial . overlap between clusters is allowed.
  • the following Example is given for the purpose of illustration only and is not to be considered as limiting to the invention disclosed herein.
  • adenosine antagonists fell into one cluster whereas adenosine agonists fell into a second cluster.
  • Monoclonal antibodies to theophylline are produced as follows:
  • mice are immunized by i.p. injection of a mixture of 50 ug of theophylline-BSA mixed with an equal volume of complete Freund's adjuvant. The mice are rested for 4-8 weeks and then boosted repeatedly with theophylline-BSA as before except for the use of incomplete Freund's adjuvant.
  • the immune response of each mouse is followed during the inoculation period in order to determine the best responder.
  • the immune response is monitored by measuring antibody titers at alternating two week intervals until a suitable titer is reached.
  • Serum samples are diluted in 25 mM Tris-HCl buffer containing lO M MgC12 and 0.1% BSA, pH 7.4 to make serum dilutions of 1:5, 1:10, 1:100, and 1:1000 (final assay dilutions are 1:25, 1:50, 1:500, and 1:5000 respectively).
  • 100 ul of diluted serum is added to wells of microtiter plates in which theophylline, conjugated to an irrelevant carrier protein, is immobilized.
  • Assay controls include wells that receive normal mouse serum in place of the mouse bleeds.
  • Serum antibodies that bind to theophylline are detected with enzyme-conjugated goat antibody to mouse immunoglobulin and an appropriate chromogenic substrate.
  • the mouse with the highest antibody titer receives one additional IV injection of theophylline-BSA.
  • the spleen is removed and fused with myeloma cells and is described as follows. Spleen cells are isolated by teasing apart the spleen in a dish of culture medium; spleen cells are dispersed by repeated pipetting until a single cell suspension is obtained. A cell count and viability determination are performed. The suspension of spleen cells is subsequently employed in a fusion step with myeloma cells.
  • the myeloma cells should be selectable, i.e. the growth of unfused myeloma cells in culture should be inhibited by a factor present in the culture medium. However, upon fusion with a lymphoid cell, the resultant cellular fusion product can grow in the presence of this factor. For this purpose, hypoxanthine, aminopterin, and thymidine are included in culture medium.
  • Preferred myeloma cell lines that are deficient in hypoxanthine (guanine) phosphoribosyl transferase include P3X63-Agul and P3X63- Ag8.653, each of which is supplied by American Type Culture Collection. Directly prior to the fusion step, the myeloma cell line is cultivated until it reaches "S" growth phase.
  • a cell mixture is made by combining a sample of the myeloma cells with a sample of the suspension of spleen cells from the best responder mouse. Best results are found using a cell mixture having a cellular composition of 1:2, i.e. one part myeloma cells, two parts spleen lymphoid cells.
  • the above cell mixture is combined with polyethylene glycol (PEG 1000) under standard conditions to form a 50% solution in culture medium.
  • the resultant cell mixture is diluted with medium, i.e. Dulbecco's minimal essential medium (DMEM) , 10% fetal bovine serum, hypoxanthine, aminopterin, and thymidine.
  • DMEM Dulbecco's minimal essential medium
  • the dilution is calculated to yield a cell mixture with approximately 1-1.5x10(6) cells per milliliter.
  • Aliquots of 0.2 illiliters of the diluted cell mixture are then pipetted into microtiter plates, preferably 96-well plates.
  • the wells of the microtiter plates include a feeder layer of mouse thymocytes.
  • the resultant fusion products are then incubated in the microtiter plates for 10-12 days.
  • Supernatants from hybridomas are screened for antibody production, antibody producing cells are diluted and screened (expansion screen) , sub-cl ⁇ ned by limiting dilution, screened, expanded again and screened again. Selected monoclonal antibody producing cells are grown and injected into mice for ascites production.
  • Antibody screening is analogous to screening of sera from immunized mice.
  • Media from hybridoma cultures is added to wells of microtiter plates in which theophylline, conjugated to an irrelevant carrier protein, is immobilized.
  • Assay controls include wells that receive an irrelevant monoclonal antibody in place of hybridoma supernatants.
  • Hybridoma antibodies that bind to theophylline are detected with enzyme-conjugated goat antibody to mouse immunoglobulin and an appropriate chromogenic substrate. This analysis, is performed for hybridoma cultures at each stage of development until final selections of the best hybridomas are made, and ascites are produced for these hybridomas. After production of the appropriate ascitic fluid, purified antibody is produced by the following method.
  • Saturated ammonium sulfate is added dropwise with continuous stirring to an equal volume of ascites fluid in a Beckman 5/8 x 3 inch polycarbonate tube, stirred for an additional 15 minutes at room temperature and .then centrifuged for 15 minutes at 10,000 rpm, 4°C. The supernatant is discarded.
  • the pellet is suspended in 1/2 volume of PBS and an equal volume of ammonium sulfate added dropwise, stirred for an additional 15 minutes and centrifuged as above.
  • the pellet is resuspended in 1/2 volume of PBS and dialyzed against 1 liter of 0.01 M phosphate buffer containing 0.2 M NaCl, pH 7.4 overnight with one change of dialysis buffer.
  • a 10 ml disposable pipette is packed with 10 ml of DEAE (250 mm hydrostatic pressure) .
  • the column is washed with 0.5M phosphate buffer, pH 7.0 then with 0.5M acetate buffer, pH 6.0 until the eluate is pH 6.0.
  • the column is washed with 0.5 M phosphate buffer, pH 7.0 until the eluate is pH 7.0 and then washed with several volumes of 0.05 M phosphate buffer, pH 7.0.
  • the column outflow tube is attached to a UV monitor (280 nm) and the monitor outflow tube to a collection vessel.
  • the dialyzed sample is transferred to the column and eluted with 0.05 M phosphate buffer, pH 7.0.
  • Collection vessels are changed when the protein begins passing through the UV monitor and collection stopped at the end of the protein peak.
  • concentration of antibody recovered from the column is determined with an assay protocol using BCA protein assay reagents from Pierce Chemical Company, Rockford, IL.
  • Standards are made up from a 2 mg/ml stock of bovine albumin powder in 0.001 M NaHC03.
  • a standard protein concentration range of 0-250 ug/ml in a total volume of 2.0 ml is made up in the same buffer as the test material in each assay.
  • 0.1 ml of each standard or test sample is pipetted into 13 x 100 mm tubes and 2.0 ml BCA working reagent (50 parts Reagent A plus 1 part Reagent 2) is added and mixed.
  • the tubes are incubated at 60°C for 30 minutes and then cooled to room temperature.
  • the absorbency is read at 562 nm against a water blank.
  • the protein • values of test samples are determined by comparing the O.D. of the test sample with the bovine albumin standard curve.
  • Adenosine Receptor Antagonist Screening A solution of the antibody prepared as described against this immunogen is allowed to react with each screening compound, i.e., an adenosine antagonist, a dissimilar drug, or compounds that are not drugs but have a tertiary structure similar to theophylline, in a separate container.
  • a solution of 120 ml of theophylline-BSA immunogen is prepared as indicated earlier is used to coat the microtiter wells of a 96 microtiter well plate. The irrelevant binding sites on the immunogen linked bovine serum albumin are blocked by the addition of bovine serum albumin solution to the microtiter plate.
  • microtiter plate is emptied and washed and each screening compound antibody mixture is added to the several wells and allowed to react.
  • the wells are emptied and washed three times with distilled water. Thereafter horseradish peroxidase conjugated goat anti-mouse antibody is added.
  • the wells are emptied again and washed three times.
  • a suitable substrate for horseradish peroxidase is added and the optical density is read at 450 nanometers. The results of the optical density readings are converted to binding affinities Kd (M) measured in moles per liter are set forth in Table 1.
  • affinities of the adenosine agonists and antagonists for the different antibodies are submitted to hierarchical cluster analysis on a IBM PC AT computer using the SYSTAT (V.2.1.) package.
  • the centroid linkage method on the Euclidean distance is utilized in the cluster analysis. Results of this assay are also set forth in Table 3.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Un procédé permet de sélectionner des composés à faible poids moléculaire utiles comme médicaments capables de se lier à un récepteur d'origine naturelle. Selon ce procédé, on utilise des anticorps monoclonaux qui imitent des éléments critiques de liaison de la structure tridimensionnelle de surface de récepteurs d'hormones ou de médicaments. Des critères de sélection permettent d'identifier et de définir la spécificité structurelle requise des anticorps monoclonaux, afin d'assurer l'imitation tridimensionnelle des éléments critiques de liaison des récepteurs. Afin de sélectionner des composés prometteurs dans le groupe analysé à des fins d'évaluation ultérieure, on utilise un procédé de regroupement hiérarchique en conglomérats.
PCT/US1991/000447 1990-01-22 1991-01-22 Anticorps de selection de composes pharmacologiquement actifs et analyse hierarchique de conglomerats WO1991010907A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US46811690A 1990-01-22 1990-01-22
US468,116 1990-01-22

Publications (1)

Publication Number Publication Date
WO1991010907A1 true WO1991010907A1 (fr) 1991-07-25

Family

ID=23858491

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/000447 WO1991010907A1 (fr) 1990-01-22 1991-01-22 Anticorps de selection de composes pharmacologiquement actifs et analyse hierarchique de conglomerats

Country Status (1)

Country Link
WO (1) WO1991010907A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004557A2 (fr) * 1994-08-03 1996-02-15 Dgi Technologies, Inc. Triages specifiques de cibles et leur utilisation permettant la decouverte de petits groupements pharmacophores moleculaires organiques
US7189582B2 (en) 2005-04-27 2007-03-13 Dade Behring Inc. Compositions and methods for detection of sirolimus
US8021849B2 (en) 2004-11-05 2011-09-20 Siemens Healthcare Diagnostics Inc. Methods and kits for the determination of sirolimus in a sample

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2805961A1 (de) * 1977-04-15 1978-10-19 Syva Co Theophyllin-antigene und -antikoerper und ihre verwendung zur bestimmung von theophyllin
US4302438A (en) * 1979-01-13 1981-11-24 Byk Gulden Lomberg Chemische Fabrik Gmbh Antigen, antiserum and immunoassay for theophylline
US4533493A (en) * 1981-08-27 1985-08-06 Miles Laboratories, Inc. Theophylline immunogens, antibodies, labeled conjugates, and related derivatives

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2805961A1 (de) * 1977-04-15 1978-10-19 Syva Co Theophyllin-antigene und -antikoerper und ihre verwendung zur bestimmung von theophyllin
US4302438A (en) * 1979-01-13 1981-11-24 Byk Gulden Lomberg Chemische Fabrik Gmbh Antigen, antiserum and immunoassay for theophylline
US4533493A (en) * 1981-08-27 1985-08-06 Miles Laboratories, Inc. Theophylline immunogens, antibodies, labeled conjugates, and related derivatives

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 90, No. 11, issued 19 October 1978, SINGH et al., "Theophylline antigens and antibodies and Their Use for Determining Theophylline", see Abstract No. 79831m; & DE,A,28 05 961. *
PHARMACOLOGICAL REVIEWS, Volume 25, No. 2, issued 1973, BERNARD F. ERLANGER, "Principles and Methods for the Preparation of Drug Protein Conjugates for Immunological Studies", pages 274-283. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004557A2 (fr) * 1994-08-03 1996-02-15 Dgi Technologies, Inc. Triages specifiques de cibles et leur utilisation permettant la decouverte de petits groupements pharmacophores moleculaires organiques
WO1996004557A3 (fr) * 1994-08-03 1996-04-25 Dgi Technologies Inc Triages specifiques de cibles et leur utilisation permettant la decouverte de petits groupements pharmacophores moleculaires organiques
US6010861A (en) * 1994-08-03 2000-01-04 Dgi Biotechnologies, Llc Target specific screens and their use for discovering small organic molecular pharmacophores
EP1028315A2 (fr) * 1994-08-03 2000-08-16 Dgi Technologies, Inc. Triages spécifiques de cibles et leur utilisation permettant la découverte de petits groupements pharmacophores moléculaires organiques
EP1028315A3 (fr) * 1994-08-03 2004-01-02 DGI BioTechnologies, Inc. Triages spécifiques de cibles et leur utilisation permettant la découverte de petits groupements pharmacophores moléculaires organiques
US8021849B2 (en) 2004-11-05 2011-09-20 Siemens Healthcare Diagnostics Inc. Methods and kits for the determination of sirolimus in a sample
US7189582B2 (en) 2005-04-27 2007-03-13 Dade Behring Inc. Compositions and methods for detection of sirolimus

Similar Documents

Publication Publication Date Title
AU2019284029B2 (en) Antibodies to paliperidone haptens and use thereof
US10793644B2 (en) Antibodies to risperidone haptens and use thereof
US7205116B2 (en) 5-Fluoro-uracil immunoassay
EP1875237B1 (fr) Immunodosage du docetaxel
EP1864129B1 (fr) Dosage immunologique de doxorubicine
CN101019024B (zh) 紫杉醇免疫测定法
US7169907B2 (en) Derivatives, immunogens, and antibodies for detecting ecstasy-class drugs
EP0098179B1 (fr) Lignées de cellules hybridomes et anticorps monoclonaux contre la théophylline
US7101980B2 (en) Derivatives, conjugates, and antibodies for detecting ecstasy-class analytes
CA3008812A1 (fr) Anticorps diriges contre la quetiapine et utilisation associee
WO1991010907A1 (fr) Anticorps de selection de composes pharmacologiquement actifs et analyse hierarchique de conglomerats
WO2008052737A2 (fr) Analogues de médicaments contre le vih immunochimiquement équivalents
WO1986001539A1 (fr) Procede de production d'anticorps monoclonaux anti-idiotypiques
CN114685342B (zh) 一种左乙拉西坦衍生物、免疫原、抗左乙拉西坦特异性抗体及其制备方法与应用
EP0390612A1 (fr) Production de médicament utilisant des anticorps à réactions non croisées
EP1671132B1 (fr) Procede de selection de composes utilisant des anticorps agissant comme agoniste, antagoniste ou modulateur allosteriques
JP2004083429A (ja) セリン−トレオニンタンパク質リン酸化酵素を認識するモノクローナル抗体

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

NENP Non-entry into the national phase

Ref country code: CA