EP1071954A1 - Phase solide de type peigne pour mesurer les analytes - Google Patents

Phase solide de type peigne pour mesurer les analytes

Info

Publication number
EP1071954A1
EP1071954A1 EP99917707A EP99917707A EP1071954A1 EP 1071954 A1 EP1071954 A1 EP 1071954A1 EP 99917707 A EP99917707 A EP 99917707A EP 99917707 A EP99917707 A EP 99917707A EP 1071954 A1 EP1071954 A1 EP 1071954A1
Authority
EP
European Patent Office
Prior art keywords
reactant
probe
reaction vessel
biomolecule
label
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Withdrawn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
EP99917707A
Other languages
German (de)
English (en)
Inventor
Zhibo Gan
Ronald Marquardt
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.)
University of Manitoba
Original Assignee
Norzyme Inc
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 Norzyme Inc filed Critical Norzyme Inc
Publication of EP1071954A1 publication Critical patent/EP1071954A1/fr
Withdrawn legal-status Critical Current

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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • the present invention relates generally to the field of biological and biochemical assays.
  • the assay of enzyme activity is one of the most frequently encountered procedures in biochemistry.
  • most enzyme assays are used to estimate the amount or activity of an enzyme present in a cell, tissue, other preparation, or as an essential part of an enzyme purification protocol.
  • the current assay methods have been developed based on the physical, chemical and immunological properties of the enzyme of interest and use detection means such as for example photometric, radiometric, high performance liquid chromatographic, and electrochemical assays. (Eisenthal, R. and Danson, M. J., in Enzyme Assays: a practical approach. (IRL Press: Oxford University, Oxford, (1993)).
  • immunoassays have been widely using in human clinical tests and therapeutics, agriculture, food, veterinary and environmental diagnostics (Deshpandes, S. in Enzyme immunoassays from concept to product development., (Chapman & Hall: New York, 1996)). While for most purposes, immunoassays are effective (Cleaveland, J. S. et al, 1990, Anal Biochem 190: 249-253.), in some cases they are not suitable, for example, in the determination of enzyme activity. This is due to the fact that binding assays for antibody and antigen (in this instance, an enzyme) only measure the concentration of an antigen (enzyme) and not its activity.
  • the assay should be suitable for a wide variety of applications, easy to use and require only a few steps for obtaining results.
  • the assay method is based on a one- step procedure for separating the reactants from the products (resultants) after completion of the reaction followed by measurement of the amount of a labeled reactant or labeled product that has been left in the reaction vessel or on the pin.
  • the device consists of two parts: one of which is a reaction vessel and other is a pin-like device (a probe) that can fit into the reaction vessel.
  • the reaction begins by insertion of the pin-like device that is coated with a reactant into the reaction vessel containing the same or other reactants than those coated on the pin-like surface. The said pinlike surface is taken out from the reaction vessel to stop the reaction.
  • the amount of the labeled products or labeled reactant remaining in the reaction vessel or on the probe can be determined according to the intensity of its label which can be fluorescent, luminescent and chromogenic molecules or radioactive tag, etc.
  • the amount of the label in the reaction vessel is directly or reciprocally proportional to the activity or amount of the bioactive substance that is to be measured.
  • the device can be used for the assay of enzymes, their inhibitors, antibodies and antigens, and receptors or lectins and their ligands.
  • a method for measuring the activity or concentration of a biomolecule comprising: providing a reaction vessel containing a sample, said sample including a biomolecule having a biological activity; providing a probe coated with a reactant, said reactant being capable of interacting with the biomolecule; adding a known quantity of a compound with a detectable label to the sample; inserting the probe into the reaction vessel such that the biomolecule and the detectable label contact the reactant and interact with the reactant such that label is bound to either the reactant or the biomolecule; removing the probe from the reaction vessel; and measuring the quantity of detectable label in the reaction vessel and/or on the probe.
  • the probe may have a shape selected from the group consisting of: pin-like; cone-like; cuboid; cylindrical; star-shaped; and spire-shaped.
  • the detectable label may be selected from the group consisting of: colorimetric label; radioactive label; luminescent label; and fluorescent label.
  • the sample may be a biological sample.
  • the biological activity may be an enzyme; a receptor; a lectin or an antibody; or a specific ligand of an antibody, a receptor, a lectin or an enzyme.
  • the reactant may be bound to the probe.
  • the sample may include an inhibitor of the biological activity of the biomolecule.
  • the sample may include a competitor of the biological activity of the biomolecule.
  • the biomolecule may be selected from the group consisting of: an enzymatic product; an enzyme; a substrate; a receptor; an inhibitor; a ligand; a lectin; a lectin-binding ligand; a receptor-binding ligand; an antibody; an antigen; a receptor; and a lectin.
  • the compound may be selected from the group consisting of: an enzymatic product; an enzyme; a substrate; a receptor; an inhibitor; a ligand; a lectin; a lectin-binding ligand; a receptor-binding ligand; an antibody; an antigen; a receptor; and a lectin.
  • a method for measuring the activity or concentration of a biomolecule comprising: providing a reaction vessel containing a sample, said sample including a biomolecule having a biological activity; providing a probe coated with a reactant, said reactant being capable of interacting with the biomolecule, said reactant including a detectable label; inserting the probe into the reaction vessel such that the reactant and the detectable label contact the biomolecule and interact with the biomolecule such that label is released from the reactant; removing the probe from the reaction vessel; and measuring the quantity of detectable label in the reaction vessel and/or on the probe.
  • the probe may have a shape selected from the group consisting of: pin-like; cone-like; cuboid; cylindrical; star-shaped; and spire-shaped.
  • the detectable label may be selected from the group consisting of: colorimetric label; radioactive label; luminescent label; and fluorescent label.
  • the reactant may be bound to the probe.
  • the sample may be a biological sample.
  • the biological activity may be an enzymatic activity or a binding affinity.
  • the sample may include an inhibitor of the biological activity of the biomolecule.
  • the sample may include a competitor of the biological activity of the biomolecule.
  • the biomolecule may be selected from the group consisting of: an an enzymatic product; an enzyme; a substrate; a receptor; an inhibitor; a ligand; a lectin; a lectin-binding ligand; a receptor-binding ligand; an antibody; an antigen; a receptor; and a lectin.
  • Ligand refers to a bioactive molecule having specific binding affinity for another biomolecule.
  • Receptor refers to a bioactive molecule that has a specific binding affinity for another biomolecule, for example, a ligand.
  • Bioactive molecule or “biologically active substance” as used herein except where otherwise stated refers to a molecule or complex having a biological activity, for example, an enzymatic activity or binding affinity for another biomolecule.
  • Probe refers to a member arranged to be reversibly inserted into a reaction mixture, as discussed below.
  • the present invention relates to a method for the detection, identification and measurement of the amount or activity of biologically active molecules via a one-step separation of reactants from products using a device comprising a probe whose surface is coated with a biologically active substance and a reaction vessel containing the reaction mixture.
  • the reaction mixture contains a bioactive molecule and may, for example, be a biological sample or a synthetic reaction mixture in which all of the components are known.
  • the assay consists of a probe and a reaction vessel.
  • the reaction vessel contains a reaction mixture which includes a bioactive molecule, for example, an enzyme or ligand, as well as possibly other compounds, for example, inhibitors, antibodies or receptors, as described below.
  • the probe has a surface and the surface of the probe is coated with a compound.
  • the compound selected for coating is either a substrate for or has binding affinity for the bioactive molecule, as described below.
  • the compound may be labeled, for example, fluorescently, luminescently, colorimetrically or radioactively, or the reaction mixture may contain a labeled substrate for or a labeled biomolecule having binding affinity for the bioactive molecule.
  • the labeled group on the compound is selected such that the activity of the bioactive molecule causes the label to be cleaved or removed from the compound and released to the reaction mixture.
  • the label on the substrate within the reaction mixture is selected such that the activity of the bioactive molecule will cause the label to be transferred from the labeled substrate to the compound coating the surface of the probe, as described below.
  • the labeled biomolecule and the biomolecule in.the reaction mixture may compete for binding to the compound on the surface of the probe, or the biomolecule in the reaction mixture may compete with the labelled compound in the reaction mixture and the compound on the surface of the probe, as described below.
  • the assay is carried out as follows: the quantity of labeled compound coated on the surface of the probe or the quantity of labeled substrate in the reaction vessel may first be determined by means known in the art according to the type of label used. The probe is then lowered into the reaction vessel such that the coated surface of the probe contacts the reaction mixture. The bioactive molecules present in the reaction mixture then either release label from the compound, transfer label from the substrate in the reaction mixture to the compound on the surface of the probe or compete for binding, as described below. Once the probe has been in contact with the reaction mixture for a pre-determined period of time, the probe is removed from the reaction mixture, thereby terminating the reaction without the need to add additional chemicals, heat the reaction mixture or centrifuge the reaction mixture. The quantity of label either in the reaction mixture or on the surface of the probe can then be determined, which can then be used to calculate reaction kinetics.
  • the probe has a substantially pinlike shape. It is of note that in other embodiments, the probe may be of other shapes, for example, cylindrical, spire-shaped, star-shaped, cuboid, or cone-shaped.
  • reaction vessel containing a reaction mixture including a bioactive molecule
  • adding a labeled compound that is a substrate for the bioactive molecule stopping the reaction, that is, stopping the biological activity of the bioactive molecule, and separating unused substrate from used substrate by washing or chromatography.
  • An alternative type of assay known in the art involves coating a reaction vessel with a compound, adding a ligand, allowing binding between the ligand and the compound to occur, washing away unbound ligand, and adding a reporter compound to detect bound ligand.
  • the surface of the probe in this embodiment a pin-like device, is coated with reactant 1 which is an acceptor of a moiety from reactant 3.
  • Reactant 3 is a substrate for reactant 2 having a biological activity. That is, in this example, reactant 1 is the compound used to coat the probe, reactant 3 is the labeled substrate and reactant 2 is the bioactive molecule. Furthermore, reactant 2 and reactant 3 are present in the reaction mixture in the reaction vessel.
  • the coated pin-like device is inserted into a reaction vessel, the labeled moiety from reactant 3 is transferred to reactant 1 due to the biological activity of reactant 2 acting on reactants 1 and 3.
  • the amount of labeled reactant 3 remaining in the reaction vessel can be directly determined without an additional step and is reciprocally proportional to the activity of reactant 2 after the pin-like device containing the labeled moiety from reactant 3 has been taken out of the reaction vessel.
  • the amount of label transferred to the probe may be determined, which is directly proportional to the activity of reactant 2.
  • reactant 4 which is an inhibitor of reactant 2.
  • reactant 4 inhibits transfer of the labeled moiety from reactant 3 to reactant 1
  • the amount of labeled reactant 3 remaining in the reaction vessel once the probe has been removed from the reaction vessel is directly proportional to the amount of reactant 4
  • the amount of labeled reactant 1 present on the surface of the probe is reciprocally proportional to the amount of reactant 4
  • the surface the probe in this embodiment, a pin-like device is coated with reactant 1 which is a labeled substrate for reactant 2 having a biological activity. That is, in this example, reactant 1 is the labeled compound and reactant 2 is the bioactive molecule. Insertion of the pin-like device containing reactant 1 into a reaction vessel containing reactant 2 and other compounds essential for the reaction results in the initiation of a reaction in which labeled products of the reaction are released into the reaction vessel. The amount of the released label in the reaction vessel can be directly measured after the pin-like device has been taken out of the reaction vessel without an additional step and the quantity of label released is proportional to the activity of reactant 2.
  • the amount of label remaining on the pin-like device can be determined, which would be reciprocally proportional to the activity of reactant 2.
  • the release of label from reactant 1 coated on the surface of the pin-like device by reactant 2 is interfered with by reactant 3 which is an inhibitor of reactant 2 and is present in the reaction mixture.
  • Insertion of the pin-like device containing reactant 1 into a reaction vessel containing reactant 2 and other compounds essential for the reaction as well as reactant 3, the inhibitor results in the initiation of a reaction in which labeled products of the reaction are released into the reaction vessel.
  • the amount of the released label from reactant 1 to the reaction vessel is reciprocally proportional to the amount of reactant 3 (inhibitor) after the pinlike device has been taken out of the reaction vessel.
  • the amount of label remaining on the pin-like device can be determined, 10 which would be directly proportional to the activity of reactant 3 (inhibitor) on reactant
  • the surface of a pin-like device is coated with reactant 1 which is a binding ligand for product 1.
  • Product 1 is formed from reactant(s) 3 linked with either a label or a binding agent capable of binding specifically to reactant 1. That is, product 1 is caused by the biological activity of reactant 2 in a reaction vessel acting on reactant(s) 3.
  • the coated pin-like device is then inserted into the reaction vessel containing product 1 and other non-reacted reactants.
  • the probe is coated with a compound (reactant 1 ) that binds or has binding affinity for a second compound (product 1 ). This second compound (product 1 ) is formed through the action of the bioactive molecule (reactant 2) and components of the reaction mixture (reactant(s) 3).
  • product 1 As product 1 is formed through the action of reactant 2, product 1 binds to reactant 1 which coats the surface of the probe.
  • the amount of labeled non-reacted reactant 3 remaining in the reaction vessel can be directly measured without an additional step and is reciprocally proportional to the activity of reactant 2.
  • the amount of labeled product 1 bound to the probe can be directly measured, which is directly proportional to the activity of reactant 2.
  • the formation of product 1 from reactants 3 due to biological activity of reactant 2 is interfered with by reactant 4 which is an inhibitor of reactant 2.
  • the assay is carried out as described above (Example V), except that in this instance, the amount of labeled non-reacted reactant 3 remaining in the reaction vessel after the pin-like device has been taken out of the reaction vessel is directly proportional to the amount of reactant 4 (inhibitor) present in the reaction mixture.
  • the amount of labeled product 1 bound to the probe can be directly measured, which is reciprocally proportional to the amount of reactant 4 (inhibitor) present in the reaction mixture.
  • reactant 1 which is a binding ligand for reactant 3 being the labeled form of reactant 2 or a labeled compound that will compete for binding with reactant 2 for binding to reactant 1 , as described below.
  • reactant 3 may be chosen such that reactant 1 and reactant 2 will compete for binding to reactant 3, as described below. Competition between reactant 2 and reactant 3 for binding to reactant 1 or between reactant 2 and reactant 1 for binding to reactant 3 are initiated when the pin-like device bound using reactant 1 is inserted into a reaction vessel containing a unknown amount of reactant 2 and a known amount of labeled reactant 3.
  • the probe is coated with a first compound and the reaction vessel contains an unknown quantity of a second compound.
  • a known quantity of a labeled third compound is added to the reaction vessel and the coated probe is inserted into the reaction mixture.
  • the labeled third compound is selected such that the third compound competes with the second compound for binding to the first compound or the first compound and the second compound compete for binding of the labeled third compound.
  • the amount of the labeled reactant 3 remaining in the reaction vessel can be directly measured without an additional step after the pin-like device has been taken out of the reaction vessel and is directly proportional to the amount of reactant 2 in the reaction vessel.
  • the amount of labeled product 1 bound to the probe can be directly measured, which is reciprocally proportional to the amount of reactant 2 in the reaction vessel.
  • labeled reactant 3 competes with reactant 2 for binding to the coated probe.
  • reactant 1 and reactant 2 compete for binding to the labeled third compound.
  • the relative amount of reactant 3 bound to the probe or remaining in the reaction vessel provides information on the amount of reactant 2 in the reaction vessel.
  • Casein is dissolved in PBS to the concentration 5 ug/ml and 100 ul/well is added to each well of the microplate blocked with skim milk.
  • the 96-pin lid is inserted into the wells of the microplate and incubated at 37° C for 3 hr, and is then taken out and rinsed with PBST. As a result of this arrangement, the probe is coated with casein.
  • the reaction is initiated by inserting the 96-pin lid coated with casein. During incubation at 37° C for 30 min, 32 P is transferred from 32 P-ATP to casein by protein kinase. The 96-pin lid is then taken out of the wells of the microplate to stop the reaction.
  • the reaction of phosphate transferring is initiated by inserting the 96-pin lid coated with casein followed by incubation at 37° C for 30 min. Protein kinase catalyzes the transfer of 32 P from 32 P-ATP to casein with the degree of transfer being reciprocally related to the concentration of the inhibitor.
  • the 96-pin lid is taken out of wells of the microplate to end the reaction.
  • proteinase K, elastase, protease XIII, papain, trypsin, pepsin, casein, dimethyl sulfoxide (DMSO), Na 2 HPO 4 , NaH 2 PO 4 , NaCI, Tween-20, citrate, ovomucoid, aprotinin are from Sigma.
  • NHS-coumarin is from Molecular Probes.
  • the 96-well microplate and 96-pin lid are from VWR Canlab. Method:
  • Fluo-casein is dissolved in PBS to the concentration 5 ug/ml and 100 ul/well is added to each well of the microplate blocked with skim milk.
  • the 96-pin lid is inserted into the wells of the microplate, incubated at 37° C for 3 hr, then taken out and rinsed with PBST.
  • the reactions are initiated by inserting the 96-pin lid coated with the fluo- casein and incubated at 37° C or room temperature for 30 min.
  • the fluo-casein is hydrolyzed by protease to release the fluorescent labels into the reaction vessel.
  • the 96-pin lid is taken out of the reaction vessel to stop the reaction.
  • the fluorescent intensity of the label in the wells of the microplate is measured with a fluorometer and is directly proportional to the activity of the protease.
  • a fixed concentration of the protease in the buffer (50 ul/well) is added to the wells containing inhibitor and the controls.
  • the inhibition of the protease by the inhibitor reduces the amount of label released into the reaction mixture.
  • the fluorescent intensity of the label in the wells of the microplate is measured with a fluorometer and is reciprocally proportional to the amount of the inhibitor.
  • telomerase activity in the presence and absence of inhibitor is assayed.
  • this is an example of Examples V and VI, binding of a synthesized product to the probe.
  • telomerase produces a nucleic acid molecule using labeled nucleotides and a primer that includes a binding agent which binds to the compound coating the probe.
  • unextended primer will bind to the probe but will not produce a signal and unincorporated nucleotides will not be able to bind the probe and will remain in the reaction mixture.
  • Tris-acetate buffer pH 8.5
  • potassium acetate ⁇ - mercaptoethanol
  • spermidine MgCI 2
  • EDTA streptavidin
  • dATP dTTP
  • fluo-dGTP fluo-dGTP
  • telomerase S10 RNase
  • biotin-oligonucleotide primer may be purchased from Boehringer Mannheim or elsewhere.
  • Inhibitor (7-deaza-dATP).
  • Streptavidin or avidin is dissolved in a buffer to the concentration 5 ug/ml and 100 ul/well is added to each well of the microplate blocked with skim milk.
  • the 96-pin lid is inserted into the wells of the microplate and incubated at 37° C for 3 hr, and is then taken out and rinsed with PBST.
  • telomerase A series of concentration of telomerase in a reaction mixture containing 50 mM Tris-acetate pH 8.5, 50 mM potassium acetate (KAc), 5 mM ⁇ -mercaptoethanol, 1 mM spermidine, 1mM MgCI 2 , 0.5-2 mM dATP, 0.5-2 mM dTTP, 1.5 uM fluo-dGTP, 1 uM biotin-oligonucleotide primer (TTAGG) 3 , are added to wells of a microplate and the mixture is incubated at 30° C for 1 hr. The reaction of DNA synthesis is stopped by adding the stop solution (10 mM Tris-HCI, pH7.5, 230 mM EDTA and 100 ug/ml RNase) at 37° C for 15 min. 16
  • telomerase A fixed activity of telomerase in the reaction mixture (50 ul/well) is added to the wells containing an inhibitor and the controls. Incubate the wells of the plate at 30° C for 1-2 hr and stop the DNA synthesis with the stop solution.
  • Na 2 HPO 4 , NaH 2 PO 4 , NaCI, dimethyl sulfoxide (DMSO) are from Sigma.
  • NHS-fluorescein is from Molecular Probes.
  • a mucus receptor from a piglet and E. coli K88 fimbriae are prepared in a laboratory. Inhibitors are from different sources.
  • Receptor is dissolved in PBS (pH 7.2) to a concentration of 5 ug/ml and 100 ul/well is added to each well of the microplate blocked with skim milk.
  • the 96-pin lid is inserted into the wells of the microplate and incubated at 37° C for 3 hr, and is then taken out and rinsed with PBST.
  • the fluorescent intensity of the fluo-fimbriae remaining in the wells of the microplate is determined using a fluorometer and is directly proportional to the amount of the receptor (competitor).
  • the fluorescent intensity of the fluo-fimbriae remaining in the wells of the microplate is determined using a fluorometer and is directly proportional to the amount of the fimbriae or E. coli cells (competitor). 5. Inhibitor of the fimbriae-receptor binding assay:
  • the reactions between the inhibitor and the immobilized receptor for binding to fluo-fimbriae or between the inhibitor and the fluo-fimbriae for binding to the immobilized receptor are initiated by inserting the 96-pin lid coated with the receptor into the wells. After incubation at 37° C for 1 hr the 96-pin lid is taken out of the vessel to stop the reaction.
  • the fluorescent intensity of the fluo-fimbriae remaining in the wells of the microplate is determined using a fluorometer and is directly proportional to the amount of the inhibitor (competitor).
  • the above-described assay has a wide range of applications due to its versatility. Furthermore, the assay does not require washing or "stopping" steps which are time-consuming and can lead to loss of signal or contamination. It is of note that in the examples above, the probe comprises a pin- shaped member and the reaction vessels are wells of a microtiter plate. However, it is to be understood that the probe may be of suitable shapes and arrangements, as discussed above, as may the reaction vessels.

Abstract

L'invention concerne un procédé d'analyse simplifiant grandement la détection et la détermination de l'identité, de la quantité et de l'activité de molécules à activité biologique. Cette analyse permet une mesure rapide et facile de la quantité et de l'activité d'enzymes, d'inhibiteurs enzymatiques, de lectines, de récepteurs et d'autres substances biologiquement actives, à l'aide d'une technique à une étape permettant d'isoler un réactif d'un produit après achèvement de la réaction. Ce procédé consiste à introduire une sonde ayant une surface extérieure recouverte d'un réactif dans un récipient de réaction contenant les composés d'intérêt. On laisse la réaction se faire pendant un temps donné, puis on retire la sonde du récipient de réaction. On peut ensuite mesurer la quantité de produits ou de réactifs marqués dans le récipient de réaction, soit directement, soit en déterminant la quantité de réactif restant sur la sonde, sans qu'il y ait besoin d'étapes supplémentaires, telles que le lavage.
EP99917707A 1998-04-16 1999-04-16 Phase solide de type peigne pour mesurer les analytes Withdrawn EP1071954A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CA 2228821 CA2228821A1 (fr) 1998-04-16 1998-04-16 Mesure de matiere bio-active par le biais d'un dispositif en forme d'aiguille
CA2228821 1998-04-16
PCT/CA1999/000364 WO1999054734A1 (fr) 1998-04-16 1999-04-16 Phase solide de type peigne pour mesurer les analytes

Publications (1)

Publication Number Publication Date
EP1071954A1 true EP1071954A1 (fr) 2001-01-31

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EP99917707A Withdrawn EP1071954A1 (fr) 1998-04-16 1999-04-16 Phase solide de type peigne pour mesurer les analytes

Country Status (4)

Country Link
EP (1) EP1071954A1 (fr)
AU (1) AU3590899A (fr)
CA (1) CA2228821A1 (fr)
WO (1) WO1999054734A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030027193A1 (en) * 2001-07-23 2003-02-06 Zhibo Gan Non-isotopic rapid method and kit for measurement of levels of therapeutic compounds
DE60225587T2 (de) 2001-11-08 2009-04-02 Aaron Diamond Aids Research Center Protease assay zur kontrolle medikamentöser therapie
GB0210535D0 (en) * 2002-05-08 2002-06-19 Novartis Ag Organic compounds

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0106855A1 (fr) * 1982-04-16 1984-05-02 Genefusion S.A. Dispositif d'analyse pour echantillons biologiques
JPH0823558B2 (ja) * 1984-11-27 1996-03-06 オ−ジエニクス リミテツド 検定装置
AT394114B (de) * 1989-07-13 1992-02-10 Immuno Ag Verfahren zur bestimmung von antigenen und/oder antikoerpern in menschlichen koerperfluessigkeiten sowie set zur durchfuehrung des verfahrens

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9954734A1 *

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CA2228821A1 (fr) 1999-10-16
AU3590899A (en) 1999-11-08
WO1999054734A1 (fr) 1999-10-28

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