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WO1986006170A1 - Analyse homogene directe - Google Patents

Analyse homogene directe Download PDF

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Publication number
WO1986006170A1
WO1986006170A1 PCT/US1986/000668 US8600668W WO8606170A1 WO 1986006170 A1 WO1986006170 A1 WO 1986006170A1 US 8600668 W US8600668 W US 8600668W WO 8606170 A1 WO8606170 A1 WO 8606170A1
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WO
WIPO (PCT)
Prior art keywords
substance
labeled
bindable
test sample
enzyme
Prior art date
Application number
PCT/US1986/000668
Other languages
English (en)
Inventor
Paul A. Liberti
Original Assignee
Immunicon 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 Immunicon Corporation filed Critical Immunicon Corporation
Publication of WO1986006170A1 publication Critical patent/WO1986006170A1/fr
Priority to DK591186A priority Critical patent/DK591186A/da

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    • 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

Definitions

  • the present invention is directed to the qualitative and quantitative determination of specific binding substances.
  • the invention enables the presence or amount of one substance of any specific binding pair of substances to be determined by means of its complementary binding substance.
  • the present invention is useful for determining whether a specific binding substance is present in a test sample in excess of a predetermined amount.
  • the invention also permits quantitative measurement of any excess of the specific binding substance present in the test sample above a predetermined amount.
  • the present invention has a variety of applications/ it is particularly useful in the determination of immunochemical binding substances- in biological fluids/ for example, the detection or quantification of antigen or antibodies in human serum. - PRIOR ART
  • an immunoassay involves the determination of an i munoreactive substance, either directly or indirectly, by means of an immune reaction between the immunoreactive substance and a labeled form of its i munospecific conjugate or other receptor system.
  • substances that are not immunogenic by themselves, such as haptens/ can be determined by immunoassay if they are bound to larger carrier substances which are capable of inducing antibody to the lower molecular weight substance.
  • Immunoassays are useful in the detection of immune reactions in blood serum and are also employed in a number of immunohistochemical methods performed on tissue.
  • Immunoassays may be carried out with all of the immunoche ical binding substances in solution/ or with the immunoreactive substance or its immunospecific conjugate affixed to a solid support, such as glass or plastic tubes/ beads, or icrotiter plates.
  • a solid support such as glass or plastic tubes/ beads, or icrotiter plates.
  • the latter is known as a solid-phase immunoassay.
  • Solid-phase immunoassay is widely used due to its simplicity of performance and the ease with which the labeled complex may be separated from the unreacted, labeled antigen or antibody.
  • One type of solid-phase immunoassay involves a direct non-competitive binding technique for determination of immunochemical binding substances.
  • an immunoassay of this type to determine antibody, for example/ antigen is immobilized on a solid support and then contacted with the test sample containing the antibody to be determined. Thereafter/ a second antibody which is labeled and reacts specifically with the first antibody is added to the test sample. Because the second antibody is' labeled the presence of the first antibody in the sample may be determined.
  • this technique commonly known as a "sandwich technique”/ specific antibody can readily be detected in blood serum.
  • Solid phase immunoassays are also commonly performed as competitive binding assays ' / based upon competition between labeled and an unlabeled forms of an immunoreactive substance for binding sites on the immunospecific conjugate.
  • the test sample is usually mixed with labeled antigen, and then contacted with the corresponding antibody bound to a solid support/ with the labeled and unlabeled antigen competing for antibody binding sites.
  • the test sample is then separated into a liquid phase and a solid phase and the relative amount of labeled antigen present in either phase is quantitatively determined.
  • the direct, competitive and saturation techniques used in performing solid-phase immunoassays each permit quantitative determinations of immunoreactive substances by way of calculations based on the extent of binding of the labeled reagent employed in the assay.
  • Radioisotopes, enzymes and various chromophoric substances are commonly employed as labels in the above-described immunoassays. Radioisotopes provide a readily measurable signal which permits the results of the assay to be determined directly. Immunoassays employing such labels are generally characterized by exceptional sensitivity and accuracy.
  • Enzymes have been proposed as labels for immunochemical binding substances, especially in assays intended for home use.
  • a notable advantage of the enzyme labels is that enzyme activity is detectable by the naked eye or by inexpensive detection equipment/ such as a colorimeter.
  • the enzyme activity is measured by using a suitable substrate for producing an enzyme-catalyzed reaction/ typically involving production or extinction of a colored compound whose light adsorption may be readily determined either qualitatively or quantitatively.
  • Immunoassays employing enzyme labels are commonly referred to as enzyme-linked immunoabsorbant assays (ELISA).
  • test strip or "dip stick” technique.
  • test strip or "dip stick” technique.
  • These assays employ a bibulous substrate or carrier/ which may be a natural or synthetic polymeric material, but is typically a cellulosic material, such as filter paper, to which is affixed the immunospecific conjugate of the immunoreactive substance being determined.
  • the enzyme label may be linked to the immunospecific conjugate bound to the support, or with a component of the immunochemical binding pair in the test sample.
  • assays are intended to eliminate the manipulative steps, e.g. washing and incubation which are normally involved in the standard ELISA technique, thereby reducing the chance of error/ so as to permit use by untrained personnel. Moreover/ because the procedure may be conducted rapidly and gives a visual result/ these assays are desirable for use in a doctor's office and in the home.
  • an ELISA-type assay employing a test strip having antibodies bound thereto is described in U.S. Patent No. 4,168,146. This assay is based on the aforementioned "sandwich technique".
  • the antibody-bearing test strip is immersed in the test sample suspected of containing the antigen to be determined. After the test sample migrates along the test strip, and any antigen present in the test sample reacts with the antibodies bound to the test strip, the test strip is contacted with a solution containing antibodies linked to an enzyme label and the enzyme activity is determined, as described above. '
  • test strip-type assay employing a combination of enzymes
  • the test strip is in the form of an i munochromatograph having a porous support permitting solvent travel, a plurality of a specific binding pair substance (e.g. antibody) and an enzyme, the latter two components being non-diffusively and uniformly bound to the support to define an immunosorbing zone.
  • a specific binding pair substance e.g. antibody
  • an enzyme-labeled specific 5 binding pair member e.g. enzyme-labeled antigen
  • the target substance being determined e.g. antigen
  • the enzyme label is preferably related to the enzyme on the immunochromatograph, such that the substrate of one is the product of the other. In practice/ the immunochromatograph is contacted with the
  • test sample for a time sufficient for the test sample to migrate across the immunosorbing zone.
  • the immunochromatograph is also contacted with the labeled specific binding pair substance in a solvent medium, so that the labeled specific binding pair ' substance binds
  • the immunosorbing zone is contacted with a development solution containing a substrate for the enzyme label to produce a measurable signal/ and the distance of the aforesaid 5 border from one end of the immunochromatograph is determined, in order to quantify the amount of target substance in the test sample.
  • test strip When used for the determination of antigen, the test strip comprises a first zone containing antigens and enzyme-linked antibodies which are capable of im unochemcially reacting with the antigens and a second zone containing a substance capable of undergoing a color forming reaction with the enzyme label linked to the antibodies/ thereby to indicate the presence of the antibodies in the second zone.
  • the antibodies are diffusively bound in the first zone/ such that they will pass from the first zone to the second zone upon reaction with antigens from the test sample migrating through the first zone/ but will not diffuse through the first zone in the absence of migrating antigens.
  • the test strip is contacted with a test sample suspected of containing the target antigen for a time sufficient for the test sample to migrate along the test strip, and the presence or absence of any color change is observed as an indication of the presence or absence of the target antigen in the test sample. It is disclosed that this assay may be adapted for quantitative determinations by providing it with multiple, separate, regions, each containing a different amount of immobilized reference antigen, the position of the color change being dependent on the concentration of the antigen in the test sample.
  • test strips proposed heretofore for implementing the ELISA technique may provide a rather uncomplicated and relatively rapid procedure for the qualitative and/or quantitative determination of immunochemical binding substances, these prior art assays do not enable the determination of the target substance in excess of a predetermined amount, or the quantitative measurement of any excess of the target substance beyond the predetermined amount.
  • the present invention provides a simple and convenient method and test kit for determining the presence of a bindable substances in a test sample in 5 excess of a predetermined amount and the quantitative measurement of any such excess.
  • an array of complementary binding substance which is capable of binding to the bindable substance and having a predetermined binding
  • test sample and the array of complementary binding substances are contacted for a time sufficient to allow binding of bindable substance in the test sample to the complementary binding substance. Thereafter/ a carrier
  • the medium containing a given amount of labeled bindable substance is contacted with the array of complementary binding substance.
  • the given amount of labeled bindable substance is such that, when added to the predetermined amount of bindable substance being
  • the binding capacity of the complementary binding substance is substantially filled.
  • the absence or significant presence of unbound labeled bindable substance is then determined to differentiate whether or not the bindable substance is present in the test
  • the binding capacity of complementary binding substance would be substantially filled by the sum total of the predetermined amount of bindable substance being determined (if present in the test sample) and the 0 given amount of labeled bindable substance that is brought into contact with the array of complementary binding substance, the significance presence of unbound labeled bindable substance indicates that bindable substance was present in the solution in excess of the predetermined amount.
  • the amount of unbound labeled bindable substance can also be determined quantitatively to give a measurement of the degree to which the bindable substance in the test sample exceeds the predetermined amount.
  • the array of the present invention is described as a homogeneous array, in that no separation of bound and free specific binding pair substance is required.
  • the test kit of the present invention comprises the substances and devices necessary to perform the above-described method.
  • the test kit includes: 1) an array of complementary binding substance on a support, the array having a predetermined binding capacity for the bindable substance being determined; and 2) labeled bindable substance in an amount sufficient/ when added to the predetermined amount, to substantially fill the binding capacity of the complementary binding substance.
  • the method and test kit of the present invention provides a simple, rapid and reliable method for determining chemical inbalances in various biological and other fluids.
  • the present invention may be used to particular advantage in medical diagnosis and in detecting drugs in relatively low concentrations in body fluids.
  • the present invention is directed to the qualitative and quantitative determination of one substance of a specific binding pair, consisting of a bindable substance and its complementary binding substance.
  • the binding pair substance being determined is referred to as the bindable substance, although in practice, either of the binding pair substances could be determined using the method and test kit of the present invention.
  • binding substance refers to any substance which reacts with a complementary binding substance based on the mutual specific binding affinity between the two substances.
  • binding substance includes, but is not limited to proteins/ hormones, both polypeptides and steroids, carbohydrates and glycoproteins. Representative examples of specific binding pairs are antigens and their antibodies/ haptens and their antibodies, hormones and their receptors/ vitamins and their receptors and toxins and their receptors/ determinations of all of which are within the scope of the present invention.
  • complementary binding substance refers to the specific binding partner of the bindable substance being determined.
  • the complementary binding substances used in the practice of this invention are generally underivitized/ which means that they are not bound to/ or otherwise conjugated with/ any other chemical moiety.
  • the complementary binding substance is provided in the form of an array on a suitable support.
  • the support can be in the form of a glass or plastic capillary/ a paper strip, polymer beads/ a column packed with a suitable matrix/ or the like.
  • affixing a complementary binding substances to such supports. Such methods employ/ for example/ covalent bonding as well as other types of affixation/ such as absorption. Any method may be employed that avoids significant diffusion of the bound substance.
  • the complementary binding substance is preferably applied to the support as a one-dimensional lattice/ by which is meant a linear array of binding sites such that target substance binding and saturation begins at one end and proceeds uniformly to the other. This may be achieved by arranging the binding substance on a three dimensional support whose length is significantly greater than width/ height or cross sectional area.
  • the binding component may be affixed to a two dimensional support whose length is significantly greater than its width.
  • the "predetermined amount" employed as the standard in carrying out the present invention may vary depending on the particular type of bindable substance in question and the nature of the assay being conducted. In the case of biological fluids/ for example/ there exists established levels of various components considered to be the normal or average amounts/ which are commonly referred to as "clinical norms". In applying the present invention as an immunoassay, the predetermined amount of bindable substances to be determined would generally be the clinical norm.
  • the method of the present invention involves the use of a labeled form of the bindable substance being determined, or a substance having the same binding affinity for the complementary binding substance as the bindable substance being determined.
  • Suitable labels include radioisotopes, enzymes and chromophoric substances/ the latter including dyes which absorb light in the U.V. or visible region of the electromagnetic spectrum, and fluroescent or phosphorescent substances.
  • Appropriate methods for linking of any of the aforesaid labels to a bindable substance of the type described above are well known to those skilled in the art.
  • An important requirement of the method of labeling the bindable substance is that it not sterically hinder or interfere with the reactive sites of either component of the specific binding pair.
  • Radioactive or phosphorescent labels rarely interferes with the stereochemical properties of chemical substances to which they are linked.
  • the labeled bindable substance may consist of antigen covalently linked to an enzyme.
  • the enzyme must be linked to the antigen or hapten in such a way that the affinity of the antigen or hapten for the antibody is not impaired, or otherwise diminished.
  • Enzymes that have exhibits enzymatic activity after in conjugated form include, catalases, perioxidases, glucuronidases, glucosidases, galactosidases, urease and oxidoreductaces, such as glucose oxidase and galactose oxidase.
  • An important aspect of the present invention is that a known relationship exists between the value of the predetermined amount (i.e. the clinical norm in the case of an immunoassay) of the bindable substance being determined, the amount of label, and the number of available binding sites of the complementary binding ' substance on the array. Since the bindable substance and the label are the same substance/ or are substances having substantially the same binding affinity for the complementary binding substance on the array/ any bindable substance present in the test sample competes equally with the labeled bindable substance for the available binding sites on the complementary binding substance.
  • the predetermined amount i.e. the clinical norm in the case of an immunoassay
  • the array of complementary binding substance has a known binding capacity for the bindable substance/ which is substantially filled by the clinical norm of bindable substance and the given amount of labeled binding substance used in carrying out the method, the presence of bindable substance in the test sample in excess of the clinical norm may be readily determined. Thus, if more than the clinical norm of bindable substance is present in the test sample, there will not be sufficient binding sites on the array to accommodate all of the unlabeled and labeled bindable substance and and an excess, or "spillover"/ of both unlabeled and labeled bindable substance will result.
  • the unlabaled and labeled bindable substance are competing for the available binding sites on the array of complementary binding substance with equal affinity/ the spillover will virtually always contain labeled bindable substance/ since, as noted above, the unlabeled and labeled bindable substance will be bound in proportion to the relative amount of each that is contacted with the array of complementary binding substance. Accordingly, the significant presence of unbound labeled bindable substance in the spillover differentiates whether or not the bindable substance in the test sample exceeds the established norm. As used herein, the expression "significant presence” refers to an amount which is two-three-times background noise/ or error level in the test system.
  • a test sample believed to contain bindable substance to be determined is contacted with the array of complementary binding substance for a time sufficient to allow binding of the bindable substance to the complementary binding substances to occur.
  • the available binding sites on the complementary binding substance are filled to the extent of the bindable substance present in the test sample.
  • the array is then contacted with labeled bindable substances in a carrier medium.
  • Suitable carrier mediums include aqueous or other polar solvents, such as ether or alcohol for biological systems. Non-polar solvents may be used in other systems where appropriate.
  • the carrier medium may be buffered to the desired pH range, and a detergent may be added to reduce non-specific binding. If the test sample contained the clinical norm of bindable substance, then the labeled bindable substance will substantially fill the remaining available binding sites of the complementary binding substance on the array.
  • the labeled bindable substance may be incorporated in the test sample for contact with the array of complementary binding substance simultanesouly with the bindable substance being determined. According to the this procedure, the labeled and unlabeled bindable substances compete directly for available binding sites on the array. In this procedure the test sample serves as the carrier * medium.
  • the number of sites provided on the array of conjugate antibody substantially equals the amount of antigen corresponding to the clinical norm and the amount of labeled antigen which is added in carrying out the method. Since the number of available binding sites on the array is known, and the clinical norm of the antigen in question is also known, the amount of labeled bindable substance employed may be varied to achieve the desired substantial filling of the array. For example, if the clinical norm for a certain antigen fills 60 binding sites on an array of antibody, then an array having, for example, 120 available binding sites may be provided and used in conjunction with an amount of labeled antigen which will substantially fill the remaining 60 binding sites.
  • the present method is adaptable for use in a physician's office or at home/ in the form of a simple test kit.
  • the test kit includes a suitable support to which is affixed an array of complementary binding substance having a predetermined number of binding sites, along with a predetermined amount of labeled bindable substance.
  • the amount of labeled bindable substance would depend on the clinical norm of the substance being tested for and the number of sites on the array. If such a test kit employed enzyme as the label/ a visible signal would be produced in the spillover thus obviating specialized detection equipment.
  • Such an assay could be performed on body fluids, such as saliva or urine by relatively untrained personnel.
  • the effluent obtained after contacting the test sample or carrier medium with the array contains unbound labeled and unlabeled antigen.
  • the amount of unbound labeled antigen in the effluent may then be measured.
  • the enzymatic activity of the effluent may be determined, for example, by measuring color intensity in a colorimeter. If a radioactive label is used, the radioactivity of the effluent may be measured.
  • the amount of labeled antigen in the effluent test solution or carrier medium can be easily calculated, since binding of the labeled and unlabeled antigen occurs proportionally to the concentration of labeled and unlabeled antigen initially present. Specific measurements may be made using a standard curve, as is well known in the art. For example, where a column is employed containing an array of antibodies providing 300 binding sites, and the clinical norm for the antigen in the test sample occupies 80 of the binding sites, 220 units of labeled antigen are added to the test solution.
  • a plot of effluent activity (labeled antigen in "spill over") plotted against test antigen present in the sample solution (in excess of 80 units) provides a curve which is substantially linear.
  • an array of antibody may be provided having a binding capacity of/ for example, 300 units of antigen. 220 units of enzyme labeled antigen would then be added to the test sample or to a separate carrier medium. Adjacent to the array of 300 sites/ on a separate portion of the same solid support/ there would be affixed an indicator substance, such as a substrate which produces a color change under the influence of the enzyme label. The sample solution would then be contacted with the array so as to fill the 300 available binding sites before reaching and contacting the indicator substance.
  • the amount of antigen present in the sample solution were not greater than the clinical norm of 80 units, then substantially all of the ant.igen, both labeled and unlabeled, would be bound to the array of antibody, and none of the antigen would reach the indicator portion of the support. If, however, the test sample were to contain more than the clinical norm of 80 units of antigen, the labeled antigen would reach the indicator substance on the support and produce an visible signal, by means of a color change.
  • This latter embodiment of the invention enables quantitative determination of the amount by which the bindable substance in a test sample exceeds the clinical norm.
  • the area of the indicator substance on the solid support which undergoes reaction with the label of the immunoreactive substance relates to the amount of the substance present in excess of the established norm.
  • the area of the indicator substance undergoing reaction with the label bears essentially the same functional relationship to the excess as the data reported above in Table I.
  • a porous cellulosic material e.g. an elongated paper strip/ as the solid support in the embodiment just described/ so as to permit the test sample to diffuse readily along the support by capillary action.
  • Immunoreactive substances and indicator substances such as enzymes may be bound directly to a paper support by methods well known in the art.
  • EXAMPLE I This example describes the making of a test device for use in the determination of DNP and DNP-derivitized proteins, in accordance with the present invention.
  • Rabbit antiserum directed against DNP was prepared by immunization of rabbits with dinitrophenylated random sequence polypeptide composed of 40% glutamic acid, 30% lysine and 30% alanine.
  • the synthetic polypeptide had 10% of its lysines conjugated with DNP.
  • DNPj_o BSA Bovine Serum Albumin
  • the titer of the antiserum with respect to DNP was determined to be 2.1 mg. antibody/ml. of serum.
  • Antiserum was coupled to Sepharose 4B by the sodium carbonte/Sepharose/cyanogen bromide activation method. Prior to coupling to Sepharose, the antiserum was decomplemented by adsorption with rabbit BSA anti-BSA precipitates, dialyzed against buffer (0.1 M NaHC03) and centrifuged in a microfuge. To achieve uniform coupling all reagents were mixed rapidly and resulted in an adsorbent slurry.
  • Micro-columns containing the adsorbent slurry were constructed using 20 microliter disposable micro-pipettes as the body of the micro-columns. A 10 to 15 cm. length of 1.5 mm. internal diameter plastic tubing was fitted to the bottom of the micro-pipette to serve as an outflow tube. Prior to fitting the pla ⁇ tic tubing over- the end of the micro-pipete a small piece of cotton or a fragment of tissue was inserted into the end of the plastic tubing. When fitted over the end of the micro-pipette, the tissue or cotton material butted against the bottom of the micro-pipette forming a barrier through which adsorbent could not pass.
  • micro-pipette was fitted with a funnel constructed by taking a standard laboratory plastic pipette tip and slicing the end to allow insertion of the micro-pipette.
  • the micro-columns were first filled with phosphate buffered saline (PBS) containing 1% bovine serum albumin and incubated for 1 hour to reduce non-specific adsorption to the sub-nanogram levels.
  • PBS phosphate buffered saline
  • the columns were filled by introducing an appropriate charge of the adsorbent slurry. Each column was brought to a bed volume of 20 icroliters by removing excess adsorbent using a needle and syringe.
  • the columns were 69 to 72 mm. high and could be filled with consistency to within 0.5 mm. Therefore/ bed volumes could be controlled to within 0.7%.
  • micro-columns were calibrated using radioactive DNPi.aBSA.
  • the D Pi.sBSA which was carefully determined by appropriate absorbance measurements at 280 and 360 nanometers, was labeled with iodine 125 by the Iodogen method.
  • An activity of 6 microCuries/Microgram was achieved and it was found, as result of binding to mini-columns (100 to 300 microliters) , that the labeled protein was undamaged by labeling, since the appropriate amounts of labeled protein were bound when diluted with unlabeled protein.
  • This example describes an experiment in which labeled DNP/BSA was added to the test sample for simultaneous delivery to the test device with unlabeled DNP/BSA.
  • Mini-columns of 460 nanogram capacity were constructed as described in Example I. Samples containing known amounts of unlabeled DNP/BSA were mixed with 200 nanograms (260,000 counts per minute) of labeled DNP/BSA and applied to the micro-columns. After delivery of the test samples seven column volumes of BSA/PBS were passed through each column, and the effluent was collected in a counting vial and counted. The results are shown in Table III, two trials being run of each sample.
  • Example II The experiment of Example II was performed using a sequential rather simultaneous delivery of the labeled and unlabeled DNP/BSA to the test device.
  • test samples were delivered to the column first in a volume of 30 microliters, followed by 20 microliters of buffer and then by 200 nanograms (260,000 counts per minute) of the labeled antigen.
  • the invention may be used for the determination of specific binding pair substances, (e.g. DDT and naturally occurring DDT-binding substances), present as hazardous contaminants in ground water in excess of standards established by federal regulation.
  • specific binding pair substances e.g. DDT and naturally occurring DDT-binding substances
  • the method and test kit of the present invention were used for assaying ground water for compliance with federal regulatory requirements/ the predetermined amount would be the standard established by the relevant regulation.

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Abstract

Procédé et kit d'essai pour déterminer la présence, dans un échantillon d'essai, d'une substance de liaison, par ex. un antigène ou un anticorps, en excédent d'une quantité prédéterminée, et pour effectuer la mesure quantitative de cet excédent.
PCT/US1986/000668 1985-04-10 1986-04-02 Analyse homogene directe WO1986006170A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DK591186A DK591186A (da) 1985-04-10 1986-12-09 Fremgangsmaade til bestemmelse af gensidig bindingsaffinitet for stofpar

Applications Claiming Priority (2)

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US72158985A 1985-04-10 1985-04-10
US721,589 1985-04-10

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WO1986006170A1 true WO1986006170A1 (fr) 1986-10-23

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Cited By (11)

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US5028535A (en) * 1989-01-10 1991-07-02 Biosite Diagnostics, Inc. Threshold ligand-receptor assay
WO1992002818A1 (fr) * 1990-08-10 1992-02-20 Purdue Research Foundation Dosage immunologique a addition sequentielle sur une matrice
EP0475783A1 (fr) 1990-09-14 1992-03-18 Biosite Diagnostics Inc. Anticorps contre les ligand-analogues et leur utilité dans les dosages ligand-recepteurs
EP0494509A1 (fr) * 1990-12-11 1992-07-15 Enzymatics, Inc. Essai, employant la signalisation analoque à digitaire, pour la présence d'un analyte conjointement avec une partie à liaison, appareil pour la mise en oeuvre de ce processus, et méthode pour la mise en oeuvre de cet essai
US5750339A (en) * 1994-11-30 1998-05-12 Thomas Jefferson University Methods for identifying fetal cells
US5939272A (en) * 1989-01-10 1999-08-17 Biosite Diagnostics Incorporated Non-competitive threshold ligand-receptor assays
WO2004061422A3 (fr) * 2002-12-31 2004-09-02 Rodi Pharma Inc Jeux ordonnes d'enzymes de liaison et procedes proteomiques haut debit
EP2189791A3 (fr) * 1998-02-04 2011-03-09 Life Technologies Corporation Microréseaux et leurs utilisations
US8585971B2 (en) 2005-04-05 2013-11-19 The General Hospital Corporation Devices and method for enrichment and alteration of cells and other particles
US8921102B2 (en) 2005-07-29 2014-12-30 Gpb Scientific, Llc Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US8986966B2 (en) 2002-09-27 2015-03-24 The General Hospital Corporation Microfluidic device for cell separation and uses thereof

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US3991174A (en) * 1970-07-20 1976-11-09 Rafa Laboratories Ltd. Method of determining concentration of luteinizing hormone in body fluid
US4039652A (en) * 1973-10-11 1977-08-02 Miles Laboratories, Inc. Column method of immunoassay employing an immobilized binding partner
US4446232A (en) * 1981-10-13 1984-05-01 Liotta Lance A Enzyme immunoassay with two-zoned device having bound antigens
US4533629A (en) * 1981-04-17 1985-08-06 Syva Company Simultaneous calibration heterogeneous immunoassay

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US3991174A (en) * 1970-07-20 1976-11-09 Rafa Laboratories Ltd. Method of determining concentration of luteinizing hormone in body fluid
US4039652A (en) * 1973-10-11 1977-08-02 Miles Laboratories, Inc. Column method of immunoassay employing an immobilized binding partner
US4533629A (en) * 1981-04-17 1985-08-06 Syva Company Simultaneous calibration heterogeneous immunoassay
US4446232A (en) * 1981-10-13 1984-05-01 Liotta Lance A Enzyme immunoassay with two-zoned device having bound antigens

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5028535A (en) * 1989-01-10 1991-07-02 Biosite Diagnostics, Inc. Threshold ligand-receptor assay
EP0378391A3 (fr) * 1989-01-10 1991-10-02 Biosite Diagnostics Inc. Des essais ligands-récepteurs utilisant un seuil
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JPS62502495A (ja) 1987-09-24
EP0218680A1 (fr) 1987-04-22

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