WO1996022533A1 - Procede pour immobiliser des haptenes sur un article de test - Google Patents

Procede pour immobiliser des haptenes sur un article de test Download PDF

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Publication number
WO1996022533A1
WO1996022533A1 PCT/US1996/000416 US9600416W WO9622533A1 WO 1996022533 A1 WO1996022533 A1 WO 1996022533A1 US 9600416 W US9600416 W US 9600416W WO 9622533 A1 WO9622533 A1 WO 9622533A1
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WIPO (PCT)
Prior art keywords
hapten
solid phase
carrier molecule
binding substance
phase surface
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Application number
PCT/US1996/000416
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English (en)
Inventor
Robert F. Zuk
Jeffrey A. Pierce
Stroughton L. Ellsworth
Original Assignee
First Medical, 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 First Medical, Inc. filed Critical First Medical, Inc.
Priority to AU46980/96A priority Critical patent/AU4698096A/en
Publication of WO1996022533A1 publication Critical patent/WO1996022533A1/fr

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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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • 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 relates generally to the production of test articles used for determining the presence of analyte in biological samples. More particularly, the present invention relates to a method for immobilizing haptens onto solid phase surfaces.
  • Specific binding assays rely on detecting an analyte using a specific binding substance which reacts with the analyte in a highly selective manner. Numerous protocols and formats for performing such specific binding assays are described in the patent, technical, and medical literature.
  • many specific binding assays employ a solid phase surface having a specific binding substance, such an antibody or antibody fragment, immobilized on the surface. The surface may then be utilized to capture analyte through direct or indirect binding of the immobilized specific binding substance to the target analyte.
  • Immobilization of the specific binding substance on the solid phase surface can be problematic in a number of respects.
  • the specific binding substance must be applied and attached to the surface under conditions which do not result in loss of binding activity, e.g. conditions which do not result in denaturing of antibodies or other binding protein.
  • a variety of otherwise desirable application techniques such as ink jet printing, are difficult to use since they can denature labile proteins, such as antibodies.
  • many antibody binding protocols which are sufficiently gentle to avoid denaturing the binding substances require multiple steps which are time consuming and labor intensive, making preparation of the solid phase surface expensive. The expense is further increased when it is desired to form a plurality of discrete, frequently very small, reaction zones on a single solid phase surface.
  • many specific binding substance immobilization techniques require considerable time to achieve maximum binding of the substance to the solid phase surface. Attempts to shorten the binding time often result in the desorption or sloughing of the substances from the test surface.
  • U.S. Patent No. 5,316,784 describes a two-step process for attaching immunologically active substances, such as antibodies, antigens, and binding proteins, to solid phase surfaces by applying a mixture of the active substance and a linking group having photoactivable groups to the surface. After allowing the mixture to absorb into the surface, the active substance is covalently attached by activating the linking group.
  • U.S. Patent No. 5,258,041 describes the use of spacer arms having hydrophobic guiding groups for attaching biomolecules to solid phase supports.
  • Methods for preparing solid phase surfaces for use as test articles in biological and other assays comprise immobilizing a hapten on the solid phase surface via a carrier molecule, typically a macromolecule such as a protein, which is capable of non-specific adherence to the surface.
  • a conjugate, of the hapten and carrier molecule typically covalently attached is applied to the surface so that the carrier molecule adheres to and the hapten is immobilized precisely at the site of application.
  • the hapten is a small molecule which will retain binding activity even under relatively harsh conditions of application that would denature antibodies and other specific binding proteins. While the carrier molecule, which may be a protein, might be denatured under such conditions, loss of biological activity of the carrier molecule is not significant since it will still provide for the necessary non-specific adherence to the solid phase surface and preservation of the attached hapten.
  • the test articles with immobilized hapten may find use in assays where the hapten is used to bind directly or indirectly to an analyte of interest, typically where the analyte has previously been bound to anti-hapten antibody.
  • the test article may be further prepared by introducing a specific binding substance, usually an antibody or antibody fragment, to the hapten via an anti- hapten linker moiety.
  • a covalently attached conjugate of the specific binding substance and the anti- hapten binding substance may be reacted with the solid phase surface of the test article under relatively mild (non- denaturing) conditions to form a non-covalent linkage between the hapten and the specific binding substance.
  • Such fabrication methods are preferably performed using printing processes where discrete droplets of a liquid medium containing the hapten-carrier molecule conjugate are applied to the solid phase surface to form precise and accurately defined reaction zones, often in complex geometric patterns which would be difficult to produce using other techniques.
  • the printing processes also allow for application of different haptens within different reaction zones, to produce test articles which are useful in multiple analyte assays.
  • ink jet printing is employed where the liquid medium is contained in a reservoir and the droplets are propelled or ejected through an aperture by known ink jet printing technologies. Both piezoelectric and thermal ink jet printing systems may be used for performing the methods of the present invention.
  • Test articles according to the present invention will have a solid phase test surface having hapten immobilized thereon via a carrier molecule.
  • the carrier molecule will usually be non-specifically attached to the solid phase test surface, but in some cases, it may be desirable to further covalently cross-link the carrier molecule to the surface (using conventional cross-linking methods employed after the initial non-covalent attachment) .
  • Such further covalent attachment has several advantages. First, cross-linking of the carrier can reduce the time necessary to achieve high affinity or avidity binding of the complex to the solid phase. Second, presence of the carrier molecule reduces the risk of denaturing the hapten which provides the active binding site in the complex. Third, the chance of desorption or other loss of the complex is greatly reduced.
  • the solid phase may be composed of any material conventionally used in assay systems, such as organic polymers, glasses, ceramics, metals, absorptive papers, and the like. The haptens and the carrier molecules will generally be as described above.
  • FIG. 1 is a schematic illustration of an ink jet printing system adapted for the printing of hapten-carrier molecule conjugates on a solid phase disk surface according to the method of the present invention
  • Fig. 2 is an electrical schematic diagram showing the components of the head drive electronics of the ink jet printing system of Fig. l;
  • Fig. 3 is an illustration of a biotin-BSA printed disk showing discrete patterns and demonstrating the retention of streptavidin binding activity
  • Fig. 4 is a graph illustrating the desorption of biotin-BSA as a function of liquid-phase adsorption time
  • Fig. 5 is a graph illustrating the amount of time required for maximal biotin-BSA binding to the acrylic test surface once spotted with the ink jet system
  • Fig. 6 is a graph illustrating the acceleration of biotin-BSA binding by heat treatment
  • Fig. 7 is a graph illustrating the results of an assay for CKMB using a solid phase test article prepared according to the present invention, wherein binding of CKMB to the test article is determined both interferometrically and via light absorption.
  • the target analyte will be a member of a specific binding pair (SBP) , including compounds, compositions, aggregations, and virtually any other substance which may be detected or reacted by immunological or equivalent techniques. That is, the analyte, or a portion thereof, will usually be antigenic or haptenic, defining at least one epitopic site, or will be a member of a naturally-occurring binding pair (e . g. , carbohydrate and lectin, hormone and hormone receptor, ligand and anti-ligand, and the like) .
  • SBP specific binding pair
  • Analytes of particular interest include antigens, antibodies, proteins, glycoproteins, carbohydrates, macromolecules, toxins, bacteria, tumor markers, and the like, which define a plurality of epitopic sites.
  • Other analytes of interest include haptens, drugs, and other small molecules, which usually define only a single epitopic binding site.
  • a non-exhaustive list of exemplary analytes is set forth in U.S. Patent No. 4,366,241, at column 19, line 7, through column 6, line 42, the disclosure of which is incorporated herein by reference.
  • cardiac markers such as troponin I, troponin T, myoglobin, and creatine kinase isozymes.
  • the analytes may be present in a wide variety of samples, where the sample is liquid, can be liquified, or can be suspended in a liquid.
  • the methods of the present invention will find their greatest use with biological specimens such as blood, serum, plasma, urine, cerebral fluid, spinal fluid, ocular lens liquid (tears) , saliva, sputum, semen, cervical mucus, scrapings, swab samples, and the like, which are frequently employed in the diagnosis and monitoring of disease and therapeutic treatments.
  • the methods of the present invention may be used with industrial, environmental, and food samples, such as water, process streams, milk, meat, poultry, fish, conditioned media, and the like.
  • pretreat the sample such as by liquification, separation, solubilization, concentration, filtration, chemical treatment, or a combination of these steps, in order to improve the compatibility of the sample with the remaining steps of the assay, as described hereinafter.
  • the selection and pretreatment of biological, industrial, and environmental samples prior to immunological testing is well known in the art and need not be further described.
  • the haptens which are to be immobilized on the solid phase surface are small molecules, typically being below 2000 Daltons, usually being below 1000 Daltons, frequently having a molecular weight in the range from 100 to 500 Daltons, and more frequently having a molecular weight in the range from 200-500 Daltons.
  • Such small molecules will generally be resistant to the denaturing conditions which may be present in the methods of the present invention, particularly when ink jet printing mechanisms are employed, and will thus remain stable and biologically active so that they can bind their specific binding partner.
  • ink jet printing mechanisms differ significantly in their designs, such mechanisms can subject the material to be deposited to a variety of conditions which can denature labile materials, such as localized heating (including boiling of the liquid medium carrying the material to be deposited) , substantial shear forces which can physically disrupt larger molecules, as well as adverse drying conditions utilized after the material has been deposited.
  • denature labile materials such as localized heating (including boiling of the liquid medium carrying the material to be deposited) , substantial shear forces which can physically disrupt larger molecules, as well as adverse drying conditions utilized after the material has been deposited.
  • the haptens should possess a high-affinity binding partner, preferably having a binding affinity of at least about 10 "8 M “1 , more preferably being at least 10 "10 M “1 , and most preferably being 10 "11 M “1 or higher.
  • the haptens should be suitable for attachment, usually covalent attachment, to the carrier molecule, as described in more detail hereinbelow.
  • Exemplary haptens include biotin (which binds to avidin, streptavidin and anti-biotin antibody with very high affinity) , fluorescein (which binds with high affinity to anti-fluorescein antibody) , dinitrophenol (which binds with very high affinity to anti-dinitrophenol antibody) , digoxin (which binds with very high affinity to anti-digoxin antibody) , luminol (which binds with very high affinity to anti-luminol antibody) , theophylline (which binds with very affinity to anti-theophylline antibody) , morphine (which binds with very high affinity to anti-morphine antibody) , and the like.
  • Carrier molecules useful in the methods of the present invention will typically be macromolecules having a molecular weight of at least about 10 kiloDaltons (kD) with no upper size limit, usually being at least about 20 kD, and typically being in the range from about 20 kD to 200 kD.
  • kD kiloDaltons
  • exemplary carrier proteins include albumins, ovalbumins, immunoglobulins, thyroglobulins, ferritin, and the like.
  • Particularly preferred carrier proteins include serum albumins, such as bovine serum albumin (BSA) , human serum albumin (HSA) , and the like.
  • the hapten and carrier molecule will be conjugated to each other to form a hapten-carrier molecule complex prior to application to the solid phase surface.
  • Such conjugation will usually be achieved through covalent binding using conventional chemistries depending on the natures of both the hapten and the carrier molecule.
  • covalent binding will be achieved using a bivalent cross-linking substance which is capable of attaching at one end to the hapten and at the other end to the carrier molecule.
  • Such substances include succinimides, carbodiimides, glutaraldehydes, diazotization coupling, and the like.
  • These and other suitable coupling chemistries are well-described in the biological and chemical literature. See, for example, Avrameus et al. (1978) Scand. J. Immunol. 8:7-23.
  • the resulting conjugates will typically have hapten numbers of at least 1, more usually of at least 2, preferably being at least 4, often being in the range from 4 to 50, and preferably being in the range
  • the hapten-carrier molecule conjugates will be present in a liquid medium suitable for direct application to the solid phase surface of the test article.
  • the liquid medium will typically be aqueous, more typically being buffered within a desired pH range, usually from pH 5 to pH 9.
  • Phosphate-buffered saline (PBS) is a suitable liquid medium for most hapten-carrier molecule conjugates.
  • liquid medium may contain other components for achieving desired purposes.
  • liquid media may include preservatives, such as sugars, intended to enhance stability of the conjugate during application, surprisingly, however, it has been found that the hapten conjugates of the present invention generally do not require preservative or other components to maintain stability of the haptens in the printing methods of the present invention.
  • preservatives such as sugars
  • the ability to dispense with preservatives is a particular advantage of the present invention.
  • the liguid medium will typically have a hapten- carrier molecule concentration in the range from about 1 ⁇ g/ml to 250 / -tg/ml, preferably from 5 ⁇ g/ml to 50 ⁇ g/ml, with viscosities equivalent to water, preferably being about 1 cp to 2 cp. It is possible to add viscosity-enhancing components, such as glycols, polyvinyl alcohols, or the like, where the viscosity is increased to about 5 to 10 cp, resulting in improved fluid dispersion characteristics from an ink jet printing head.
  • viscosity-enhancing components such as glycols, polyvinyl alcohols, or the like, where the viscosity is increased to about 5 to 10 cp, resulting in improved fluid dispersion characteristics from an ink jet printing head.
  • viscosity-enhancing reagents can interfere with protein adsorption particularly to plastics where it will interfere with non-specific hydrophobic interactions between a protein and a plastic surface.
  • the addition of such viscosity-enhancing components can decrease the amount of active hapten-carrier molecule adsorbed onto the solid phase surface, and the addition of viscosity enhancing components is not generally preferred at present.
  • a preselected volume of the liquid medium (typically from 200 pi to 100 ⁇ l) containing the hapten-carrier molecule conjugates may be applied to the solid phase surface in any manner that results in an initial non-covalent attachment of the carrier molecule to the surface.
  • the liquid medium may be applied by pipetting, including micropipetting (volumes in the range from about 200 pi to 100 ⁇ l) , spraying, printing, dipcoating, spin coating, stamping, or the like.
  • the preferred and exemplary method for applying the liquid media comprises printing using a conventional or modified ink jet printing mechanism where the liquid medium is initially contained in a reservoir and dispensed therefrom in discrete liquid droplets (typically each having a volume in the range from 10 pi to 1 ⁇ l) using piezoelectric, thermal, or other dispensing mechanisms.
  • the fluid to be printed (which is the liquid medium in the case of the present invention) is ejected through a small aperture under pressure and transformed into uniform droplets by one of various known ink jet printing mechanisms.
  • a preferred class of ink jet printer relies on an evaporative volume increase produced by thermal cycling of the liquid medium.
  • ink jet printing mechanisms are available commercially from Hewlett-Packard, Palo Alto, California.
  • Other ink jet printing mechanisms which may find use rely on vibration of a piezoelectric crystal or thermal effect to produce the droplets, where droplets may be charged electrostatically and deflected by a control system, typically a computer.
  • the resulting pattern of applied liquid medium can be precisely and accurately controlled.
  • the ink jet printing mechanism will typically be combined with a mechanism for supporting the solid phase surface to be derivatized.
  • the solid phase can be automatically relocated relative to the ink jet printing head in order to print the liquid medium (which contains the hapten-carrier molecule conjugate) to different locations on the solid phase surface.
  • a plurality of different reaction zones can be formed on the solid phase surface.
  • reaction zones having a variety of different activities can be formed on a single solid phase surface.
  • the solid phase surface employed in the methods of the present invention will include at least a single reaction zone, often including two or more reaction zones, frequently including from 3 to 100 reaction zones, and often including from 4 to 50 reaction zones.
  • the reaction zones may be specific for a single analyte, or different reaction zones may be specific for different analytes. It will be appreciated, of course, that the reaction zones may be capable of directly or indirectly, competitively or non-competitively binding, or providing any other type of binding compatible with the assay formats of the present invention.
  • the reaction zones are usually optically flat to facilitate spectrophometric, interferometric, fluormetric or other interrogation thereof.
  • the reaction zones will usually have a minimum area of 0.01 mm 2 , usually being from 0.1 mm 2 to 100 mm 2 , and preferably from 0.1 mm 2 to 5 mm 2 .
  • a plurality of circular reaction zones having areas in the range from 0.1 mm 2 to 5 mm 2 may be formed on a single flat surface of a disk substrate.
  • the total number of droplets will usually be in the range from 1 to 10,000, preferably being from 500 to 2,500, for an approximately 1 mm diameter spot.
  • the substrates which provide the solid phase surface may be composed of a variety of conventional materials, including organic polymers, glass, ceramics, metals, and the like.
  • substrates will preferably be transparent or translucent, and will be composed of organic polymers.
  • An exemplary material is methylmethacrylate polymer.
  • the surface may be treated to enhance adherence of the carrier molecule to the surface.
  • the solid phase surface may be at a temperature above room temperature, typically at least 50°C, and often 90°C, or higher, to provide substantially accelerated and enhanced adherence of the carrier molecule to the surface.
  • Such high temperature treatment is typically performed for a relative short period sufficient to maximize binding of the complex to the surface, usually from 10 minutes to 1 hour. While such conditions would inactivate many antibodies and other specific binding materials, the hapten-carrier molecule complexes of the present invention will remain active and able to bind their binding partners even after exposure to heat and other potentially denaturing conditions.
  • covalently cross-link the carrier molecules to the solid phase surface by subjecting the adsorbed surface to suitable conditions, e.g., light, radiation, heat, cross-linking reagents, or the like. It should be noted that such covalent cross-linking of the molecule occurs after initial immobilization (usually non-covalent attachment) has been effected.
  • suitable conditions e.g., light, radiation, heat, cross-linking reagents, or the like.
  • the hapten-carrier molecule will be further derivitized to include a cross-linking moiety, preferably a photo-activatable group such as a benzophenone of fluorinated aryl azide.
  • a cross-linking moiety preferably a photo-activatable group such as a benzophenone of fluorinated aryl azide.
  • the complexes will be highly substituted with the reactive groups on the carrier molecule (typically from 1 to 25, preferably from 5 to 15 substitutions per molecule) and high doses of the ultraviolet radiation can be applied to the surface (typically from 100 to 1,000 ⁇ W/cm 2 at 6 inches from the surface) .
  • the hapten-carrier molecule complex of the present invention has been found to be significantly less susceptible to such denaturation. The formation of multiple links between the carrier molecule and the plastic surface will not significantly reduce the binding activity of the hapten.
  • the hapten and carrier molecule may be selected for optimal ultraviolet light tolerance, preferably both having low absorption in the UV range around 280nm.
  • the resulting test article may be used directly in certain assay formats.
  • assays which rely on introducing an anti-hapten binding substance to a target analyte using antibody or other binding substance specific for the target analyte can utilize the test article having the immobilized hapten as a capture device.
  • avidin bound to a target-analyte specific antibody may be reacted with the test sample.
  • the resulting complex of analyte, antibody and avidin may then be captured using a test article having biotin hapten immobilized on its test surface.
  • the presence of the target analyte can be confirmed using antibody attached to a label in a conventional "sandwich" assay format.
  • test articles of the present invention may alternatively be further derivatized to have a specific binding substance of interest immobilized to the test surface.
  • immobilization may be readily accomplished by reacting the solid phase surface under mild (non-denaturing) conditions with a solution containing a specific binding substance, such as an antibody or antibody fragment, covalently attached to the anti-hapten binding substance.
  • a specific binding substance such as an antibody or antibody fragment
  • the specific binding substance is precisely and accurately bound to the reaction zone(s) defined by the immobilized hapten under mild conditions which do not result in loss of activity of the specific binding substances.
  • Such test articles are useful in a variety of conventional assay formats which employ direct binding between the immobilized specific binding substance and a target analyte.
  • FIG. 1 A system for ink jet printing of haptenated macromolecules is shown in Figs. 1 and 2.
  • the system includes a HP Model 51616A Ink Jet Head 10 (Hewlett Packard Ink Jet
  • the resulting assembly was mounted on X-axis and Y-axis micrometer slides 20 and 22 to facilitate positioning the head precisely over an acrylic disc D on which the solutions were to be printed.
  • the acrylic disc D Prior to printing, the acrylic disc D was installed on a round disc mounting platen 24 via insertion on a center locating pin.
  • the disc mounting platen 24 was attached to a motor shaft of stepper motor 26 (Parker-Compumotor, Harrison City, PA, Model # S57-51-MO) , allowing the disc to be rotated under computer control.
  • the stepper motor 26 was mounted on a Z-axis slide 30 which allowed a stepper motor 28 (Parker-Compumotor Model # S57-51- MO) to drive the platen 24 in the Z direction.
  • Fig. 2 shows details of the head drive electronics 31 (see Fig. 2) .
  • Stepper motors were controlled using two S-6 Series Microstep stepper motor controllers 32 (Parker-Daedal, Harrison City, PA) .
  • the stepper motor controllers 32 were interfaced to computer 34 using an AT-MI0-16X DAQ acquisition board 36 (National Instruments Corporation, Austin, Texas) .
  • Control software was written to control the motor motions using a LabVIEW software package (National Instruments Corporation, Austin, Texas) .
  • Fig. 2 shows details of the head drive electronics
  • the purpose of the head drive electronics is to (1) supply a controlled impulse of energy to a selected thermal resistor 40 inside the ink jet head, (2) control the rate at which pulses are input to the head, and (3) control the number of energy pulses delivered to the head in a sequence thus controlling the amount of fluid deposited in one spot on the acrylic disc D.
  • the circuit functions as follows.
  • the ink jet printing technology relies upon heating the fluid contained in small chambers inside the ink jet head. This heating is accomplished by passing electrical current through small resistors 40 embedded in the fluid chambers.
  • the HP Model 51616A Ink Jet Head 10 has thermal heating resistor values of approximately 65 ohms.
  • Each of the 12 orifices in this head has a heating resistor 40 localized above it.
  • the 12 resistors are connected internal to the head in the network shown in Fig. 2. Internal wiring resistances result in the parasitic resistances also shown in Fig. 2. All chamber heating resistors are connected to one common line.
  • MOSFET transistor 42 (BUZ71; Siliconix, Santa Clara, CA) is turned on under control from NPN transistor 44 (2N3904; Motorola, Phoenix, AZ) when a high logic state is output from monostable multivibrator 46 (74LS123; Texas Instruments, Dallas, Texas) .
  • the monostable multivibrator 46 controls the time that MOSFET transistor 42 is allowed to stay on allowing electrical current to pass through the chamber heating resistor 40 in the ink jet head 10.
  • Resistor 48 (20 kQ) is adjusted to control the pulse period and tune the head for optimal jetting of the solution of interest. A pulse width between 3 and 3.5 microseconds was found to be optimal for jetting the solutions of interest.
  • the common node of the ink jet head is supplied with a 23 volt DC potential from power supply 50 (Model LPS152, Leader Instruments, Hauppauge, NY) . This voltage is held constant under all operating conditions using the monostable multivibrator 46 to control the energy input to the chamber heating resistors 40.
  • the number of pulses of energy supplied to the head 10 to form one spot is controlled by 12 bit pre- loadable binary counter 52 which was loaded with the number of droplets to be printed using 12 SPST switches 54.
  • a 2 KHz clock source 56 is input to the counter 52 and is used to count down to zero from the pre-loaded number. The resulting output occurs at a 1 KHz rate, half the input clock rate.
  • the maximum rate at which the HP 51616A ink jet head 10 has been specified to be driven is 1.25 Khz, though it can be operated up to 3 to 4 Khz for accelerated deposition rates. Even higher jetting rates cause inconsistent jetting and damage to the head (resistor failure) .
  • the line connected from the counter 52 to the computer interface 36 is used to command the head drive electronics 31 to deposit a spot on the acrylic disc D after the stepper motors 26 and 28 have positioned the disc D in the appropriate position.
  • 35 mm acrylic disks used for substrate material were obtained from the Germanow-Simon company. They were machined from Hesalite HTC material and were 1.1 or 1.4 mm thick with a high-tolerance 2.4 mm center hole. This material has optical properties that are sufficient for interferometric and/or fluorescent interrogation and protein binding characteristics enabling immunoassay development on it. Disks were shipped from the vendor laminated on both sides to minimize scratching during machining, transport, and storage. Prior to ink jet spotting, laminates were removed, and the disks were (occasionally washed with mild detergents and) rinsed with deionized water both to quench static electricity build-up and to remove debris remaining from the machining process.
  • Bovine serum albumin was covalently attached to biotin moieties (biotinylated) starting with 60 mg of a 10 mg/mL solution of BSA in phosphate buffered saline (PBS, 10 mM, pH 7.4,). The BSA was then conjugated at a molar ratio of ca. 10:1 NHS-LC-biotin to BSA.
  • a molar ratio of ca. 10:1 NHS-LC-biotin For 60 mg of BSA, 4.1 mg of NHS-LC-Biotin (Pierce #94052374) was dissolved in 60 ⁇ L of DMF (Pierce #931026155) and added to the 6 mL of BSA in PBS.
  • This reaction mixture was placed in an ice bath for 2 hours followed by placing the mixture in a 12,000 MWCO dialysis tubing and dialyzing against 0.1 M phosphate buffer (pH 7.0, with at least two 1 L changes) at 4°C to remove unreacted biotin. Following dialysis and hapten number assessment, 0.1% NaN 3 was added to the concentrated B-BSA to discourage microbial growth. Hapten numbers in the range from 2 to 9 were obtained. Before spotting these solutions using the ink jet system, an aliquot of a concentrated biotin-BSA solution was diluted to 40 ⁇ g/mL in PBS and degassed.
  • the biotin-BSA has been further derivatized to include a benzophenone moiety that may be activated with ultraviolet light to form a covalent bond with the acrylic test surface.
  • a benzophenone moiety that may be activated with ultraviolet light to form a covalent bond with the acrylic test surface.
  • 1.0 mL of biotin-BSA (5 mg/mL in PBS) was combined with 68 ⁇ L of LC-SPDP (Pierce #21652, 10 mg/mL in DMF) and allowed to react for two hours at room temperature, followed by overnight dialysis vs. PBS.
  • Fig. 3 shows that liquid volumes in the range of 200 pL to 200 nL, with corresponding spot sizes in the 0.05 (1 droplet) to 1.0 mm (1000 droplets) range, may easily be achieved in a desired pattern using this embodiment of ink jet technology.
  • the use of multiple printing heads allows the formation of complex patterns of different materials which can be deposited simultaneously onto the test surface.
  • acrylic test disks were typically spotted with 40 ⁇ g/mL B-BSA in PBS in 32 or 64 circumferentially-spaced spots per revolution (at 0.4 inches radius) with a drop-per-spot setting ensuring 50% coverage of surface along the circumference.
  • Liquid-phase adsorption as an example Classically, protein adsorption is performed under stagnant liquid-phase conditions, where binding between the protein and the surface is achieved over a several hour time scale (at room temperature) and the tightness of binding, or resistance to desorption, increases with adsorption time ( Figure 4) .
  • Biotinylated-BSA see "C. Preparation of Biotinylated BSA solutions" above. This material was diluted to 40 ⁇ g/mL in PBS before use.
  • Assay Diluent 0.1% BSA (Pentax) , 0.05% Tween® 20
  • wash Buffer 0.1% Tween® 20 (Mallinckrodt) in 10 mM PBS.
  • SA-AP Conjugate Streptavidin-alkaline phosphatase (SA- AP, Pierce, Product # 21324) was reconstituted to 1 mg/mL and frozen. Prior to use, the stock was further diluted in the 1:1000 to 1:1000000 range with Assay Diluent.
  • PNPP Kit a tablet of PNPP (Sigma, N-2770) was reconstituted in the suggested buffer immediately prior to use.
  • Rings Rings several millimeters thick and 3/8 in (I.D.) diameter were affixed to the clean, unspotted, 35 mm acrylic test articles (Germanow-Simon) with double-stick tape to facilitate containment of liquid materials above the test surface.
  • the metal PNPP substrate (diluted as instructed) was then added to the wells (150 ⁇ L) and allowed to react for 20 minutes without agitation.
  • Adsorption after ink jetting normal drying
  • the protein-binding process after ink jet-based deposition of protein-based solutions onto the acrylic disk actually consists of a least two phenomena - visual drying of the spotting fluid and the associated process of adsorption (or covalent linking, with associated UV-curing chemistry) of a portion of the spotted B-BSA to the acrylic surface.
  • Disk drying time depends on spot size, fluid composition, temperature, and humidity. For water-based vehicles spotted at ambient conditions in the 32 spot format (ca. 0.2 ⁇ L per spot) , the crusting time is on the order of 10 to 30 minutes.
  • Adsorption, as indirectly measured by SA-HRP-DAB assay after ink jet deposition (Figure 5) occurs on a longer (several hour) time scale that is not unlike that for liquid phase adsorption ( Figure 4) .
  • Assay Diluent 0.1% BSA (Pentax) , 0.05% Tween® 20 (Mallinckrodt) in 10 mM PBS, 0.2 ⁇ m filtered (Gelman acrodisc) .
  • SA-HRP Conjugate Streptavidin-horse radish peroxidase (SA-HRP, Pierce, Product No. 21127) was reconstituted to 1 mg/mL and frozen. Prior to use, the stock was further diluted in the 1:1000 to 1:1000000 range with Assay Diluent.
  • Rings One with a diameter similar to the outer diameter of the disk and about 2 mm thick, and the other with an approximately 0.5 inch diameter (and similar thickness) were affixed with double stick tape. This allowed containment of liquid between the rings in order to perform the assay.
  • the metal DAB precipitating enzyme substrate was then added to the disk and allowed to react for a set time in the range of 10 to 30 minutes without agitation. 5) The reaction was stopped by Dl-water washing the disks to remove the metal DAB solution. After washing, some disks were dried with heat (typically, a hair dryer) .
  • Heating in the 70°- to 902 c regime, with the upper temperature limited by the softening of the acrylic surface
  • Heating accelerates the protein binding process from hours to minutes.
  • heat treating aged disks in a similar fashion has little, if any, influence on the eventual interferometric signal ( Figure 6) .
  • hours of ambient drying/adsorption are apparently functionally equivalent to minutes of heating. This suggests that either a similar phenomena is occurring due to the heating process (time - temperature superposition) or different mechanisms (such as crosslinking, etc. for the heated samples) are occurring but the surface area for protein - plastic interactions governs maximal binding.
  • Biotinylated-BSA see "C. Preparation of Biotinylated BSA solutions" above. This material was diluted to 40 ⁇ g/mL in PBS before use.
  • Assay Diluent 0.1% BSA (Pentax) , 0.05% Tween® 20 (Mallinckrodt) in 10 mM PBS, 0.2 ⁇ filtered (Gelman acrodisc) .
  • Wash Buffer 0.1% Tween® 20 (Mallinckrodt) in 10 mM PBS.
  • SA-HRP Conjugate Streptavidin-horse radish peroxidase (SA-HRP, Pierce, Product #21127) was reconstituted to 1 mg/mL and frozen. Prior to use, the stock was further diluted in the 1:1000 to 1:1000000 range with Assay Diluent.
  • o-Phenylenediamine Dih ⁇ drochloride tOPD) substrate A solution of 0.4 mg/mL OPD was made by dissolving a 15 mg tablet of OPD (Sigma) into 37 mL of 0.1M citrate- phosphate buffer, pH 5.0. Immediately prior to use, 10 mM hydrogen peroxide was added (37 ⁇ L of 30% solution, Sigma) as an activating agent. Sulfuric acid (2N) was added in an in an equal volume to OPD to stop the reaction at the desired time.
  • Teflon Rings Teflon Rings several millimeters thick and 0.25 inches in inner diameter were affixed to the clean, unspotted, 35 mm acrylic test articles (Germanow-Simon) with double-stick tape to facilitate containment of liquid materials above the test surface. After the protein adsorption step in the assay, the rings were removed and a fresh ring and double-stick tape was affixed to the original double-stick tape. This eliminates assay activity due to "wall effects.”
  • Teflon rings were then replaced with new ones and a new piece of double-stick tape.
  • a microplate reader was used to determine absorbance at 490 nm.
  • a preferred embodiment of this invention is to determine CKMB levels in suspected AMI patients. This method, assayed spectrophotometrically or interferometrically, is able to rapidly detect CKMB in clinically-significant levels ( Figure 7) .
  • Assay Diluent 0.1% Bovine Serum Albumin (BSA) (Pentax) , 0.05% Tween® 20 (Mallinckrodt) in 10 mM PBS, pH 7.4, 0.2 ⁇ m filtered (Gelman acrodisc) .
  • BSA Bovine Serum Albumin
  • Blocking Buffer 0.3% BSA (Pentax), 0.05% Tween® 20 (Mallinckrodt) in 10 mM PBS, pH 7.4; 0.2 ⁇ m filtered (gelman acrodisc) .
  • Wash Buffer 0.05% Tween® 20 (Mallinckrodt) in 10 mM PBS, pH 7.4.
  • Streptavidin-antiCKMB Conjugate Streptavidin/antiCKMB conjugate was prepared using S-acetylthioglycolic acid N- hydroxysuccinimide ester (SATA, Pierce #26102) , a heterobifunctional linking reagent from Sigma and Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Pierce #22320) from Pierce.
  • the purified antiCKMB-SATA and streptavidin-SMCC were mixed at the protein molar ratio of 1:3, resulting in the total reaction volume of 6mL.
  • the conjugation reactin was initiated by adding 1 M hydroxylamine to a final concentration of 100 mM and incubated over 18 hours at 4°C. The reaction was stopped by adding 100 mM N-ethylmaleimide (NEM) from Aldrich Chemical at 1 mM final concentration in the reaction mixture and incubating for 15 minutes at room temperature. After the incubation with NEM, the conjugation reaction mixture was concentrated 3-fold, and the final volume was reduced to 2 mL using a Centricon- 100 concentrator from Amicon. Then the concentrated mixture was purified using a Bio-Gel® A-5m column from Bio-Rad to isolate streptavidin-antiCKMB conjugate.
  • NEM N-ethylmaleimide
  • AntiCKMM-Horse Radish Peroxidase (HRP) Conjugate AntiCKMM-HRP conjugate was made in two steps as follows. First, goat antiCKMM antibody (BiosPacific #G31520) was conjugated to SATA (Sigma) at a molar ratio of SATA to antibody of 20:1. 4 mg of antibody required 0.1156 mg of SATA, which was obtained by adding 5.2 mg of SATA to 500 ⁇ L of DMF (Pierce) for a 10.4 mg/mL solution. The
  • the HRP/SMCC and goat antiCKMM/SATA were each placed on a PD-10 column (Pharmacia) which had been equilibrated in PBS, pH 7.3 to remove the free SATA and SMCC.
  • the goat antiCKMM was eluted with 3 mL of PBS while the HRP was eluted with 2.8 mL of PBS. This 5.8 ml volume was placed together and 580 ⁇ L of 1M hydroxylamine, pH 7.0 (Sigma) was added to start the conjugation which continued at 4°C overnight.
  • the reaction was then stopped by adding 64 ⁇ L of a 12.5 mg/mL solution of NEM (Aldrich) in DMF and incubating for 15 min. at room temperature. The 6+ mL were then concentrated in a Centricon 30 concentrator to 2 mL and fractionated over a 100 cm Bio-Gel® A-5M column.
  • CKMB Calibrators CKMB (Fortron, #1-028) was diluted to prepare 0, 10, 100 ng/mL calibration solutions.
  • Enzyme substrate was added at room temperature.
  • the OPD coloring solution (200 ⁇ L) was transferred to a microplate and read with microplate reader (Molecular Devices, E M ⁇ ) at 490 nm.
  • the DAB disks were dried and measured with the interferometer.

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Abstract

Ce procédé, qui sert à préparer des articles de test, consiste à immobiliser un conjugué de molécules porteuses d'haptènes sur une surface en phase solide. La fixation d'un haptène s'effectue par l'intermédiaire de la molécule porteuse qui se colle par adsorption non covalente à la surface en phase solide. L'haptène est une petite molécule qui conserve son activité de fixation même dans des conditions relativement hostiles qui risqueraient de dégrader des substances plus labiles, telles que des anticorps ou d'autres protéines de liaison. De telles protéines de liaison labiles peuvent ensuite être introduites dans la phase solide par liaison avec une substance de liaison anti-haptène dans des conditions douces. La molécule porteuse peut éventuellement être attachée par covalence à la surface en phase solide après une position non covalente initiale, par exemple par réticulation induite par rayonnement.
PCT/US1996/000416 1995-01-18 1996-01-16 Procede pour immobiliser des haptenes sur un article de test WO1996022533A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2764988A1 (fr) * 1997-06-20 1998-12-24 Bio Merieux Compose conjugue, reactif et kit le contenant, et utilisations dans un procede de fixation d'un materiel biologique
US6136610A (en) * 1998-11-23 2000-10-24 Praxsys Biosystems, Inc. Method and apparatus for performing a lateral flow assay
WO2003016911A1 (fr) * 2001-08-16 2003-02-27 Institut für Chemo- und Biosensorik Münster E.V. Support pour biomolecules immobilisees et procede pour immobiliser des biomolecules
US6528323B1 (en) 1999-06-14 2003-03-04 Praxsys Biosystems, Inc. Bidirectional lateral flow test strip and method
WO2004084195A1 (fr) * 2003-03-18 2004-09-30 Nanomagnetics Ltd Production de pellicules minces de nanoparticules
US7605004B2 (en) 2001-07-18 2009-10-20 Relia Diagnostic Systems Llc Test strip for a lateral flow assay for a sample containing whole cells
US8003407B2 (en) 2004-07-29 2011-08-23 Relia Diagnostic Systems, Llc Lateral flow system and assay
DE19924643B4 (de) * 1999-05-28 2017-02-23 Roche Diagnostics Gmbh Verfahren zur Herstellung von Protein-beladenen Mikropartikeln
WO2017093226A1 (fr) 2015-11-30 2017-06-08 F. Hoffmann-La Roche Ag Immuno-essai pour la détermination d'anticorps modifiés de la région fc

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US4096138A (en) * 1975-12-08 1978-06-20 Scherr George H Immunological test procedure
US4791067A (en) * 1987-06-25 1988-12-13 Fisher Scientific Co. Agglutination immunoassay for hapten involving monoclonal antibody of IgA class reagent
US4960692A (en) * 1986-03-18 1990-10-02 Fisher Scientific Company Assay employing binding pair members on particles and on a filter or membrane
US5045480A (en) * 1989-02-09 1991-09-03 Miles Inc. Gel particles having hapten moieties bound thereto as immunoassay reagent
US5063081A (en) * 1988-11-14 1991-11-05 I-Stat Corporation Method of manufacturing a plurality of uniform microfabricated sensing devices having an immobilized ligand receptor
US5279955A (en) * 1991-03-01 1994-01-18 Pegg Randall K Heterofunctional crosslinking agent for immobilizing reagents on plastic substrates
US5316784A (en) * 1989-07-14 1994-05-31 Boehringer Mannheim Gmbh Process for the production of a solid phase matrix coated with an immunologically active substance
US5362655A (en) * 1988-08-29 1994-11-08 Boehringer Mannheim Gmbh Process for the determination of a specifically bindable substance
US5468649A (en) * 1994-02-15 1995-11-21 Abbott Laboratories Process for labeling acridinium to microparticles and application in an instrument

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4096138A (en) * 1975-12-08 1978-06-20 Scherr George H Immunological test procedure
US4960692A (en) * 1986-03-18 1990-10-02 Fisher Scientific Company Assay employing binding pair members on particles and on a filter or membrane
US4791067A (en) * 1987-06-25 1988-12-13 Fisher Scientific Co. Agglutination immunoassay for hapten involving monoclonal antibody of IgA class reagent
US5362655A (en) * 1988-08-29 1994-11-08 Boehringer Mannheim Gmbh Process for the determination of a specifically bindable substance
US5063081A (en) * 1988-11-14 1991-11-05 I-Stat Corporation Method of manufacturing a plurality of uniform microfabricated sensing devices having an immobilized ligand receptor
US5045480A (en) * 1989-02-09 1991-09-03 Miles Inc. Gel particles having hapten moieties bound thereto as immunoassay reagent
US5316784A (en) * 1989-07-14 1994-05-31 Boehringer Mannheim Gmbh Process for the production of a solid phase matrix coated with an immunologically active substance
US5279955A (en) * 1991-03-01 1994-01-18 Pegg Randall K Heterofunctional crosslinking agent for immobilizing reagents on plastic substrates
US5468649A (en) * 1994-02-15 1995-11-21 Abbott Laboratories Process for labeling acridinium to microparticles and application in an instrument

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998059241A1 (fr) * 1997-06-20 1998-12-30 Bio Merieux Procede de mise en evidence d'un materiel biologique cible, phase de capture, phase de detection et reactif les contenant
AU748402B2 (en) * 1997-06-20 2002-06-06 Bio Merieux Method for isolating a target biological material, capture phase, detecting phase and reagent containing them
FR2764988A1 (fr) * 1997-06-20 1998-12-24 Bio Merieux Compose conjugue, reactif et kit le contenant, et utilisations dans un procede de fixation d'un materiel biologique
US6632615B2 (en) 1997-06-20 2003-10-14 Bio Merieux Method for isolating a target biological material, capture phase, detecting phase and reagent containing them
US6136610A (en) * 1998-11-23 2000-10-24 Praxsys Biosystems, Inc. Method and apparatus for performing a lateral flow assay
DE19924643B4 (de) * 1999-05-28 2017-02-23 Roche Diagnostics Gmbh Verfahren zur Herstellung von Protein-beladenen Mikropartikeln
US6528323B1 (en) 1999-06-14 2003-03-04 Praxsys Biosystems, Inc. Bidirectional lateral flow test strip and method
US7229839B2 (en) 1999-06-14 2007-06-12 Relia Diagnostic Systems, Llc Bidirectional lateral flow test strip and method
US7605004B2 (en) 2001-07-18 2009-10-20 Relia Diagnostic Systems Llc Test strip for a lateral flow assay for a sample containing whole cells
WO2003016911A1 (fr) * 2001-08-16 2003-02-27 Institut für Chemo- und Biosensorik Münster E.V. Support pour biomolecules immobilisees et procede pour immobiliser des biomolecules
WO2004084195A1 (fr) * 2003-03-18 2004-09-30 Nanomagnetics Ltd Production de pellicules minces de nanoparticules
US8003407B2 (en) 2004-07-29 2011-08-23 Relia Diagnostic Systems, Llc Lateral flow system and assay
WO2017093226A1 (fr) 2015-11-30 2017-06-08 F. Hoffmann-La Roche Ag Immuno-essai pour la détermination d'anticorps modifiés de la région fc
KR20180082597A (ko) * 2015-11-30 2018-07-18 에프. 호프만-라 로슈 아게 Fc 영역 변형 항체의 측정을 위한 면역 분석
CN108351361A (zh) * 2015-11-30 2018-07-31 豪夫迈·罗氏有限公司 用于测定Fc区修饰的抗体的免疫测定法
AU2016363638B2 (en) * 2015-11-30 2022-04-21 F. Hoffmann-La Roche Ag Immunoassay for the determination of Fc-region modified antibodies
US11561229B2 (en) 2015-11-30 2023-01-24 Hoffmann-La Roche Inc. Immunoassay for the determination of Fc-region modified antibodies
KR102628570B1 (ko) 2015-11-30 2024-01-23 에프. 호프만-라 로슈 아게 Fc 영역 변형 항체의 측정을 위한 면역 분석

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