WO1999040438A1 - Dispositifs et procedes de mesure des titres d'anticorps immunoprotecteurs - Google Patents

Dispositifs et procedes de mesure des titres d'anticorps immunoprotecteurs Download PDF

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Publication number
WO1999040438A1
WO1999040438A1 PCT/US1999/001511 US9901511W WO9940438A1 WO 1999040438 A1 WO1999040438 A1 WO 1999040438A1 US 9901511 W US9901511 W US 9901511W WO 9940438 A1 WO9940438 A1 WO 9940438A1
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WIPO (PCT)
Prior art keywords
conjugate
antibody
zone
sample
signal
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PCT/US1999/001511
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English (en)
Inventor
John A. Cutting
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Synbiotics Corporation
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Publication of WO1999040438A1 publication Critical patent/WO1999040438A1/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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • the present invention relates to a device for determining the immune status of vertebrates, more particularly, for determining the immune status of cats, dogs and birds.
  • the present invention also relates to an apparatus and method for determining immune status and for formulating customized vaccines. Both human and veterinary vaccines have greatly reduced the incidence of contagious and lethal disease.
  • the efficacy of a veterinary vaccine is determined by a controlled challenge of a small group of animals with the appropriate virulent pathogen, at some time after the vaccine has been administered.
  • the longest period post-vaccination before challenge has been one year, although it has frequently been only a matter of weeks.
  • the manufacturers of veterinary vaccines have recommended annual revaccination and, since 1970, annual revaccination has become the norm within the industry.
  • the number of vaccines that are available for cats, dogs and commercial poultry has steadily increased over the years to the point where there are six or more different entities for which they are being vaccinated. With the annual revaccination practice, a cat or dog may over its lifetime receive 10-15 combination vaccinations.
  • type 1 reactions are local reactions such as discomfort, pain, and stinging on injection
  • type 2 reactions are systemic reactions such as depression, lethargy, listlessness, inalletence, or pyrexia occurring 6 to 12 hours after injection and generally lasting 6 to 24 hours
  • type 3 reactions are hypersensitivity usually expressed as vomiting, angioneurotic edema, erythema, and cyanosis shortly after vaccination
  • type 4 reactions are miscellaneous reactions including delayed vomiting and diarrhea, alopecia, seizures, and abortion.
  • CMV Canine parvovirus
  • Adenovirus types I and II (CAV)
  • CDV Canine distemper virus
  • CPIV Parainfluenza virus
  • CMV Canine coronavirus
  • Kittens and older cats are usually vaccinated against:
  • FCV Calicivirus
  • Feline leukemia virus FeLV
  • Rabies virus RSV
  • the level of protective immunity that develops following first and subsequent regimens of vaccination will vary from animal to animal. Factors that influence the extent of the developed immunity are: breed, size, sex, age and, state of health at vaccination and subsequently.
  • Some of these antibody levels are known by direct determination as a result of animals withstanding a challenge with the virulent organism for which disease would develop in animals with lesser titers of antibody. In other cases, these levels are inferred, as a result of being unable to successfully revaccinate in the presence of the particular titer of antibody.
  • an animal is successfully revaccinated, there is an anamnestic response and the animal immediately commences to produce higher titers of the antibody specific for the immunogen with which it was vaccinated. This effect does not occur, and the vaccine does not induce additional immunity, when the existing antibody titers are above a certain threshold titer.
  • HI Hemagglutination inhibition
  • N Neutralization for infectivity in tissue culture or animals
  • RID Radial immunodiffusion
  • Radioimmunoassay Only one company is known that currently produces diagnostics for the determination of antibody titers for specific pathogens infecting cats and dogs.
  • the device is called Biogal-lmmunocomb® and is manufactured by Biogal Gated Labs, Kibbutz Galed, M.P. Megiddo 19240, Israel.
  • a chromatographic assay device for use with immunoassays.
  • the device comprises: (1 ) a first opposable component comprising a sample preparation means adapted to receive a sample to be assayed, and a chromatographic medium; and (2) a second opposable component comprising a second application means and an absorbing means separated from the second application means.
  • a sample preparation means adapted to receive a sample to be assayed, and a chromatographic medium
  • second opposable component comprising a second application means and an absorbing means separated from the second application means.
  • addition of a sample to the first application means causes the sample to be applied to a first end of the chromatographic medium and wick along the medium toward a second end of the medium.
  • the first and second opposable components can be brought into opposition so as to cause the second application means to come into operable contact with the second end of the chromatographic medium so as to apply a specific binding partner to the second end of the chromatographic medium.
  • the chromatographic medium contains a detection zone containing a specific binding partner to the analyte immobilized thereto against diffusion.
  • EP 0,306,336 discloses an assay device comprising a housing, a piece of bibulous material enclosed in the housing for capturing a first member of a specific binding pair in a zone within the bibulous material, and a liquid absorbent material in liquid receiving relationship with the bibulous material.
  • the combination of the bibulous material and the absorbent material provides for capturing a specific binding pair member in a zone and transporting liquid away from the zone by capillary action.
  • This device is a compact system devised for convenient on site testing of a variety of analytes, but is not suitable for determining immune status of a vertebrate because the assay device of EP 0,306,336 is designed only for qualitative analysis.
  • EP 0,291,194 discloses an analytical test device useful in pregnancy testing.
  • the device comprises a hollow casting constructed of moisture-impervious solid material containing a dry porous carrier which communicates directly or indirectly with the exterior of the casting such that a liquid test sample can be applied to the porous carrier, the device also containing a labelled specific binding reagent for an analyte which labeled specific binding reagent is freely mobile within the porous carrier when in the moist state, and unlabelled specific binding reagent for the same analyte which unlabelled reagent is permanently immobilized in a detection zone on the carrier material and is therefore not mobile in the moist state, the relative positioning of the labelled reagent and detection zone being such that liquid sample applied to the device can pick up labelled reagent and thereafter permeate into the detection zone, and the device incorporating means enabling the extent to which the labelled reagent comes into the detection zone to be observed.
  • EP 0,284,232 discloses a solid phase assay device for determining the presence or absence of an analyte in a liquid sample.
  • the device comprises a test strip having a first and second portion and being arranged on the strip in the same plane in a manner such that material can flow by capillary attraction from the first portion to the second portion.
  • the first portion has a tracer movably supported therein wherein the tracer comprises a ligand specific for the analyte when the device is configured for a sandwich assay, or an analogue thereof when the device is configured for a competitive assay, conjugated to a nonsoluble particulate marker and being the site for addition of the sample.
  • the second portion has immobilized therein a binder which is specific for the analyte when the device is configured for a sandwich assay and is specific for the analyte and ligand when the device is configured for a competitive assay; the binder being present in an amount such that the tracer bound in such portion is visible.
  • U.S. Patent 4,703,017 discloses a solid phase assay for an analyte wherein binder is supported on a solid support, such as nitrocellulose, and the tracer is comprised of ligand labelled with a colored particulate label.
  • the assay has a high sensitivity, and the tracer is visible on the support under assay conditions, whereby tracer can be determined without further treatment.
  • U.S. Patent 4,861,71 1 discloses a solid diagnostic device for the quantitative determination of substances of biological affinity in biological fluids. A process is also described in which the biological fluid is brought into contract with specific functional sectors situated beside one another and containing suitable reagent components, and one or more substances of biological affinity are detected in such functional sectors which contain, for each substance to be detected, at least one combination partner of biological affinity, attached to a solid phase.
  • U.S. Patent 5,578,577 discloses improved specific binding assay methods, kits and devices utilizing chromatographically mobile labelled materials.
  • Methods and devices are provided utilizing colloidal particle labelled specific binding materials which are chromatographically mobile and capable of producing visually detectable signals, and enzyme labelled materials which are dried onto a chromatographic medium in the presence of a meta-soluble protein and are capable of being rapidly resolubilized in the presence of an appropriate solvent.
  • One aspect of the present invention provides a method of determining the presence of an immunoprotective level of antibody in a vertebrate, comprising the steps of: a) providing a chromatographic device having a first detection zone and a second detection zone;
  • another method of the present invention for determining whether a vertebrate has an immunoprotective level of antibody generally comprises the steps of: a) mixing a known volume of sample containing an antibody from the vertebrate with a predetermined amount of a first specific binding conjugate capable of binding specifically to an amount of the antibody corresponding to the immunoprotective level, the specific binding conjugate and the antibody forming a antibody-conjugate complex; b) applying the mixture of the sample and the specific binding conjugate to a sample application zone on a chromatographic medium, wherein the chromatographic medium additionally comprises a first and a second detection zone, the first detection zone contains an amount of immobilized second specific binding conjugate capable of binding specifically to at least the amount of the first specific binding conjugate applied to the sample application zone, the second detection zone contains a third specific binding conjugate immobilized thereto, the third binding conjugate is capable of binding specifically to the antibody; c) allowing the mixture to move through the chromatographic medium past the first and second detection zones on the chromatographic
  • a chromatographic device for determining the presence of an immunoprotective level of antibody in a vertebrate.
  • the device comprises: a chromatographic medium; a sample application pad on the chromatographic medium; and a first detection zone and a second detection zone on the chromatographic medium, wherein the first and second detection zones contain the same immobilized antigen capable of binding specifically to the antibody, and the first detection zone contains an amount of antigen capable of binding to an amount of antibody corresponding to the immunoprotective level of the antibody.
  • the device may further comprise a second opposable component comprising: a second absorbing material containing a binding compound capable of binding specifically to the antibody, the compound being labelled with a detectable label and in a form that can be resolubilized by the addition of an aqueous buffer to the second absorbing material, wherein the location of the second absorbing material is opposite the second end of the chromatographic medium when the first and second opposable components are brought into apposition; an aperture located opposite the second detection zone of the chromatographic medium when the first and second opposable components are brought into apposition; and a third absorbing material located opposite the first end of the chromatographic medium when the first and second opposable components are brought into apposition.
  • a second opposable component comprising: a second absorbing material containing a binding compound capable of binding specifically to the antibody, the compound being labelled with a detectable label and in a form that can be resolubilized by the addition of an aqueous buffer to the second absorbing material, wherein the location
  • Another aspect of the present invention provides a method for determining the immune status of a vertebrate.
  • the method comprises the steps of: providing an immunochromatographic medium having a sample catch zone capable of specifically binding a target analyte in a blood sample from the vertebrate, and at least a first control zone; mixing a predetermined amount of the sample with a first signal-generating conjugate capable of specifically binding to the target analyte so that substantially all the analyte is bound to the first conjugate forming an analyte- conjugate complex; passing the complex through the immunochromatographic medium to the sample catch zone, so that substantially all the complex is immobilized in the sample catch zone; passing a second signal-generating conjugate containing the same signal generator as the first conjugate and capable of binding to the control zone, so that a predetermined amount of the second conjugate is bound to the first control zone; measuring the signal intensity generated by the complex in the sample catch zone and the signal intensity generated by the second signal-generating conjugate in the first control zone, so that the concentration of the
  • the device comprises: an elongated immunochromatographic membrane having a first end, a second end, and an upper surface; a sample catch zone in the membrane capable of specifically binding to a target analyte in a blood sample of the vertebrate; a first control zone in the membrane capable of specifically binding a predetermined amount of a universal signal-generating conjugate; a second control zone in the membrane capable of specifically binding a predetermined amount of the universal signal-generating conjugate.
  • the chromatographic device may further comprises: a sample pad for introducing and filtering the blood sample so that only plasma or serum in the sample can pass the sample pad, the sample pad being located above the upper surface near the first end of the immunochromatographic membrane and in fluid communication with the membrane; a conjugate pad for storing the universal signal-generating conjugate and a binding conjugate capable of specifically binding to the analyte, the binding conjugate also containing the same signal generators as the universal signal-generating conjugate, the conjugate pad being located between the sample pad and the upper surface of the immunochromatographic membrane, wherein the conjugate pad receives the plasma or serum from the sample pad and mixes it with the conjugates stored in the conjugate pad when in use.
  • the system comprises: a chromatographic device for determining the presence of an immunoprotective level of antibodies in a vertebrate; an apparatus for preparing corresponding univalent vaccines and formulating them into the multicomponent vaccine; and an interface between the chromatographic device and the apparatus for receiving immune status information from the chromatographic device and sending the information to the apparatus to direct automatic preparation of the multicomponent vaccine.
  • the apparatus comprises: a vial for receiving the univalent vaccines and a complementing buffer to form the multicomponent vaccine; WO 99/40438 .., .,. PCT/US99/01511
  • a plurality of vaccine rehydration units for rehydrating the univalent vaccines and sending the rehydrated vaccines to the vial; a dispensing device connected to the vial for delivering the buffer to the vial; a manifold connecting the vaccine rehydration unit to the vial; wherein the vial and the rehydration unit are normally sealed.
  • the vaccine rehydration unit comprises: a capsule containing a lyophilized univalent vaccine fraction, the capsule having an upper end, a lower end, and a cylindrical body; a collapsible sachet containing an amount of water for rehydrating the lyophilized univalent vaccine fraction, the sachet having a first needle at its lower portion for engaging the upper end of the capsule and delivering the water therein into the capsule; a second needle connected to a tubing, which is connected to the manifold, for delivering the rehydrated vaccine fraction from the capsule to the vial, the a second needle adaoted to engage the lower end.
  • Another aspect of the present invention provides a method for determining the immune status of a vertebrate and formulating a multicomponent vaccine suitable for the immune status.
  • the method comprises the steps of: a) determining the presence of an immunoprotective level of target antibodies in the vertebrate; b) providing N univalent vaccines, each in a concentration N times their customary concentration and in a volume of 1/N a predetermined volume, wherein N is the maximum number of the possible univalent vaccine components; c) mixing the univalent vaccines against each pathogen for which the antibody level is less than the immunoprotective level and complementing the mixture with a buffer to add to the predetermined total volume.
  • step a) can be conducted by the steps of: providing a plurality of chromatographic devices, each having a sample application zone, a first detection zone capable of specifically binding to one of the target antibodies, and a second detection zone capable of binding to the same antibody; applying a predetermined volume of blood sample obtained from the vertebrate to the sample application zone of each of the chromatographic devices; allowing the sample to move through the chromatographic device past the first detection zone and then the second detection zone, wherein an amount of the antibody corresponding to the immunoprotective level of the antibody is bound to the first detection zone and at least a portion of the remaining antibody which passes the first detection zone is bound to the second detection zone; and observing the second detection zone for the bound antibody in each of the chromatographic devices, wherein the presence of the bound antibody indicates that the vertebrate has an immunoprotective level of the antibody.
  • step a) can be conducted by the steps of: providing a plurality of immunochromatographic media, each having a sample catch zone capable of specifically binding one of the target antibodies in the blood sample from the vertebrate, and a control zone; mixing a predetermined amount of the sample with a first signal-generating conjugate capable of specifically binding to one of the target antibodies so that substantially all said one of the antibodies is bound to the first conjugate forming an antibody-conjugate complex on each of the immunochromatographic media; passing each said complex through each corresponding immunochromatographic medium to the sample catch zone, so that substantially all the complex is immobilized in the sample catch zone; passing a second signal-generating conjugate containing the same signal generator as the first conjugate and capable of binding to the control zone, so that a predetermined amount of the second conjugate is bound to the control zone of each of the immunochromatographic media; measuring the signal intensity generated by the complex in the sample catch zone and the signal intensity generated by the second signal-generating conjugate in the control zone, so that the concentration of the antibody in the blood sample is quantitative
  • Still another aspect of the present invention provides a water-soluble conjugate.
  • the conjugate comprises a dextran polymeric carrier molecule to which are covalently attached at least a first molecular species and a second molecular species, each molecular species being attached via a linking group derived from divinyl sulfone, the attachment of each the linking group to the polymeric carrier molecule being via a covalent linkage formed between one of the two vinyl groups of a divinyl sulfone molecule and a reactive functionality on the carrier molecule, and the attachment of a molecular species to the linking group being via a covalent linkage formed between the other vinyl group originating from the divinyl sulfone molecule and a functional group on the molecular species, wherein said first molecular species comprises a targeting species capable of selective binding to, or selective reaction with, a complementary molecular or a complementary structural region of a material of biological origin.
  • Such a device for determining a plurality of immunoprotective levels of antibodies comprises: a) a plurality of chromatographic media comprising separate lanes; b) a plurality of sample application zones on the chromatographic media; and c) a plurality of first and second detection zones on the chromatographic media, wherein the detection zones are substantially smaller than the media, wherein the first and second detection zones on each of the separate lanes contains the same immobilized antigen capable of binding specifically to an antibody, wherein each of the first detection zones contains an amount of antigen capable of binding to an amount of antibody corresponding to a minimum immunoprotective level of the antibody and wherein each of the separate lanes contains a different antigen so that a separate antibody assay can be conducted simultaneously in each lane.
  • a device for the simultaneous determination of a variety of antibodies further comprises a first side.
  • the first side comprises: a) the chromatographic media comprising first and second ends and conducting material in contact with the first and second ends; b) the sample application zones in contact with the first ends of the chromatographic media; and c) absorbing material in contact with the second ends of the chromatographic media.
  • the aforementioned device with a first side preferably further comprises a second side.
  • the second side comprises: a) absorbing material containing specific binding partners capable of binding specifically to the antibodies, the partners being labelled with a detectable label in a form that can be resolubilized by the addition of an aqueous buffer to the absorbing material containing the binding partners, wherein the location of the absorbing material containing the binding partners is opposite the second ends of the chromatographic media of the first side; b) apertures, located opposite the second detection zones of the chromatographic media of the first side, for viewing the second detection zones; and c) absorbing material, located opposite the first ends of the chromatographic media of the first side.
  • a plurality of parallel immunochromatographic membranes can be employed. Each of the immunochromatographic membranes is adjusted to target one specific analyte or antibody.
  • the immunochromatographic membrane described in this section and the chromatographic medium described in the previous sections can be installed in a single device in a parallel manner. They can be adjusted to target the same analyte or different analyte.
  • FIGURE 1 is a drawing of one embodiment of an immunochromatographic device of the present invention for the determination of the protective antibody status of a patient for a particular antigen;
  • FIGURE 2 shows the avidin-biotin linked conjugates for use in present invention;
  • FIGURE 3 is a drawing of an immunochromatographic device with an internal standard;
  • FIGURE 4 depicts the process of the simple manual preparation of a customized polyvalent vaccine;
  • FIGURE 5 depicts a sachet and vial system to ensure the delivery of the correct volume of water for rehydration
  • FIGURE 6 depicts a system for joining two or more vials so that their contents can be mixed while retaining a sterile barrier
  • FIGURE 7 depicts the process for preparing a customized polyvalent vaccine with joined vials
  • FIGURE 8 depicts the process for preparing a customized polyvalent vaccine with joined vials
  • FIGURE 9 depicts the assembly of three components into a vaccine rehydration unit for use in an automatic method for preparing vaccines
  • FIGURE 10 depicts a module for the automatic preparation of a customized polyvalent vaccine.
  • the present invention describes how the immune status of a vertebrate can be more easily and precisely determined and how patient-specific vaccine "cocktails" can be automatically formulated.
  • the simple methods and devices of the present invention can be used in the office or laboratory of a doctor or veterinarian, or in the field, at the time of scheduled revaccination to determine the immune status of the patient for each of the pathogens for which revaccination is scheduled as well as to fully automate the formulation of the customized vaccine.
  • the basic method for revaccinating a vertebrate involves the steps of: a) determining whether the antibody level in the vertebrate against a plurality of pathogens is immunoprotective, i.e., whether a vertebrate has an antibody titer above or below a certain predetermined threshold limit; b) mixing vaccine components against each pathogen for which the antibody level is less than the immunoprotective level in a predetermined amount; and c) vaccinating the animal with the mixed vaccine components.
  • This basic inventive concept of the present invention can be carried out with a variety of devices and can be used for the determination of the pre-vaccination immune status of cats, dogs, birds and other vertebrates.
  • Appropriate devices include chromatographic assay devices, icrowell assay devices, and nonserological immunity assay devices. These devices and related methods are described below.
  • Chromatographic assay devices and methods employing such devices are particularly useful in the present invention to determine the immune status.
  • chromatographic assay devices have been used to determine the presence of certain analyte (such as antibody) in a particular sample.
  • analyte such as antibody
  • just knowing whether an antibody exists in a vertebrate is not enough to determine the immune status of the animal.
  • the total concentration of an antibody has to be measured and compared with its presumptive minimally-protective titer in order to determine the immune status for each target antibody, such a procedure will be time consuming, inconvenient, and impractical for a stand alone test in the office or laboratory of a doctor or veterinarian, or in the field.
  • the immune status of a vertebrate can be easily determined without the need to measure the total concentration of an antibody in the circulation of the vertebrate.
  • the methods of the present invention focus on determining whether antibody levels fall above or bellow a predetermined level for each target antibody, e.g. the presumptive minimally-protective antibody titers as discussed in the background section. Once this minimally-protective level is known for the entities for which the animal is to be vaccinated, determining the immune status can be performed by simply determining whether those antibodies exist above that level.
  • the approach for determining immune status according to the chromatographic assay method of the present invention comprises two basic steps:
  • step (b) If the target antibody is detected in step (b), the animal is determined to have the antibody in an amount above the minimally protective level and no revaccination is necessary.
  • Step (a) can be accomplished in a variety of ways.
  • a predetermined amount of a target antibody in a sample can be removed or separated from the rest of the sample through chemical and physical means.
  • the separation is achieved by binding a predetermined amount (corresponding to the minimally-protective level) of the target antibody to a specific binding pair immobilized to a zone within a chromatographic medium.
  • a sample containing a target antibody is passed through a first zone in a chromatographic medium.
  • a predetermined amount of specific binding pair member which is capable of specifically binding to the target antibody, is immobilized to the first zone so that the predetermined amount of the target antibody is bound and immobilized by the specific binding pair member, and the rest of the sample passes through the first zone.
  • a second zone is provided in the chromatographic medium downstream the first zone to receive the remaining antibody which passes through the first zone with the sample.
  • the antibody reaching the second zone is detected. If the antibody is detected in the second zone, it is a definite indication that the test animal has the antibody above its minimally-protective level and revaccination of the animal is unnecessary and possibly harmful. If no antibody is detected in the second zone, the animal needs to be revaccinated.
  • a variety of means can be used to detect the antibody in the second zone.
  • a binding pair member capable of specifically binding to the antibody is immobilized to the second zone so that part or all of the antibody entering the second zone will be bound to the binding pair member and immobilized. Then a liquid containing labeled binding reagent capable of specifically binding to the antibody is passed through the second zone and bound to the antibody captured in the second zone.
  • the labels in the reagent indicate the presence of the antibody in a predetermined way.
  • any conventional labels in the art can be used, such as monoclonal or polyclonal antibody directed against the immunoglobulins of the species for which the immune status is being determined.
  • These anti-antibodies may be labeled with a colloidal metal such as gold, or with colored latex beads, or with microparticulate metals or carbon. Alternately they may be labeled with latex polymers to which dyes are conjugated.
  • enzymes that can generate an insoluble colored complex when it contacts a specific substrate are: Horseradish Peroxidase/4-Chloro-1-Naphthol or, Alkaline Phosphatase.5-Bromo-4-Chloro-3- Indolyl Phosphate plus, Nitro Blue Tetrazoiium.
  • enzymes that can generate an insoluble colored complex when it contacts a specific substrate
  • examples of this enzyme/substrate pair are: Horseradish Peroxidase/4-Chloro-1-Naphthol or, Alkaline Phosphatase.5-Bromo-4-Chloro-3- Indolyl Phosphate plus, Nitro Blue Tetrazoiium.
  • other molecules which universally bind to immunoglobulins such as protein A or protein G, may be used to detect the antibodies of interest, when they are conjugated with one or more of the same substances as was indicated for the anti-antibodies.
  • proteins including ferritin, phycoerythrins, phycocyanins and phycobilins; enzymes, including horseradish peroxidase, alkaline phosphatase, glucose oxidases, galactosidases and ureases; toxins; drugs; dyes; fluorescent, luminescent, phosphorescent and other light- emitting substances; metal-chelating substances, including iminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylenediaminetetraacetic acid (DTPA) and desferrioxamine B; substances labelled with a radioactive isotope; and substances labeled with a heavy atom; substances labelled with a radioactive isotope of an element selected from the group consisting of hydrogen, carbon, phosphorus, sulfur, iodine, bismuth, yttrium, technetium, palladium and samarium; substances labelled with a radioactive isotope of an element selected from
  • the minimally-protective level of an antibody in a sample is separated by mixing the sample with a predetermined amount of an antigen or other binding compound capable of binding specifically to the antibody.
  • the amount of the antigen should be such that the amount of the antibody corresponding to the minimally-protective level is bound to the antigen. Then the mixture is fed to a chromatographic medium or a chromatographic column so that the antibody bound to the antigen travels in the medium or column slower than the unbound antibody because of its size or interaction between the binding compound and the medium and, thus, is separated from the unbound antibody. Then the unbound antibody can be detected in a detection zone with any suitable means.
  • a sample is first mixed with a predetermined amount of antigen or other binding compound capable of binding specifically to the target antibody in the sample.
  • the predetermined amount of the antigen should be such that an amount of the antibody corresponding to the minimally protective level is bound to the antigen.
  • the mixture is allowed to travel through a first zone in a chromatographic assay device.
  • the first zone contains a first specific binding pair member immobilized thereto in such amount that it is enough to bind all the antigen or binding compound in the mixture.
  • a second zone is provided downstream the first zone in the direction of sample flow.
  • the second zone contains a second specific binding member capable of specifically binding to the target antibody, therefore, the antibody passing from the first zone will be bound to the second specific binding member and immobilized to the second zone. Then a labeled specific binding reagent capable of specifically binding to the antibody is applied to the second zone, and bound to the antibody present in the second zone. Therefore, any antibody reaching the second zone will be indicated by the presence of the label.
  • the appropriate amount of a specific binding pair member needed to bind the amount of an antibody which corresponds to the minimal protective level can be precisely and readily determined by known methods.
  • step (b) the portion of the antibody in a sample corresponding to the amount above the minimally- protective level may be detected in a variety of ways.
  • the important feature is that only the presence of the target antibody needs to be detected in this step. This allows the use of highly sensitive detection means and, thus, results in high accuracy. Furthermore, determining whether an antibody is present is much easier than determining the absolute amount of an antibody present, which makes the assay much simpler and the results more reliable. Because the amount of binding pair member (or binding partner, binding entity) capable of specifically binding to a certain amount of a specific antibody can be determined precisely in advance, the method of the present invention converts a normally quantitative analysis into an on-site qualitative analysis. Once determined, the parameters can be standardized for each target antibody and the device can be produced on a large scale. It should be noted that the method and the device of the present invention are not limited to determine antibody levels. Obviously, they can also be used to determine levels of other analytes such as antigens in a sample taken from an animal.
  • the antibodies dealt with in the present invention include, but not limited to, antibodies specific to the group of immunogenic live, attenuated, killed, whole or part pathogens: canine parvovirus (CPV), canine adenovirus types I and II (CAV), rabies virus (RV), canine distemper virus (CDV), canine parainfluenza virus (CPIV), Leptospirosis species, canine coronavirus (CCV), Bordatella brochiseptica, Borellia burgdorferi, canine heartworm, feline panleukopenia parvovirus (FPLV), feline calicivirus (FCV), feline leukemia virus (FeLV), feline rhinotracheitis virus (FRV), Chlamydia psittaci, feline infectious peritonitis virus (FIPV), feline immunodeficiency virus (FIV), Haemobartonella felis, Bartonella henselae, ringworm and fleas.
  • CPV
  • FIGURE 1 is a drawing of one embodiment of a stand alone test (STAT) immunochromatographic device which can be used in the present invention for the determination of the protective antibody status of a patient for a particular antigen.
  • the immunochromatogaphic device 12 comprises a flat housing of moisture impermeable material such as plastics with a first side 1 and a second side 2, separated by a hinge region 9.
  • a chromatographic medium strip 3 is provided on the second side 2.
  • line 4 and 5 are perpendicular to the sample flow, or to the longitudinal axis of the strip 3.
  • a sample pad 6 is attached to one end of the strip 3 for receiving the sample and filtering out red blood cells if a whole blood sample is used. Also on the strip 3, at the opposite end of the sample pad 6, is a sump pad 10. On the first side 1 of the device is a conjugate pad 7 upon which is dried a colored conjugate directed against the patient immunoglobulins. A transparent window 8 and an absorbent pad 11 are also provided on the first side 1 of the device 12.
  • sample and chase buffer are applied to the sample pad 6.
  • buffer is also applied to the conjugate pad 7 so as to rehydrate the conjugate dried thereon.
  • Plasma wicks along the chromatographic medium strip 3 past antigen lines 5 and 4.
  • a predetermined amount of the specific antibody is bound by the antigen at line 5 and the rest of the antibody is bound by the antigen at line 4.
  • Excess plasma is allowed to run into the sump pad 10. Then the first side 1 is folded over onto the second side 2 and retained by a closure (not shown).
  • the absence of signal in the window, or for a very weak signal, is an indication that the animal is due for revaccination against the pathogen involved. Although a strong signal may develop on the line 5 it will not be seen, thus the possibility of confusion is eliminated.
  • the chromatographic method described above entails the use of an immunochromatic device in which, following its operation, a signal is seen by the operator only when the antibody levels in the sample exceeded the minimum requirements for protection.
  • a quantitative signal for the purpose of making judgments about immune status In this procedure, three or more separate signals are developed in the observation window following operation of the device. These individual signals will all develop as a result of specific binding of colored conjugates. There may be one or more specificities in the conjugate, but the color will in all cases depend on the same signal generator.
  • One of these signals is specific for antibodies against the pathogen for which immune status is being determined. It will occur at a site where catch antigen has been bound to the chromatographic membrane as a stripe placed orthogonally to the direction of flow on the membrane. This signal will develop if the sample contains antibody against the test entity and it will be proportional to the concentration of such antibodies in the test sample.
  • Two other signals will also be developed on orthogonal stripes. Their purpose is to act as internal controls for the device.
  • the materials placed on these stripes will be unrelated to any entity in prospective samples and, thus will not be reactive with any entity in prospective samples. They will bind conjugate when contacted with it.
  • the materials on these two stripes will be at different concentrations: a high and a low level. They will be adjusted so that the signals that result when the device is operated correspond to the high and low limits (over a linear range), for the antibody signal that the device is designed to measure.
  • This method and device provide an internal standardization to control for sample to sample variation.
  • a variety of signal generating conjugates and formulating methods can be employed. CHROMATOGRAPHIC DEVICE WITH INTERNAL STANDARDIZATION AND UNIVERSAL CONTROL SIGNAL
  • a chromatographic device with internal standardization and a universal control signal is provided in accordance with the present invention.
  • FIGs. 3a and 3b an embodiment of such an assay device is shown.
  • this device has a longitudinal immunochromatographic membrane 300.
  • the membrane 300 is an elongated rectangular sheet made of nitrocellulose.
  • a conjugate pad 304 is provided on the surface of the immunochromatographic membrane 300 adjacent a first end of the membrane 300, and is in fluid communication with the membrane 300.
  • a sample pad 302 is provided on top of the conjugate pad 304 and in fluid communication with the conjugate pad 304.
  • the lower surface of the sample pad 302 is in touch with the upper surface of the conjugate pad 304, and the lower surface of the conjugate pad 304 is in touch with the upper surface of the immunochromatographic membrane 300.
  • a low control zone 306, a high control zone 308, and a sample catch zone 310 are provided along the length of the immunochromatographic membrane 300.
  • the sample catch zone 310 contains materials that are able to bind all the WO 99/40438 .., g . PCT/US99/01511
  • the low and high control zone 306 and 308 contains the same materials, which can bind the conjugate for generating control signals in the two zones, but at predetermined and different concentrations.
  • those zones 306, 308, and 310 have a linear shape and are substantially perpendicular to the longitudinal direction of the immunochromatographic membrane 300.
  • the sequence at which those three zones 306, 308, and 5 310 are arranged along the longitudinal direction of the immunochromatographic membrane may vary.
  • the sample catch zone 310 is located between the low control zone 306 and the high control zone 308 with the low control zone 306 being closest to the sample pad 302.
  • a sump pad 312 is provided on the upper surface of the immunochromatographic membrane 300 near its second end for receiving the sample and the conjugate that pass through the membrane 300.
  • the assay is performed by adding a sample of blood or serum to the sample pad 302. Cells are normally retained by the sample pad 302 while the plasma or serum pass through to the conjugate pad 304.
  • Two types of conjugates are used.
  • Type (a) conjugate is signal generator-containing conjugate capable of specifically binding the target analyte.
  • Type (a) conjugate will not be bound the control zone 306 and 308 when it passes through the control zone.
  • Type (b) conjugate contains the same type of signal generators as type (a) conjugate and can be bound 5 to the control zone 306 and 308 when it passes the control zone.
  • type (b) conjugate is not capable of binding the target analyte. Because type (b) conjugate does not interact with specific analyte and serves to generate a internal standard signal with the same signal generator, it is also referred as universal-signal-generator conjugate. Both type (a) and type (b) conjugate are pre-prepared and stored in the conjugate pad 304 in such an amount that all the target analyte in the sample will be bound by the type (a) conjugate and a desired signal intensity will be 0 developed in the two control zones 306 and 308 by type (b) conjugate or the universal-signal-generator conjugate.
  • the signals to be generated at zones 306 and 308 are made to simulate a high and a low level of antibody-specific signal as would generate at zone 310.
  • the maximum signal that could develop at zone 310 would be identical to, or somewhat below, that which generates at zone 308.
  • the signal that generates at zone 310 is linearly proportionate to the specific antibody concentration in the sample.
  • the numeric values of the signals that 5 develop at zones 306 and 308 are determined by, for example, an optical instrument with calculating abilities, then the numeric value of the signal generated at zone 310, can be converted into an antibody concentration in the sample, by simple interpolation.
  • the system could be used without detection instrumentation.
  • Liquid is provided to rehydrate the colored (in the case the signal is visible color) conjugates of type (a) and (b) in the conjugate pad 304.
  • a dextran-based signal-generating conjugate is used.
  • the reactive groups, the molecular weight ranges, the macromolecu.es that can be conjugated and the methodology for conjugation 5 are described in the PCT application WO 93/01498, which is incorporated herein by reference in its entirety.
  • the presnet invention also provides a dextran-signal-generator-avidin type of conjugate which is an improvement based on the conjugates desclosed in WO 93/01498. Detailed discussion of the dextran-signal-generator-avidin type of conjugate will be given later.
  • the liquid sample and the rehydrated conjugates of type (a) and (b) are mixed within the conjugate pad 304 where the type (a) conjugate reacts with the target analyte in the sample.
  • the resulting mixture is allowed to flow 5 along the immunochromatographic membrane 300.
  • this mixture passes over the low control zone 306 and the high control zone 308 to which a predetermined amount of biotin is attached, certain amount of the type (b) or universal-signal-generator conjugate will be bound by each control zone 306 and 308.
  • the amount of the type (b) conjugate being bound to the control zone 306 and 308 is determined by the amount of the biotin immobilized to the control zone 306 and 308.
  • the two control zones are configured so that the low control zone 306
  • the sample 10 immobilizes the biotin at a concentration lower than that in the high control zone 308. This results in the development of high and low control signal intensities in the two zones 306 and 308, respectively.
  • the target analyte for example an antibody of interest
  • the target analyte will first be bound to the type (a) conjugate, and then bound to the sample catch zone 310 together with the type (a) conjugate which is bound to the target analyte.
  • the sample-conjugate mixture continues to flow along the immunochromatographic membrane
  • the amount of type (b) conjugate applied in the conjugate pad 304 should be enough to saturate both low control zone 306 and high control zone 308.
  • the amount of type (a) conjugate applied to the conjugate pad 304 should be enough to bind all the target analyte in the sample.
  • the signals from the two control zones 306 and 308 give two points in an intensity-concentration curve for a specific type of signal generator. If the amount of the universal-sig ⁇ al-generator conjugate to be bound to the
  • a calibration line can be obtained from the two points.
  • the third signal from the sample catch zone 310 can be calibrated with the calibration line. Because the third signal intensity is substantially proportional to the amount of the signal generators captured in the sample catch zone 310, and is comparable to the signal generated in the control zones 306 and 308, the two point calibration line can be used to
  • the 25 quantitatively measure the amount of the type (a) conjugate bound to the sample catch zone 310.
  • the amount of the target analyte in the sample can be determined from the amount of the type (a) conjugate bound to the sample catch zone 310.
  • Step 1 involves activating a dextran polymer
  • the signal-generating species can be selected from: proteins, including ferritin, phycoerythrins, phycocyanins and phycobiiins; enzymes, including horseradish peroxidase, alkaline phosphatase, glucose oxidases, galactosidases and ureases; toxins; drugs; dyes; fluorescent, luminescent, phosphorescent and other light-emitting substances; metal-chelating substances, including iminodiacetic acid, ethylenediaminetetraacetic acid (EDTA),
  • proteins including ferritin, phycoerythrins, phycocyanins and phycobiiins
  • enzymes including horseradish peroxidase, alkaline phosphatase, glucose oxidases, galactosidases and ureases
  • toxins drugs
  • dyes fluorescent, luminescent, phosphorescent and other light-emitting substances
  • metal-chelating substances including iminodiacetic acid,
  • DTPA diethylenediaminetetraacetic acid
  • desferrioxamine B substances labelled with a radioactive isotope selected from hydrogen, carbon, phosphorus, sulfur, iodine, bismuth, yttrium, technetium, palladium and samarium; and substances labeled with a heavy atom selected from Mn, Fe, Co, Ni, Cu, Zn, Ga, In, Ag, Au, Hg, I, Bi, Y, La, Ce, Eu and Gd.
  • the dextran polymeric carrier can be selected from carboxymethyl-dextrans, starches, hydroxyethyl- starches, hydroxypropyl-starches, glycogen.
  • step 2 avidin, a protein that specially binds up to four molecules of vitamin biotin, is covalently attached to the free divinyl groups at regular intervals along the length of the dextran molecule 21, forming a complex 22.
  • This complex 22 can be specifically bound to biotin and is used as a universal signal-generating (type (b)) conjugate.
  • Complex 22 can be further modified into a type (a) conjugate by obtaining the requisite specificity for binding to an analyte. This can be done by mixing the complex 22 with an analyte binding reagent 23 that has been derivitized with biotin as shown in step 3 of FIGURE 2.
  • the analyte binding agent may be selected from the following species and has one or more biotin groups attached thereto: antigens; haptens; monoclonal and polyclonal antibodies; gene probes; natural and synthetic oligo- and poiynucleotides; natural and synthetic mono-, oligo- and polysaccharides; lectins; avidin and streptavidin; biotin; growth factors; hormones; receptor molecules; protein A and protein G.
  • the reaction of the biotin-derivitized analyte binder 23 and the signal generating complex 22 can be terminated by the addition of excess biotin as shown in step 4, forming a conjugate 24 (type (a)) which is capable of specifically binding a target analyte but will not bind to the biotin in control zones 306 and 308, because the excess biotin binds to all remaining free binding sites on the avidin molecules. Subsequently, any unbound biotin can readily be removed.
  • a conjugate 24 type (a)
  • the dextran polymer carrier is described in WO 93/01498.
  • the reaction between avidin and dextran is also discussed in WO 93/01498.
  • the polymeric dextran used in this invention may have a peak molecular weight of about 500,000, or about 2,000,000, or in any one of the following ranges: about 1000 to 20,000, about 20,000 to 80,000, about 80,000 to 5,000,000, or about 5,000,000 to 40,000,000.
  • Reactive vinyl groups on the dextran polymer are in the range of 1 to 5,000 //moles per gram of the polymer.
  • Each dextran polymer molecule may have 1 to 10000 molecular species of molecular weight about 2000 or below covalently attached thereto, or have 1 to 1000 molecular species of molecular weight about 2000 or above covalently attached thereto.
  • the molecular species can be a signal-generating group or an avidin groups.
  • the molar ratio of the attached avidin groups to the dextran polymer molecules is in the range 1 to 30, preferably 2 to 14.
  • the reactive groups on the molecules that are to be conjugated to the activated divynalated dextran are nucleophilic groups such as -0 , - S , -OH, -SH, primary amines and secondary amines.
  • the reaction to perform the conjugation of the macromolecule to the activated polymer can be performed at room temperature in an aqueous buffer at above pH 5.0.
  • PROCEDURE CONTROL This invention proposes the use of the dextran-signal-generator-avidin universal signal reagent as a universal procedural-control signal generator. It can be used in a lateral flow device as described above. The required condition is achieved by the use of a biotin conjugate positioned at the procedural control site on the diagnostic substrate.
  • the signal generating conjugate for the diagnostic comprises two types: the universal procedural-control signal generator conjugate (type (b)) and the analyte-specific signal generator conjugate (type (a)) that can be prepared from the universal signal generator conjugate.
  • the analyte-specific signal generator conjugate can only bind to analyte that is intended to be bound to the analyte specific site in a sample catch zone and not to the biotin at the procedural-control site in a control zone, because all of its receptors for biotin have been saturated during the final steps of preparing the analyte-specific signal generator conjugate.
  • the universal procedural- control signal generator conjugate cannot bind to the analyte specific site in the sample catch zone since it lacks any specificity for the analyte-specific site but, it is designed to bind to the procedural control site in the control zone because its component avidin molecules have free receptors for binding biotin that is present at that site.
  • the universal procedural-control signal generator conjugate described above can be used without modification in all membrane-bound diagnostic tests. It can be used to obtain a semi-quantitative result by direct visual inspection or by instrument reading.
  • the universal procedural-control signal generator conjugate can develop more than one localized signal in the same device. These signals are of different intensities and span a range that corresponds to that range of sample signals for the range of analyte concentrations that the diagnostic can detect. This correspondence permits the quantification of an unknown sample when run on the diagnostic and the results are interpreted by a reading and calculating device.
  • the procedural-control signal or internal standard signal produced does not vary in intensity and is independent of the sample composition.
  • one important aspect of this invention is to provide a means to achieve a universal control signal and internal standardization in membrane-bound diagnostics so that the analyte- specific signal resulting form the performance of a diagnostic test on a sample can be quantified more precisely.
  • Stand alone test (STAT) diagnostics are usually designed so that when the user performs the test on a sample, a signal is generated that is an indicator to the user that the test was performed correctly. This signal is the procedural control signal and it is generated whether or not the sample that was tested contains the analyte that the test is designed to detect.
  • the substrate upon which the test is performed must thus contain at least two distinct regions for the binding of substances that can lead to the generation of signal.
  • At least one of these regions must uniquely bind the analyte that is to be detected.
  • the presence of bound analyte is indicated by a signal generator that binds only to analyte. This binding to analyte may occur either prior to the analyte contacting the specific binding site on the substrate as discussed in the previous section, or subsequent thereto.
  • the other region on the substrate (the procedural control zone) to which a universal signal- generating conjugate can become attached is located either downstream or upstream of the analyte-specific binding region.
  • the procedural control zone can be located in such a position that the universal signal- generating conjugate traverses the procedural control zone only after it has traversed the analyte-specific binding zone (sample catch zone).
  • the signal thus generated ensures that the analyte-specific binding region had the opportunity to bind signal generator and, by inference, would have done so if analyte had been present in the sample.
  • the nature of the binding entity at the procedural control site has been problematic. It usually has to be different with each test type. In addition, it needs to be different depending on whether the analyte detected is an antibody or an antigen. Also, under certain circumstances the signal generators for the analyte- specific site and the procedural control site need to be different with each type having a uniquely different binding specificity.
  • the analyte that is to be detected is an antigen
  • the analyte-specific signal-generating conjugate is usually linked to an antibody in order to achieve specificity of binding.
  • This analyte-specific signal-generating complex may also serve to generate signal at the procedural control zone, by incorporating at the procedural control zone a reagent that specifically binds either the antibody or the signal generator itself of the analyte-specific signal-generating conjugate.
  • This reagent can be an antibody. However, its specificity must be changed if either the signal generator or the species from which the conjugate-antibody is derived, are changed.
  • an antibody that binds to a gold conjugate would not be expected to bind to a dextran/dye conjugate nor would a monoclonal antibody, specific for feline immunoglobulins, be expected to bind to canine immunoglobulins.
  • the analyte-specific signal-generating conjugate may either be linked to the same antigen as that which binds the antibody at the analyte specific zone (sample catch zone) or, it may be linked to a reagent that binds to antibody.
  • the antibody binding reagent may either be an anti-immunoglobulin antibody or it may be a protein such as protein A or protein G that can bind to the Fc portion of immunoglobulins.
  • the binding entity at the procedural control zone can be an antibody either against the antigen or against the signal- generating conjugate.
  • the binding entity at the procedural control zone can either be an anti-immunoglobulin or, either protein A or protein G.
  • an additional major problem with those methods relates to the variation in signal strength that results.
  • the dynamic range of the concentration of analyte that may be detected extends over two or three orders of magnitude, the amount of conjugate that arrives at the procedural-control zone can vary greatly depending on the amount of analyte in the sample.
  • a sample containing a high concentration of analyte will result in a strong signal at the analyte specific zone and a weak signal at the procedural-control zone.
  • a sample with low concentration of analyte will result in a weak signal at the analyte-specific site and a strong signal at the procedural-control site.
  • conjugates of the present invention provide the solutions to these problems.
  • the value of this type of conjugate in a diagnostic test is that the signal that is generated following the binding to the analyte of interest is greatly enhanced over that obtainable with usual conjugates in which the analyte binding molecule is linked to one or a few signal generators. The result is that the sensitivity of the diagnostic is greatly enhanced.
  • Serological titration can also be performed in microwell plates.
  • the antigen of interest is used to coat the microwells and these are then incubated with serial dilutions of the test serum. After washing away nonbound antibody and other serum proteins, the amount of bound antibody in each well is then determined by first reacting with an anti-immunoglobulin enzyme-conjugate and then, following a wash step to remove unbound conjugate, permitting the enzyme of the bound conjugate to develop a colored product from a colorless substrate during a final incubation step.
  • the plate is then read in an optical reading device and the titration of the antiserum is determined (in optical density units), by multiplying the optical density of that well whose value does not exceed the upper limits of the range for the optical device, by the dilution factor of the sample in the well (see Tijssen, P., Practice and Theory of Enzyme Immunoassays, Elsevier, New York, 1987.). Determination as to whether the antiserum had titers at a protective level can be made by comparison with the optical density units obtained for antisera having a minimum protective titer.
  • serum-antibody titers do not correlate well with the level of protection. This is particularly true with herpes viruses where protection is primarily dependent on cell-mediated immunity.
  • the level of protection can be determined from the level of helper T-cell memory cells.
  • white blood cells are prepared from a sample of whole blood, and these are then incubated with the organism (or a representative antigen or, an rDNA polypeptide derived from the organism), for which immunity is being determined. If there are specific memory T-cells in the white blood cell preparation they will respond by producing gamma interferon. The amount of gamma interferon will be proportional to the number of memory T-cells and thus to the degree of protective immunity in the animal from which they came.
  • This interferon can be measured directly in a quantitative ELISA which uses an ultrasensitive enzyme-conjugate, such as that offered by the technology described in International publication No. WO 93/01498.
  • the newly induced mRNA for the interferon can be quantified by quantitative cDNA-based PCR.
  • VACCINE FORMULATION After the immune status of an animal is determined according to the methods and devices described above, the vaccines needed are formulated accordingly. Therefore, another aspect of the present invention is a resolution of the problem of preparing selected multicomponent vaccines.
  • the present invention discloses an improved and simple device and method for preparing patient-specific multicomponent vaccines.
  • Antibody titration and the customizing of vaccines represent two basic functions of this aspect of the present invention. These two functions are integrated in a single system that is able to determine antibody titers from a single sample of blood and then automatically prepare the appropriate customized vaccine. Such a system can fine-tune the patient's vaccine requirements to a greater degree than previous methods and devices. This is because the determined antibody titers would not be cut-off values as previously described, but absolute values as determined by the system.
  • the system can also employ different antibody assay methods for different antibodies. That is, it can also be designed to employ the optimal assay method for each antibody.
  • This aspect of the present invention entails as a first step the sterile preparation of the suspensions of each vaccine type for one dose of vaccine, in a volume of 1/n ml (where n is the maximum number of vaccine components), prior to freeze-drying.
  • n is the maximum number of vaccine components
  • the next step is the determination of the immunoprotective level of a plurality of antibodies utilizing any chromatographic devices or other assay devices discussed previously. The information from the chromatographic device regarding immunoprotective levels of antibodies is conveyed via an interface between the chromatographic device and the apparatus for preparing a multicomponent vaccine.
  • the aforementioned steps can be performed manually, however, manual sample processing usually is not suitable for use in the office or laboratory of a doctor or veterinarian. Thus, one object of the present invention is to provide a device for the automation of these steps.
  • the solution to the problem regarding preparing selected multicomponent vaccines in the present invention is to first produce the individual vaccine components at between 6X and 10X of their customary concentration. All of the components are then provided to the end user in a single device in separate sterile compartments at a volume of 1/n ml, where n is the maximum number of components that could be administered. Also provided on the device are separate sterile compartments of diluent at volumes of 1/n, 2/n, 3/n, 4/n and, 5/n ml.
  • a mixing apparatus can also be provided in the device whereby the contents of any compartment can be mixed with that of any other in a mixing system such as a mixing chamber.
  • any required vaccine can be formulated on the device, based upon information generated by the chromatographic devices or other assay devices of the present invention and conveyed through an interface, so as to contain the necessary vaccine components in a final sterile volume of 1 ml. After formulating the custom vaccine, the unused entities on the device can be discarded.
  • the interface part conveys the information from the chromatographic device part and directs the apparatus for preparing a multicomponent vaccine to select the contents of the compartments containing A, B and C and mix them in the mixing chamber along with the contents of the compartment containing 3/6 ml of diluent, to yield a polyvalent vaccine containing components A, B and C in a volume of 1 ml.
  • Another patient may require components D, E and F in its customized vaccine.
  • compartments containing these components would be mixed with that containing 3/6 ml of diluent to prepare its requisite vaccine.
  • Any other vaccine cocktail is prepared in an analogous fashion whereby the contents of the compartment containing the requisite components are mixed with the contents of the compartment containing the volume of diluent that is the complement of 1 ml.
  • N is the size of the population from which all possible different groups of size n are selected. It is highly impracticable to have each of these combinations preformulated in the standard 1 ml doses so as to provide for the needs of all revaccination scenarios. The alternative of mixing the appropriate monovalent vaccines that are each produced in 1 ml is also unacceptable. Such oversize vaccines of up to 6 ml could not be administered to a small cat, dog or bird.
  • Solution vaccine components are encountered where the immunogenic fractions are either killed components, rDNA-derived or, synthetic.
  • the components are freeze-dried and the end user is required to rehydrate the vaccine "cake" with diluent immediately prior to vaccination.
  • the living organisms that are to be freeze-dried are normally suspended in buffered solutions that have a pH and osmolarity that is compatible with living tissue and thus the diluent that is supplied for rehydration of the "cake” is normally sterile water. This follows, since the only material removed during the freeze-drying operation is the water component of the organism suspension.
  • FIGURE 4 depicts the processes in the simple manual preparation of a customized polyvalent vaccine.
  • FIGURE 4a shows six different monovalent vaccine components 402 (A through F), from which it is desired to prepare a customized vaccine.
  • Each component 402 includes a vial 404 and a rubber stopper 406 for sealing the vial 404.
  • FIGURE 4b three components 402 (A, C & E) have been selected as the components for the polyvalent vaccine and, to each vial 404 the correct volume of water has been added from a syringe 408. Sterility has been maintained by piercing the rubber stopper 406 with the syringe needle and by not removing the stopper 406.
  • FIGURE 4c the rehydrated vaccines are removed from their vials 404 by inversion of the vials 404 and withdrawal of the fluid into fresh sterile syringes 408.
  • FIGURE 4d depicts the pooling of the three fractions in a sterile vial 404.
  • FIGURE 4e the calculated volume of buffer has been added to make a unit dose of the combination vaccine.
  • FIGURE 5 depicts a means to ensure the delivery of the correct volume of water for rehydration. This is achieved by providing the necessary volume of water in a single-use delivery system.
  • FIGURE 5a illustrates a sachet 502 of water provided with an affixed syringe needle 504 which in turn is surrounded by a cap 506 so as to retain sterile integrity.
  • the sachet 502 is made of a flexible material, such as polyethylene or other plastic and, with emptying, it collapses.
  • FIGURE 5b depicts the removal of the protective cap 506 form the tube 508.
  • FIGURE 5c depicts the dispensing of the water from a sachet 502 into a vial 404 containing lyophilized vaccine.
  • the vial 404 is vacuumed first. Because the vial 404 is sealed under vacuum, when the sachet needle 504 is inserted through the stopper 406 into the vial 404, water is drawn into the vial 404 and the sachet 502 collapses.
  • FIGURE 6 depicts an assembly for joining two or more vials 404 so that the contents from the two vials can be mixed while retaining a sterile barrier.
  • This assembly includes a double-headed syringe-type needle 602 and a special stopper 604 for the vaccine vial 404.
  • a double-headed syringe-type needle 602 and a special stopper 604 for the vaccine vial 404.
  • different numbers of the stopper 604 and the double-headed needle 602 can be used.
  • the connection between the double-headed needle 602 the domed stopper is based on an adaptation of a threaded Luer lock that is used to join narrow tubing and syringe needles to syringes.
  • FIGURE 6a depicts the elements of a threaded Luer lock 603.
  • the Luer lock 603 includes a female receptacle 606 defining a cavity.
  • the cavity has an elongated cylindrical shape.
  • At one end of the cavity there is a flange 612 extending away from the cavity.
  • the Luer lock 603 also includes a male juncture 608 extending outwardly from the base of a syringe 614.
  • the juncture 608 is adapted to fit into the cavity defined by the female receptacle 606.
  • the juncture 608 is further surrounded by a sheath 610 which is also secured to the base of the syringe 614.
  • the sheath 610 preferably has a cylindrical shape and is threaded on its inner surface for engaging the flange 612.
  • the double headed needle 602 of FIGURE 6b resembles the male juncture 608 and the cylindrical sheath 610, where two of them have been joined back-to-back by eliminating the syringe 614 base.
  • the male juncture 608 has been replaced with a syringe-type needle 608a.
  • the stopper 604 for connecting a plurality of the vaccine vials 404 is depicted in FIGURE 6c. The stopper
  • the 604 has a cylindrical body defining a cavity 616.
  • One end of the cylindrical body is open to the cavity 616 and adapted to sealably engage the opening of the vial 404.
  • the other end of the cylindrical body is closed.
  • a circular projection 618 extending outwardly along the outer periphery of the cylindrical body is provided intermediate between the closed end and the open end for facilitating the sealing and holding the stopper 604 in position when engaged with the vial 404.
  • Two Luer-type female receptacles 606 are attached to the cylindrical body at locations between the closed end and the open end. Preferably, the two receptacles 606 positioned diagonally opposite each other.
  • the receptacle 606 can be made of plastics such as polyethylene or polypropylene and annealed to the stopper 604 which can be made of rubber.
  • the receptacle 606 is not in fluid communication with the vial 404 when the stopper 604 engages the vial 404.
  • the needle 602 will move towards the depression 620 at the base of the receptacle 606 and just pierce into the cavity 616 of the stopper.
  • FIGURE 6d depicts a vial 404 closed with a stopper 604 through which one end of a double-headed needle 602 has pierced.
  • FIGURE 7a shows the three vaccine components A, C & E that have been rehydrated using the water from collapsible sachets 502.
  • FIGURE 7b shows the three vials 404 joined together using the double-headed needles 602. This is done with a needle 602 joining component A to C and another needle 602 joining component C to E.
  • component E has another double-headed needle 602a attached to the second receptacle 606 on its stopper 604.
  • the needle 602a is different from the standard double-headed needle 602 in that its distal end does not have a surrounding threaded sheath 610.
  • FIGURE 8a the needle 602a is used to pierce a standard stopper 406 of a vial 404, so that the conjoined components A, C & E are at a slight angle from the vertical as depicted.
  • This causes the rehydrated liquid vaccines 624 in each vial 404 to be collected at the stopper 604 directly above the lower pierced female receptacle 606.
  • the vial 404 with the standard stopper 406 is sealed under vacuum, the liquids in the components A, C & E are drawn into it.
  • the requisite amount of buffer is added to the vial 404, to complete the preparation of the customized vaccine.
  • FIGURE 9a depicts three components of a vaccine rehydration assembly 900.
  • the three components are a sachet unit 500 for rehydration of a vaccine fraction, a capsule unit 901 for containing a lyophilized vaccine fraction and, a needle-tubing unit 904 for delivering a rehydrated vaccine fraction. When assembled, as described below, they form the vaccine rehydration assembly 900.
  • the sachet unit 500 includes a collapsible sachet 502 for containing the correct volume of water for rehydration of the vaccine fraction.
  • a needle 504 is attached to the sachet 502 for transferring the water from the sachet 502 to the capsule unit 901.
  • the needle 504 is surrounded by a sheath 908 which is attached to the sachet 502 at one end.
  • sheath 908 does not need to be threaded on its inner surface.
  • the sheath 908 can be of any proper shape. In one embodiment, it is made cylindrical.
  • the sheath 908 is made of rigid plastics although other suitable materials such as metals also can be used.
  • the sheath 908 has an ear 910 extending away from and along at least a portion of the outer periphery of the sheath 908.
  • the ear 910 can be made of any suitable shape, such as a rod or flange, and of the same material as the sheath 908, such as rigid plastics. It is intended for holding or moving the sachet unit 500 in a mechanical device. Instead of being located at the end of the sheath 908, the ear 910 also can be provided at other location along the sheath 908.
  • the capsule unit 901 includes a capsule 902 having a cylindrical body 912 with two hemispherical ends 914.
  • the capsule 902 can be also made of other suitable shapes.
  • a male coupling 916 is attached at the apex of each hemispherical end 914 at the apex of each hemispherical end 914 at the apex of each hemispherical end 914 at the apex of each hemispherical end 914.
  • the coupling 916 is a cylindrical tube extending away from the capsule 902 along the longitudinal axis of the capsule 902.
  • the coupling 916 also has an ear 918 at its proximal end adjacent to the hemispherical end 914.
  • the ear 918 extends away from and along at least a portion of the outer periphery of the sheath 908.
  • the ear 918 can be attached to other locations along the coupling 916.
  • the capsule body 912 can be made of rigid plastics such as polypropylene, or of glass, such that it is noncollapsible under vacuum.
  • the hemispherical ends 914 can also be made, in part, of a rigid plastic or, totally of rubber.
  • the portion of the hemispheres 914 connected to the base of the male coupling 916 must be made of a material that is piercable by a syringe-type needle, such material can be rubber.
  • the male coupling 916 and its ear 918 may be made of the same rigid material as the sheath 908 and the ear 910 on the sachet 502.
  • the needle-tubing unit 904 includes a needle 920 secured to and surrounded by a sheath 922.
  • the sheath 922 has a cylindrical shape and can be made of the same material as the sheath 908.
  • the needle 920 is attached to and in fluid communication with a flexible tubing 906.
  • one end of the sheath 922 has a hole for passing the needle 920 and, if desired, is sealed around the needle 920.
  • the needle 920 can be secured to the hole with any conventional means such as using a glue.
  • an ear 923 is attached similar to that described for the sachet.
  • the ear 923 extends away from and along at least a portion of the periphery of the sheath 922.
  • the internal diameter of the sheaths 908 and 922 on the sachet 502 and the needle-tubing unit 904 can be made identical.
  • the sheaths 908 and 922 are intended to accept the male couplings 916 on the capsule 902.
  • the male coupling 916 has an outer diameter that is slightly less than the inner diameter of the sheath 908 or 922.
  • the needle 504 on the sachet 502 and the needle 920 of the needle-tubing unit 904 form a tight coupling with the inner surface of the male coupling 916.
  • FIGURE 9b depicts the combination of the three components described above into a vaccine rehydration assembly 900 for use in the automatic method.
  • the capsule 902 When assembled, the capsule 902 would contain lyophilized monovalent vaccine and the sachet 502 the necessary amount of water to rehydrate the vaccine.
  • the junction between the sachet needle 504 and the male coupling 916 into which it fits is sealed with a wax or an easily displaced glue so as to prevent leakage of water during storage or handling.
  • the ears 910 and 918 on the sachet 502 and its male coupling 916 as well as the ears 923 and 918 on the needle-tubing unit 904 and its male coupling 916 are brought close to each other so that the needle 504 engages one coupling 916 and the needle 920 engages another coupling 916, but separated by spacers 926, respectively.
  • the spacer can be any suitable shape and material, such as a cylindrical (or other sectional format) rigid elongated structure.
  • the spacer 926 is intended to prevent the accidental piercing of the capsule 902 by the needle 504 on the sachet 502 or the needle 920 on the needle-tubing unit 904.
  • rigid drivers 928 placed against the free surfaces of the sachet ear 910 and the ear 923 of the needle-tubing unit 904.
  • its proximal driver 928 can be used to cause penetration of the capsule 902 by the needle 504 or 920 as a result of pushing the abutted ear 910 or 923 towards the capsule 902.
  • FIGURE 10 shows an elevation view of a module 100 for the automatic preparation of a customized polyvalent vaccine, positioned as it would be in the instrument which performed the preparation.
  • the module comprises a rigid matrix 102 for supporting and positioning a number of components.
  • the components on the matrix 102 include a vial 404 for the preparation of the vaccine, vaccine rehydration units 900, a dispensing device 104 such as a syringe that can accurately deliver a volume of buffer to complement the volume of the mixed univalent vaccines, and a tubular manifold 106.
  • the matrix 102 can be made of plastics or other inexpensive and disposable material.
  • the components are attached to the matrix 102 or molded into it. In stead of using a matrix 102, the above mentioned components also can be arranged in other forms.
  • the vial 404 in which the vaccine is prepared must be easily removable from the matrix 102 after the vaccine formulation process is complete.
  • a standard flat stopper 406 of the vial 404 is modified to have two short rigid tubes 108 secured to its outer surface.
  • the tube 108 is intended to act as guide for penetrating syringe-type needles as described below.
  • the tube 108 is perpendicular to the outer surface of the stopper 406.
  • the needle-tubing unit 904 of each vaccine rehydration unit 900 is connected to the tubular manifold 106.
  • the manifold 106 is also connected to a syringe type needle 110, that is adapted to be inserted into one of the tubes 108 on the outer surface of the vial stopper 406.
  • the needle 110 has an ear 112.
  • the ear 112 can be a part of a sheath 111 surrounding the needle 110, similar to that previously described for the needles in the vaccine rehydration unit 900, or the sheath 111 can be sealed around the needle
  • the ear 112 extends away from and along at least a portion of the periphery of the needle 110 or the sheath
  • a spacer 114 is engaged with the lower surface of the ear 112, and a driver 1 16 is engaged with the upper surface of the ear 112.
  • the spacer 114 is intended to prevent accidental penetration of the needle 110 into the vial stopper 406 and the driver 116 is to effect controlled penetration after the removal of the spacer 114.
  • the movement of the driver 1 16 and the spacer 114 can be mechanically or electrically controlled.
  • the movement of the needle 110 or the needles in the vaccine rehydration unit 900 also can be controlled by any other conventional means instead of the spacer and the driver.
  • the dispensing device 104 comprises a syringe which is connected, at its injection outlet through a flexible tubing 118 to another needle 120.
  • the needle 120 has a controlling spacer 122 and a driver 124 configured similarly to those of the needle 110.
  • the needle 120 is adapted to be inserted into the second tube 108 on the surface of the vial stopper 406 and, at the time of assembly, this junction is sealed with a wax or easily displaced glue so as to prevent evaporation of water from the buffer in the dispensing device (syringe) 104.
  • the dispensing device 104 also has its own spacer 126 and driver 128.
  • the spacer 126 is positioned between the syringe body 130 and the syringe piston handle 132 against one surface of the disk-shaped handle cap 134.
  • the operation of automatic preparation of a polyvalent vaccine is as follows. The instrument performing the preparation is directed to select the required monovalent entities or, it chooses them as a result of having previously determined the immune status of the prospective vaccinee. The instrument then removes the sachet spacer 926 located between the sachet unit 500 and the capsule unit 901 from the appropriate vaccine rehydration units 900 and it then activates the corresponding sachet driver 928 causing the sachet needle 504 to penetrate the selected vaccine capsule 902.
  • each capsule 902 is under vacuum, water is drawn into it from its collapsible sachet 502.
  • complete rehydration of the chosen monovalent fractions is effected by providing a means for the gentle shaking of the module 100.
  • the spacer 926 located between the capsule 902 and the needle-tubing unit 904 of the selected vaccine rehydration unit 900 is removed and the corresponding driver 928 is activated causing penetration of the needle 920 into the end of the appropriate capsule 902.
  • the spacer 114 at the end of the manifold 106 is removed and the driver 116 is activated forcing the needle 1 10 into the vial cap 406.
  • the rehydrated monovalent vaccines are drawn out of their capsules 902 and through the manifold 106 into the vial 404.
  • the needle spacer 122 is removed and the driver 124 forces the needle 120 penetrating into the vial stopper 404 through tube 108.
  • the correct volume of buffer to complement the vaccine dose is then driven or drawn into the vial from the dispensing device 104.
  • the vacuum can be applied to the capsule 902 and the vial 404 in any proper conventional ways. For example, a needle with one end connected to vacuum source can be inserted into the vial 404 through its stopper 406 or into the capsule 902 through the portion where the coupling 916 is connected or the portion made of rubber.
  • the vacuum source can be a syringe, a vacuum pump, or simply a bigger container under high vacuum. If desired, the release of vacuum or a positive pressure can be also provided through a needle connected to a sterile gas source.

Abstract

L'invention porte sur un procédé permettant de déterminer la présence d'un niveau immunoprotecteur d'anticorps chez un vertébré consistant à placer un échantillon de sang du vertébré dans un chromatographe puis à l'y faire passer par une première puis une deuxième zone de détection. La première zone de détection contient une quantité d'antigènes pouvant se fixer à une quantité d'anticorps correspondant au niveau immunoprotecteur minimal de l'anticorps. La présence de l'anticorps cible dans la deuxième zone de détection indique un niveau immunoprotecteur d'anticorps. L'invention porte également sur une classe de conjugués générateurs de signaux de haute sensibilité comprenant un polymère support de dextran-avidine et servant de standard interne dans un autre procédé d'essai de l'invention. L'invention porte en outre sur procédé et un système permettant de déterminer l'état d'immunisation d'un vertébré et de formuler automatiquement un vaccin multicomposant à la demande.
PCT/US1999/001511 1998-02-03 1999-01-25 Dispositifs et procedes de mesure des titres d'anticorps immunoprotecteurs WO1999040438A1 (fr)

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Cited By (22)

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WO2000031539A1 (fr) * 1998-11-23 2000-06-02 Praxsys Biosystems, Inc. Methode et dispositif d'analyse en flux lateral
WO2005029073A2 (fr) * 2003-09-19 2005-03-31 Zbx Corporation Procedes et dispositif de dosage chromatographique
WO2006109311A2 (fr) * 2005-04-15 2006-10-19 Ramot At Tel Aviv University Ltd. Biocapteurs electrochimiques bases sur la canalisation d'enzymes
US7682619B2 (en) 2006-04-06 2010-03-23 Cornell Research Foundation, Inc. Canine influenza virus
US8003407B2 (en) 2004-07-29 2011-08-23 Relia Diagnostic Systems, Llc Lateral flow system and assay
WO2012003617A1 (fr) * 2010-07-05 2012-01-12 深圳大学 Dispositif de détection immunochromatographique et son procédé de détection
WO2012040514A3 (fr) * 2010-09-23 2012-05-31 Biocept, Inc. Procédés et réactifs pour l'amplification de signal
WO2012099897A1 (fr) 2011-01-18 2012-07-26 Symbolics, Llc Analyses à écoulement latéral utilisant des éléments en deux dimensions
WO2014012077A1 (fr) 2012-07-13 2014-01-16 Genisphere, Llc Essais à écoulement latéral utilisant des dendrimères d'adn
WO2014015076A1 (fr) 2012-07-18 2014-01-23 Symbolics, Llc Essais à écoulement latéral à l'aide de caractéristiques bidimensionnelles
CN104237512A (zh) * 2014-10-23 2014-12-24 武汉科前生物股份有限公司 一种免疫胶体金试纸条及制备方法及应用
WO2015038978A1 (fr) 2013-09-13 2015-03-19 Symbolics, Llc Measures d'écoulement latéral utilisant des motifs d'essai bidimensionnel et de lecture de signal témoin
WO2016028733A1 (fr) * 2014-08-18 2016-02-25 Kansas State University Research Foundation Test rapide pour déterminer la nécessité d'une revaccination
CN105510577A (zh) * 2015-11-27 2016-04-20 上海艾瑞德生物科技有限公司 一种快速多点定标定量检测血液多种分析物的方法
US9414813B2 (en) 2009-02-16 2016-08-16 Express Diagnostics Int'l, Inc. Device for assaying analytes in bodily fluids
WO2016164365A1 (fr) 2015-04-06 2016-10-13 Bludiagnostics, Inc. Dispositif d'essai pour détection d'une substance à analyser dans un échantillon de salive et procédé d'utilisation
WO2018039047A1 (fr) 2016-08-23 2018-03-01 Qoolabs, Inc. Dosage à écoulement latéral pour évaluer l'expression de protéines recombinées ou l'expression de gènes reporters
CN107860920A (zh) * 2017-11-01 2018-03-30 杭州微瑞科技有限公司 狂犬病毒抗体快速定量检测卡及使用方法
CN109232734A (zh) * 2017-07-10 2019-01-18 洛阳普莱柯万泰生物技术有限公司 特异性结合犬腺病毒的单克隆抗体、药物组合物、试剂盒及其应用
CN110568204A (zh) * 2019-06-10 2019-12-13 青岛海润检测股份有限公司 一种一步法定量检测犬瘟热病毒-犬细小病毒抗体的化学发光试剂盒
CN110596395A (zh) * 2019-07-30 2019-12-20 中国疾病预防控制中心传染病预防控制所 快速检测汉赛巴尔通体抗体的胶体金检测卡及其试剂盒
US10670596B2 (en) 2013-02-26 2020-06-02 Astute Medical, Inc. Lateral flow assay with test strip retainer

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US6136610A (en) * 1998-11-23 2000-10-24 Praxsys Biosystems, Inc. Method and apparatus for performing a lateral flow assay
US8354270B2 (en) 1998-11-23 2013-01-15 Relia Diagnostic Systems Method and apparatus for performing a lateral flow assay
WO2000031539A1 (fr) * 1998-11-23 2000-06-02 Praxsys Biosystems, Inc. Methode et dispositif d'analyse en flux lateral
EP1992951A3 (fr) * 1998-11-23 2009-09-23 Praxsys Biosystems, LLC. Procédé et appareil pour effectuer une analyse biologique à écoulement latéral
WO2005029073A2 (fr) * 2003-09-19 2005-03-31 Zbx Corporation Procedes et dispositif de dosage chromatographique
WO2005029073A3 (fr) * 2003-09-19 2008-01-17 Zbx Corp Procedes et dispositif de dosage chromatographique
US8003407B2 (en) 2004-07-29 2011-08-23 Relia Diagnostic Systems, Llc Lateral flow system and assay
WO2006109311A2 (fr) * 2005-04-15 2006-10-19 Ramot At Tel Aviv University Ltd. Biocapteurs electrochimiques bases sur la canalisation d'enzymes
WO2006109311A3 (fr) * 2005-04-15 2007-05-31 Univ Ramot Biocapteurs electrochimiques bases sur la canalisation d'enzymes
US7682619B2 (en) 2006-04-06 2010-03-23 Cornell Research Foundation, Inc. Canine influenza virus
US10076314B2 (en) 2009-02-16 2018-09-18 Express Diagnostics Int'l, Inc. Device for assaying analytes in bodily fluids
US9414813B2 (en) 2009-02-16 2016-08-16 Express Diagnostics Int'l, Inc. Device for assaying analytes in bodily fluids
US9462998B2 (en) 2009-02-16 2016-10-11 Express Diagnostics Int'l, Inc. Device for assaying analytes in bodily fluids
WO2012003617A1 (fr) * 2010-07-05 2012-01-12 深圳大学 Dispositif de détection immunochromatographique et son procédé de détection
WO2012040514A3 (fr) * 2010-09-23 2012-05-31 Biocept, Inc. Procédés et réactifs pour l'amplification de signal
US9347946B2 (en) 2010-09-23 2016-05-24 Biocept, Inc. Methods and reagents for signal amplification
EP3187876A1 (fr) 2011-01-18 2017-07-05 Symbolics, LLC Dosages à écoulement latéral ayant des éléments bimensionnels
US11016090B2 (en) 2011-01-18 2021-05-25 Symbolics, Llc Lateral flow assays using two dimensional features
WO2012099897A1 (fr) 2011-01-18 2012-07-26 Symbolics, Llc Analyses à écoulement latéral utilisant des éléments en deux dimensions
US9874576B2 (en) 2011-01-18 2018-01-23 Symbolics, Llc Lateral flow assays using two dimensional features
US9851366B2 (en) 2011-01-18 2017-12-26 Symbolics, Llc Lateral flow assays using two dimensional features
WO2014012077A1 (fr) 2012-07-13 2014-01-16 Genisphere, Llc Essais à écoulement latéral utilisant des dendrimères d'adn
US9651549B2 (en) 2012-07-13 2017-05-16 Genisphere, Llc Lateral flow assays using DNA dendrimers
US9874556B2 (en) 2012-07-18 2018-01-23 Symbolics, Llc Lateral flow assays using two dimensional features
WO2014015076A1 (fr) 2012-07-18 2014-01-23 Symbolics, Llc Essais à écoulement latéral à l'aide de caractéristiques bidimensionnelles
US10670596B2 (en) 2013-02-26 2020-06-02 Astute Medical, Inc. Lateral flow assay with test strip retainer
US9599615B2 (en) 2013-03-13 2017-03-21 Symbolics, Llc Lateral flow assays using two dimensional test and control signal readout patterns
WO2015038978A1 (fr) 2013-09-13 2015-03-19 Symbolics, Llc Measures d'écoulement latéral utilisant des motifs d'essai bidimensionnel et de lecture de signal témoin
WO2016028733A1 (fr) * 2014-08-18 2016-02-25 Kansas State University Research Foundation Test rapide pour déterminer la nécessité d'une revaccination
US10156569B2 (en) 2014-08-18 2018-12-18 Kansas State University Research Foundation Rapid test to determine the necessity of revaccination
CN104237512A (zh) * 2014-10-23 2014-12-24 武汉科前生物股份有限公司 一种免疫胶体金试纸条及制备方法及应用
WO2016164365A1 (fr) 2015-04-06 2016-10-13 Bludiagnostics, Inc. Dispositif d'essai pour détection d'une substance à analyser dans un échantillon de salive et procédé d'utilisation
CN105510577A (zh) * 2015-11-27 2016-04-20 上海艾瑞德生物科技有限公司 一种快速多点定标定量检测血液多种分析物的方法
WO2018039047A1 (fr) 2016-08-23 2018-03-01 Qoolabs, Inc. Dosage à écoulement latéral pour évaluer l'expression de protéines recombinées ou l'expression de gènes reporters
CN109232734A (zh) * 2017-07-10 2019-01-18 洛阳普莱柯万泰生物技术有限公司 特异性结合犬腺病毒的单克隆抗体、药物组合物、试剂盒及其应用
CN109232734B (zh) * 2017-07-10 2021-10-22 洛阳普泰生物技术有限公司 特异性结合犬腺病毒的单克隆抗体、药物组合物、试剂盒及其应用
CN107860920A (zh) * 2017-11-01 2018-03-30 杭州微瑞科技有限公司 狂犬病毒抗体快速定量检测卡及使用方法
CN110568204A (zh) * 2019-06-10 2019-12-13 青岛海润检测股份有限公司 一种一步法定量检测犬瘟热病毒-犬细小病毒抗体的化学发光试剂盒
CN110568204B (zh) * 2019-06-10 2022-04-01 青岛海润检测股份有限公司 一种一步法定量检测犬瘟热病毒-犬细小病毒抗体的化学发光试剂盒
CN110596395A (zh) * 2019-07-30 2019-12-20 中国疾病预防控制中心传染病预防控制所 快速检测汉赛巴尔通体抗体的胶体金检测卡及其试剂盒

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