WO1994024560A1 - Dosage immunologique - Google Patents

Dosage immunologique Download PDF

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Publication number
WO1994024560A1
WO1994024560A1 PCT/GB1994/000788 GB9400788W WO9424560A1 WO 1994024560 A1 WO1994024560 A1 WO 1994024560A1 GB 9400788 W GB9400788 W GB 9400788W WO 9424560 A1 WO9424560 A1 WO 9424560A1
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WIPO (PCT)
Prior art keywords
antibodies
solid phase
antigen
sample
immobilized
Prior art date
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PCT/GB1994/000788
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English (en)
Inventor
Richard Julian Stuart Duncan
Urszula Beckford
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International Murex Technologies Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB939307659A external-priority patent/GB9307659D0/en
Priority claimed from GB939307732A external-priority patent/GB9307732D0/en
Application filed by International Murex Technologies Corporation filed Critical International Murex Technologies Corporation
Priority to JP6522904A priority Critical patent/JPH07508102A/ja
Priority to EP94912615A priority patent/EP0646241A1/fr
Priority to SK1535-94A priority patent/SK153594A3/sk
Priority to BR9404967A priority patent/BR9404967A/pt
Priority to AU65086/94A priority patent/AU6508694A/en
Publication of WO1994024560A1 publication Critical patent/WO1994024560A1/fr
Priority to GB9424925A priority patent/GB2282884A/en
Priority to FI945857A priority patent/FI945857A/fi

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates to immunoassays.
  • Testing of samples of body fluids and of solid samples, for example, of cells or tissue, obtained from a body for the presence of various organisms, in particular bacteria, viruses, parasites and other pathogenic (infectious) organisms, is carried out routinely. Testing is carried out in two main contexts. One is testing of samples of body fluids and of solid samples for the purpose of diagnosing disease, monitoring the course of disease and/or monitoring treatment in an individual. This type of testing is often called “clinical” testing. A clinical laboratory typically carries out tests for a large number of different organisms.
  • pathogen is used herein to denote a disease-causing organism.
  • Another major type of testing is the screening of donated blood in order to maintain a supply of blood and blood products free from pathogenic contamination.
  • the regulatory authorities in each country specify the pathogens for which testing is to be carried out.
  • screening is currently mandatory for HIV, hepatitis C (non-A non-B hepatitis), hepatitis B and syphilis.
  • HTLV non-A non-B hepatitis
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • HTLV hepatitis B
  • hepatitis B an antigen assay for hepatitis B surface antigen (HBsAg)
  • HBc antibodies to hepatitis B core antigen
  • Blood screening is carried out on a large scale, and it is par ⁇ ticularly important that the results are obtained quickly, since blood has a relatively short shelf-life.
  • assays capable of detecting more than one pathogen in a single test sample have been proposed.
  • Antibody assays for determining two sub-types of the same pathogen, in particular, for HIV-l and HIV-2 (HIV-1+2), and also for HTLV-I and HTLV-II (HTLV-I+II) are on the market. It is to be noted, however, that there is often considerable cross- reactivity between sub-types of the same species, for example, the degree of immunological cross-reactivity between HIV-l and HIV-2 is such that the HIV-l tests initially on the market did, in fact, detect a very large proportion of HIV-2-containing blood.
  • EP-A-0 484 787 proposes combination antibody assays for viral pathogens that may occur in donated blood (blood viral pathogens), in particular, a combination assay for HIV and HCV (hepatitis C virus).
  • the proposed heterogeneous phase assay involves the use of multiple peptides coated on a solid surface to capture the relevant antibodies. The resulting antigen-anti ⁇ body complexes may then be detected.
  • Each additional pathogen to be detected requires the presence of at least one additional immobilized antigen.
  • current commercially available assays comprise four different antigens.
  • For a combination HIV-1+2/HCV assay at least six antigens are required.
  • EP-A-0 286 264 proposes coating a solid phase with a peptide capable of binding to an HIV antibody and coating a solid phase with an antibody specific to HBsAg.
  • the two solid phases may be different, or both the peptide and the antibody may be coated on the same solid phase.
  • WO91/10747 proposes improvements to the type of assay described in EP-A-0 286 264, the improvements being in the nature of the peptides used for coating and for detection of the HIV antibody, and in the nature of the detection system.
  • the problems of and resulting from co-coating multiple antigens on a solid surface for a combination antibody assay are overcome in a simple and elegant manner by using an immunoglobulin capture format.
  • the immunoglobulin capture format has been known as such for many years; it was described in 1979 by Duermeyer . et al in Journal of Medical Virology 4:25-32 for the detection of specific IgM antibodies in an ELISA for hepatitis A.
  • the principle of the described assay for a sample obtained from a human is that a solid phase, for example, the wells of a microtitre plate, are coated with anti-human IgM, sample is added to the coated wells and incubated, the wells are washed and then antigen is added to the wells and incubated, and finally any bound antigen is detected using enzyme-labelled antibody.
  • WO88/07680 discloses the application of the immunoglobulin capture technique to the determination of antibodies in body fluids other than blood, in particular saliva and urine.
  • a commercially available HIV-1+2 assay for saliva and urine uses the IgG capture technique described.
  • W089/12231 which describes an anti ⁇ body capture assay for multiple antibody detection, expresses concern regarding lack of specificity caused by non-immunological protein-protein interactions between antigens and the Fc region of immunoglobulins, and proposes an immunoglobulin capture assay in which the antibodies coated on the solid surface are anti-Fc antibodies. Immunoglobulins are therefore captured specifically at the Fc region, which is thereby made unavailable for non ⁇ specific protein-protein interactions.
  • Sensitivity is also regarded as a potential problem with immunoglobulin capture assays, for example, the immunoglobulins are not captured selectively, so there is no enrichment of the specific antibody species under investigation. If that specific antibody species is present in very small amounts, both in absolute terms and relative to the other species of antibody present in the serum sample, it would be expected that the detection of the antibody under investigation would be difficult and unreliable and, in particular, that the assay would lack sensitivity.
  • the present inventors have found that, using the immunoglobulin capture technique, antibodies to two or more different pathogens in samples under investigation can be detected simultaneously with excellent sensitivity and specificity.
  • the present invention provides the use of the immunoglobulin capture technique in the determination of antibodies to two or more different pathogens in a single test sample.
  • the present invention provides a method for determining specific antibodies to two or more different pathogens in a liquid test sample, which comprises
  • each antigen being capable of binding selectively to an antibody specific for one of the pathogens under investigation, each antigen being provided with means capable of providing, directly or indirectly, a detectable signal, and
  • the immobilized antibodies may be directed against IgG, IgM or IgA, or a mixture of antibodies against different classes of immunoglobulin may be used. A mixture of anti-IgG and anti-IgM antibodies is particularly preferred.
  • the assay of the present invention a "combination assay” has unlimited versatility because of the use of a universal solid phase for the capture of antibodies.
  • the user can chose which antibodies and hence which pathogens are to be detected in a sample under investigation simply by using the appropriate antigen reagents for detection. This is in complete contrast to the conventional assays that use antigens to capture antibodies, where a different antigen-coated solid phase is required for each combination of antibodies under investigation.
  • a universal solid phase is a major advantage both o the manufacturer and to the user of assays for multiple pathogens, particularly blood screening assays and clinical assays.
  • a universal antibody- coated solid phase for example, coated beads or coated wells of microtitre plates, can be used for antibody testing for any and all combinations of pathogens because the selectivity of any particular antibody assay is determined by the combination of antigen reagents used for detection.
  • This versatility is an advantage for the manufacturer of assays because a universal antibody-coated solid phase may be used for all combination antibody assays, whether they are for blood screening or for clinical testing. At present, the mandatory requirements for antibody testing of donated blood vary from country to country. Different antigen-coated solid phases are therefore required for different territories. The provision of a universal antibody-coated solid phase for all territories reduces manufacturing costs significantly. For clinical testing, a different antigen-coated solid phase is required for each and every different combination antibody assay. Again, provision of a universal antibody-coated solid phase reduces manufacturing costs significantly.
  • a further advantage to the manufacturer is the reduction in the number of components to be coated on the solid phase and hence a reduction in manufacturing costs.
  • Assays using antigens to capture antibodies require multiple antigens for combination antibody assays for as few as two pathogens. For example, at least six different antigens are required for a combination HCV/HIV-1+2 assay. The more pathogens to be detected, the more antigens must be coated on the solid phase. According to the present invention, however, it is not necessary to immobilize additional antibodies on the solid phase in order to detect additional pathogens; the presence of anti-IgG alone enables the detection of multiple pathogens. Furthermore, the antibodies used for coating are generally cheaper than the high purity antigens required for successful coating, especially for multiple antigen coating.
  • the versatility of the combination assay of the present invention is also an advantage for the user because, for example, testing for any particular combination of pathogens may be done simply by using the appropriate combination of antigen reagents. This is particularly useful in the clinical laboratory since only one, universal antibody-coated solid phase need be purchased and stored to enable testing for any desired combination of pathogens.
  • the ability to test a sample of donated blood in one assay for all the blood viral antibodies laid down by any particular national regulation is a major advance, providing blood banks with very substantial savings in both time and money.
  • the time saving is important not just in saving the time of technicians and hence being a further cost saving, it is also important in view of the relatively short shelf-life of whole blood.
  • the immunoglobulin capture assay of the present invention has excellent specificity and sensitivity. We have obtained results for samples containing a mixture of antibodies of interest that are substantially the sum of the results obtained for the corresponding serum or plasma samples each containing only one of the antibody species. It could not have been predicted that, in the combination assay of the present invention, each of a plurality of antibodies, directed to different organisms, reacts as if it were the only antibody species present. The observation of an additive effect with regard to the number of antibody species detected is an unexpected but major practical advantage. It is also unexpected that the assay of the present invention, which can be conducted using a conventional assay format, for example, a microtitre plate, is so sensitive.
  • An assay of the present invention may be an IgG assay, an IgM assay or, for special purposes, an IgA assay.
  • a mixture of antibodies to immunoglobulins of different classes may be used for coating the solid phase. It is particularly preferred to use a mixture of anti-IgG and anti-IgM antibodies.
  • a further important advantage of the assay of the present invention compared with assays using coated antigens for antibody capture is that, by using immobilized anti-IgM, early infection may be detected reliably. This is important both for blood screening and for clinical investigations.
  • IgM is the first class of immunoglobulin to be produced in response to infection. Specific IgM antibody levels rise in the first few weeks after infection, then specific IgG is produced a little later. The IgG antibody response is persistent and may last for many years, even for life.
  • IgM can be detected in conventional assays where an antibody of interest is captured onto an antigen-coated surface and the presence of captured antibody is detected using, for example, labelled anti- human-IgM antibodies. In practice, however, such IgM assays generally lack specificity because IgM is "sticky", that is to say, IgM tends to bind non-specifically to the capturing antigen.
  • the labelled anti-IgM antibody used for detection of antigen-IgM complex cannot distinguish between specific antigen-IgM complexes and non-specifically bound IgM. For this reason, conventional assays using immobilized antigens generally use anti-IgG anti ⁇ bodies for detection i.e. the assays are for IgG only.
  • anti-IgM antibodies may be coated on the solid phase. Those antibodies then capture IgM immunoglobulins specifically from the sample, and the captured IgM is detected specifically by means of the labelled antigen.
  • the system is not "sticky" and that specificity is not a problem as it is with the conventional antigen-capture assays for the detection of IgM antibodies.
  • the antibodies immobilized on the solid phase may be polyclonal antibodies, for example, polyclonal anti-human antibodies, which will contain antibodies to all immunoglobulin classes.
  • polyclonal antibodies directed against a particular class of immunoglubulin may be used, for example, polyclonal anti-IgG and polyclonal anti-IgM antibodies.
  • Polyclonal antibodies may be purified, for example, by affinity chromato- graphy. If desired, monoclonal antibodies may be used.
  • Anti- immunoglobulins may be specific for the immunoglobulin gamma, mu or alpha chains of IgG, IgM and IgA, respectively.
  • Polyclonal and monoclonal antibodies used for coating the solid phase may be prepared by methods known per se.
  • anti- immunoglobulins are available commercially, for example, rabbit, sheep and goat polyclonal anti-IgG and anti-IgM immunoglobulins, and mouse monoclonal anti-IgG and anti-IgM immunoglobulins. If a mixture of different antibodies is used for coating, the proportions of the different antibodies in the mixture may be varied as desired.
  • the present invention is not limited to the investigation of samples from humans and also finds veterinary applications. Accordingly, the antibodies used for coating should be directed against immunoglobulins of the species from which the sample under investigation is obtained. In the case of samples from humans, for example, a coating antibody should be an anti-human antibody.
  • a particularly advantageous feature of the assay of the present invention is that a universal antibody- coated solid phase means is used regardless of the number or nature of pathogens under investigation.
  • the antibodies used as coating antibodies are directed against one or more classes of immunoglobulins, especially against IgG and IgM, and capture a representative proportion of immunoglobulins of the respective classes from the sample.
  • the antigen reagents can be provided ready mixed, or the user can use separate antigen reagents in any desired combination. If used individually, the antigen reagents may be contacted simultaneously or sequentially in any order with the solid surface carrying the captured immunoglobulins. It is generally more convenient to add the desired mixture of reagents in one step.
  • an antigen to be used in an assay of the invention may be any antigenic entity that interacts selectively with the antibody under investigation.
  • an antigen may be a peptide or polypeptide, and may be a synthetic or recombinant antigen, or a purified antigen from cell culture, for example, from a viral lysate. It may be particularly useful in certain cases, for example, HCV, to use a recombinant fusion polypeptide that com ⁇ prises more than one antigenic region of a particular organism.
  • An antigen may itself be labelled with means capable directly or indirectly of providing a detectable signal to enable any immunoglobulin-antigen complex to be detected.
  • the labelled antigen is called an "antigen conjugate".
  • the detectable signal may be optical, radio-active or physico- chemical, and may be provided directly by labelling the antigen, for example, with a dye, coloured particle, radiolabel, electroactive species, magnetically resonant species or fluorophore; or indirectly by labelling the antigen with an enzyme itself capable of giving rise to a measurable change of any sort.
  • the signal may result from agglutination, a diffraction effect or a birefringement effect involving the antigen conjugate.
  • the means capable indirectly of providing a detectable signal comprises an antibody that binds to the antigen.
  • That antibody (Abl) may be provided with direct or indirect label means as described above for the antigen (single antibody detection system) , or it may be labelled by means of yet another antibody (Ab2), which binds to Abl and is itself labelled directly or indirectly as described above for the antigen (dual antibody detection system) .
  • a single or dual antibody system for detection of captured antibody-antigen complex is particularly useful if it is difficult to produce a suitable antigen conjugate.
  • the single antibody detection system has the advantage of involving fewer washing and incubation steps, but the disadvantage that it is necessary to produce an antibody conjugate (labelled antibody Abl) for each antigen to be detected.
  • the dual antibody detection system has the advantage that, with appropriate choice of Abl and Ab2, it is possible to use one antibody conjugate (labelled antibody Ab2) for the detection of all captured antibodies as follows: when each anti- body (Abl) is raised in the same animal species, the second antibody (Ab2) can be an anti-species antibody. For example, if each antibody Abl is a sheep antibody, then a labelled anti-sheep antibody can be used as Ab2.
  • the advantage to the user of requiring only one antibody conjugate for detection of all analytes generally outweighs the disadvantage of the need for an extra incubation and extra washing steps, particularly when an automated system is used.
  • the advantage of requiring only one antibody conjugate is another significant advantage to the manufacturer.
  • a labelling system that is particularly preferred for an antigen or antibody conjugate is an enzyme labelling system, particularly one in which the antigen or antibody is conjugated to an enzyme that catalyses a detectable colour change in the presence of a suitable substrate.
  • This format the enzyme-linked immunoassay or ELISA, is widely used commercially, and automated instruments are available for carrying out such assays using, for example, microtitre plates, the proprietary "bead” or “IMX” (Trade Mark) systems, or using hollow rods or pipette tips, and computer soft ⁇ ware is available to process the results obtained.
  • Examples of typical enzyme systems are those using alkaline phosphatase, ⁇ -galactosidase, urease or peroxidase, for example, horse-radish peroxidase.
  • the solid phase on which the antibodies are captured is, for example, beads or the wells or cups of microtitre plates, or may be in other forms, for example, as solid or hollow rods or pipettes, or particles, for example, from O.l ⁇ m to 5mm in diameter. (Such particles are often called "latex" particles, regardless of the material of which they are made.)
  • a solid phase may be of a plastics or polymeric material, for example, of nitrocellulose, polyvinyl chloride, polystyrene, polyamide, polyvinylidine fluoride or other synthetic polymers.
  • Particles may additionally be of natural polymers, for example, latex or protein. Microtitre plates and beads are used extensively for both blood screening and clinical testing and are widely available commercially.
  • solid phases that may be used include membranes, sheets and strips, for example, of a porous, fibrous or bibulous material, for example, of nylon, polyvinyl chloride or another synthetic polymer, of a natural polymer, for example, cellulose, of a derivatized natural polymer, for example, cellulose acetate or nitrocellulose, or of glass fibres.
  • Paper for example, diazo- tized paper may be used.
  • Films and coatings, for example, of fibrous or bibulous material, for example, as described above, may be used as the solid phase.
  • a solid phase for example, a membrane, sheet, strip, film or coating, may be incorporated in a device for the determination of multiple or, more generally, single samples.
  • test device means for carrying out an immunoassay comprising a solid phase, generally a laminar solid phase, for example, a membrane, sheet, strip, coating, film or other laminar means, on which are immobilized antibodies to one or more classes of immunoglobulin.
  • the immobilized antibodies are preferably present in a defined zone. called herein the "antigen capture zone”.
  • An assay device may incorporate the solid phase within a rigid support or a housing, which may also comprise some or all of the reagents required for carrying out an assay.
  • Sample is generally applied to an assay device at a predetermined sample application zone, for example, by pouring or dripping the sample on the zone, or by dipping the relevant part of the device into the sample. If the sample application zone is at a different site from the antibody capture zone, the arrangement of device is generally such that antibodies in the sample migrate to the antibody cap ⁇ ture zone.
  • the required reagents are then applied in the app ⁇ ropriate order at designated application zones, which may or may not be the same as the sample application zone.
  • the arrangement of a device is generally such that the reagent(s) migrate to the antibody capture zone, where any antigen-antibody complex formed is detected. All or some of the reagents required for an immunoassay may be incorporated within a device, in liquid or dry form. If so, a device is generally arranged such that interactions between different parts of the device, which interactions may occur automatically during the operation of the device or may be brought about by the user of the device, bring the various reagents into contact with one another in the correct sequence for the immunoassay to be carried out.
  • a wide variety of assay devices are described in the literature of immunoassays. Examples of membrane devices are described in U.S. Patents Nos. 4,623,461 and 4,693,984. Depending on their design and their speed of action, some assay devices are called “dipsticks” and some are called “rapid assay” devices. A “rapid assay” device generally provides a result within ten minutes of the application of sample.
  • a typical microtitre plate or bead assay requires incubation steps, and generally takes at least an hour to provide a result.
  • assay devices are generally more expensive than microtitre or bead format assays, they have particular uses in clinical testing, for example, when a result is required rapidly, for example, in the case of emergency surgery.
  • Assay devices have the particular advantage that they can be used without the need for sophisticated laboratory facilities or even without the need for any laboratory facilities. They may there ⁇ fore be used for "on the spot” testing, for example, in an emergency room, in a doctor's surgery, in a pharmacy or, in certain cases, for home testing. They are particularly useful in territories where laboratory facilities are few and far between.
  • the combination assay of the invention is particularly useful for screening donated blood.
  • the assay may therefore be used to test for antibodies to at least two viruses selected from HIV, for example, HIV-l, HIV-2 and HIV subtypes, for example, HIV-l subtype 0; HCV; hepatitis B (antibodies to core antigen); HTLV, for example, HTLV-I and HTLV-II; CMV; EBV (Epstein Barr virus); and optionally also for syphilis.
  • HIV for example, HIV-1+2; HCV; HTLV, for example, HTLV- I+II; and hepatitis B core (HBc); for example: HIV and HCV; HIV and HTLV; HTLV and HCV; HIV, HCV and HTLV; HIV, HCV and HBc; HIV, HTLV and HBc; HTLV, HCV and HBc; HIV, HCV, HTLV and HBc.
  • Syphilis may be included in any of the above combinations.
  • the combination of choice will be influenced by specific national regulations. In some cases, donated blood may also be tested for further pathogens, for example, for rubella.
  • the requirements for blood screening are reviewed as new pathogenic agents (new organisms and new subtypes of known organisms) are discovered, and the mandatory assays are extended to cover those pathogens. For example, it is currently required in most countries to test for both HIV-l and HIV-2. The recent discovery of HIV-l subtype 0 has led to the requirement that assays must also detect that subtype. Accordingly, the combinations of pathogens to be detected will almost certainly enlarge with time.
  • the present invention includes assays for such combinations of pathogens.
  • the tube/bead two component system is used, further versatility can be introduced, for example, after contacting the tube and bead with the sample, the bead may be removed and con ⁇ tacted with antigen( ⁇ ) to one or more different species of pathogen and the tube contacted with a different antigen or combination of antigens.
  • a further variant of the tube/bead two component assay format is to immobilize one or more classes of anti-immunoglobulin on one of the components and to immobilize anti-hepatitis B surface antigen (anti-HBsAg) on the other component, for example, anti- IgG and optionally also anti-IgM may be coated on the inner surface of the small tubes and anti-HBsAg on the beads, or vice versa.
  • the beads and the tubes may then be used together in a combined HBsAg/multiple antibody assay, especially an assay for HBsAg, HIV, HCV and optionally one or more further analytes selected from hepatitis B (core) , HTLV, and syphilis.
  • Peptides and polypeptides that interact specifically with HIV-l and HIV-2 are well known and are described, for example, in EP-A- 0 347 148.
  • the antigen to be used for detection of hepatitis B core anti ⁇ bodies is a core antigen.
  • DNA and predicted protein sequences for various hepatitis B serotypes have been published, for example, by Ono et al (1983) Nuc. Acids Res ⁇ , 1747, and suitable peptide and polypeptide core antigens, both synthetic and recombinant, may be derived from published sequences.
  • Hepatitis C does not, at present, appear to have one immunodominant epitope, and it is preferable to test for anti ⁇ bodies to more than one region.
  • Antigens from the core and envelope regions are preferred structural antigens.
  • Non-structural antigens may be selected, for example, from the NS3, NS4 and NS5 regions.
  • An HTLV antigen may be, for example, a p21e or gp46 recombinant protein or a peptide derived from p21e or gp46 (see, for example, US Patent No. 4,743,678).
  • Purified p21e is available from Cambridge Biotech Corporation, 1600 East Gude Drive, Rockville, Md 20850-5300, U.S.A and gp41 from Repligen Corporation, 1 Kendal Square, Building 700, Cambridge, Ma 02139, U.S.A.
  • CMV antibodies For detection of CMV antibodies, there may be used purified cultured CMV core proteins, for example, p66, or recombinant ppl50, which is dominant in Western blots.
  • CMV core proteins for example, p66, or recombinant ppl50, which is dominant in Western blots.
  • EBV antibodies For detection of EBV antibodies, there may be used capsid or early antigen.
  • Purified T. pallidum antigen may be used for the detection of syphilis antibodies.
  • a combination assay according to the present invention may be used in clinical diagnosis as a preliminary, screening test for several pathogens. If the test is positive, then a separate test for each pathogen may be carried out. If the result is negative, no further action is required. Overall, time and money is saved.
  • An example of such a combination is that used for the so-called "TORCH” screening of pregnant women in many countries.
  • the combination of pathogens under investigation varies from country to country, in analogy to blood screening requirements, but is generally selected from rubella, toxoplasmosis, CMV and herpes. It is particularly advantageous to use a mixture of immobilized anti-IgG and anti-IgM antibodies to ensure that recent infection is detected.
  • a combination assay of the present invention presented in an assay device format, especially "rapid assay" device format, is particularly useful when a screening result is required urgently, for example, in the case of emergency surgery.
  • a rapid assay for two or more different blood viral pathogens for example, as described above, for example, for HIV-1+2 and HBc will inform a surgeon if special precautions are required.
  • An assay device for combinations of pathogens of interest is of use, for example, for "on the spot” testing in doctors' surgeries to assist with diagnosis and can, overall, save time and money in both clinical laboratory testing and in the avoidance of repeat visits to the surgery. Furthermore, assay devices are useful for testing in situations where laboratory facilities are not readily available, for example, in rural areas and in developing countries. Examples of combinations of pathogens are CMV and HIV; and TB and HIV.
  • the versatility of the use of a universal antibody-coated solid phase for the detection of a plurality of antigens can be exploited in a further manner in clinical testing.
  • the object is generally to identify the pathogen(s) in a sample.
  • the conventional micro ⁇ titre plate or bead format it is customary to prepare aliquots of the various samples and to run a series of assays, each for a different pathogen.
  • the conventional assays use antigen-coated means to capture pathogen-specific antibodies. Accordingly, it is necessary to use a different antigen-coated means, generally either beads or a microtitre plate, for each pathogen.
  • the antigen-coated means is generally presented with other reagents, for example, positive and negative controls, wash solutions and diluents, in the form of a kit. The user therefore has to have a large number of different kits.
  • a universal antibody- coated means for example, microtitre plate
  • a universal antibody- coated means can be used for the simultaneous but separate determination of each of a plurality of pathogens simply by the use of the appropriate antigen reagents.
  • a series of different samples may be tested at the same time, some for one pathogen, some for another. It is particularly advantageous to test aliquots of a single sample simultaneously but separately for different pathogens, for example, to test each aliquot in a separate well of a microtitre plate for a different pathogen. This results in savings of time and money, and also reduces the risk of mistakes through loss or confusion of samples.
  • the present invention also provides a method for determining simultaneously but separately, antibodies specific to two or more different pathogens in liquid test samples, which comprises
  • each unit comprising a solid phase having immobilized antibodies to one or more classes of immuno-globulin, whereby immunoglobulins of the respective class or classes present in each sample are captured on the solid phase, the immobilized antibodies being of the same class or classes in all units of the module,
  • each unit that has previously been contacted with sample with an antigen capable of binding selectively to an antibody specific for one of the pathogens under investigation, each antigen being provided with means capable of directly or indirectly providing a detectable signal, and (iii) determining any resulting immunoglobulin-antigen complex on the solid phase.
  • Each unit may be, for example, one of the wells of a microtitre plate, or a vessel containing beads.
  • each unit may be a defined region on a membrane, strip, sheet, film or coating, especially when presented in an assay device.
  • Devices having predetermined regions for capture of components from a sample under investigation are known per se.
  • an assay strip device for a simultaneous but separate assay according to the present invention may comprise, for example, a strip of bibulous material having a band of capture immunoglobulin across the width of the strip, called herein the antibody capture zone.
  • a band is preferably divided into sections by areas not coated with capture immunoglobulin.
  • Each section of immobilized immunoglobulins may be considered to be a unit antibody capture zone.
  • An assay device for a simultaneous but separate assay is, for example, as descibed above in relation to combination assays.
  • an assay device of the present invention for a simultaneous but separate assay for different pathogens provides particular advantages for both manufacturer and user.
  • a major advantage is that one device, provided with a number of defined antibody capture zones, can be supplied together with a plurality of antigen reagents for simultaneous testing for a number of different antibodies in a sample.
  • the manufacturer has the advantage that only one device need be made for all analytes.
  • the user too, has the advantage of requiring only one device, and has the further advantage of free choice of the combination of analytes to be determined.
  • a simultaneous but separate assay of the present invention for different pathogens may be carried out for the investigation of any particular combination of pathogens in a sample.
  • a sample from a patient suffering from hepatitis may be tested simultaneously for hepatitis A, hepatitis B anti-core antibodies and HCV; a sample from a patient with non ⁇ specific urethritis may be tested for gonorrhoeae, chlamydia and Candida.
  • each unit having immobilized antibodies of the same class or classes may have a different class of immunoglobulin immobilized thereon.
  • a series of simultaneous assays for a particular antibody carried out over a period of time using such a two-unit set on samples obtained from the same individual is particularly useful for detecting early infection and for following the course of a disease, especially for following seroconversion, for example, of HIV or HCV.
  • a set of three units, one having immobilized anti- IgG, one having immobilized anti-IgM and the third having immobilized anti-IgA is particularly useful for determining if a baby has HIV: a baby's blood has both the baby's own antibodies and maternal antibodies, so using conventional tests it is difficult to determine if the baby does have its own HIV anti- bodies, which are indicative of infection.
  • a further example of the use of more than one class of antibody is in the so-called "TORCH” test carried out in many countries on pregnant women.
  • the pathogens are selected from rubella, toxoplasmosis, CMV and herpes.
  • the combination of choice varies from country to country.
  • the rubella test should include an IgM assay in order to detect a recent infection, which is potentially dangerous. Accordingly, for a "TORCH” assay involving rubella, at least one of the units in the set used should have an anti-IgM coating.
  • a module comprising two or more units having immobilized antibodies against different classes of immunoglobulins is itself part of the present invention.
  • the present invention provides a module comprising two or more units for use in a simultaneous but separate assay for different pathogens, the units comprising immobilized antibodies against immunoglobulins, at least one unit having having immobilized antibodies directed against a different class of immunoglobulins from the immobilized immunoglobulins in the other units, for example, one unit may have immobilized anti-IgM and one or more units may have immobilized IgG.
  • a module comprises three units, one unit having immobilized anti-IgG, one unit having immobilized anti-IgM and the third unit having immobilized anti- IgA.
  • a module comprising two or more units may be present in an assay device, for example, as an arrangement of two or more antibody capture zones, for example, an IgG capture zone and an IgM capture zone.
  • an assay device may be used for a simultaneous but separate assay for different pathogens, for example, for any of the particular uses described above.
  • a module may comprise two or more sets of units (each set comprising identical units).
  • a series of sets of different units may be presented as a module.
  • a set of units may be presented as a strip of microwells.
  • Such strips may be assembled as desired into modules comprising two or more sets of units, for example, a module may comprise two strips of microwells, one coated with anti-human IgG and one coated with anti-human IgM.
  • a third strip, having immobilized anti-human IgA may be added. Strips of microwells i.e. sets of units, may be assembled as desired to form a conventional microtitre plate.
  • a plate may have alternate rows or columns of anti- IgM and anti-IgG coated microwells, for example, for following seroconversion.
  • anti- IgM and anti-IgG coated microwells for example, for following seroconversion.
  • the liquid test sample may be any body fluid, for example, blood, plasma, serum, saliva, urine, cerebro- spinal fluid, milk, lymph fluid or tears.
  • a solid sample for example, of cells or tissue may be brought into liquid form for testing, for example, as tissue exudate.
  • the pathogens of interest are so-called blood viral pathogens (HIV, hepatitis B and hepatitis C, optionally also HTLV, CMV and EBV) , and also syphilis.
  • blood viral pathogens HAV, hepatitis B and hepatitis C, optionally also HTLV, CMV and EBV
  • syphilis a very much wider range of pathogens are of interest, including the above organisms and also other viruses, bacteria and other types of pathogenic organisms.
  • Rubella measles, Herpes (simplex and genitalis), Chlamydia, Gonorrhoeae, hepatitis A, chickenpox, mumps, human parvovirus, Mycobacteria tuberculosis, Mycobacteria leprae, Mycobacteria avium, Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis (antigen/toxins), Actinomycetes (for example, Streptomyces, Nocardia, Rhodococcus) , Salmonella typhi, Yersinia enterocolifica, Helicobacter pylori, Campylobacter jejuni, Pseudomonas mallei and pseudomallei, Pseudomonas aeruginosa, Legionella pneumophila and spp, Francisella tolarensis, Brucella melitenis, Mycoplasma pneumoniae, Leptospira
  • the immobilized anti-immunoglobulins may be of one or more classes, that is to say, of the IgG, IgM and IgA classes.
  • IgG is the most abundant immunoglobulin in serum.
  • IgM is the first immunoglobulin produced in response to infection, so provides an earlier indication of infection than does IgG.
  • IgG or IgM may be used alone, according to the purpose of the assay, or a mixture of anti-IgG and anti-IgM may be used in one unit for a combination assay or anti-IgG and anti-IgM may be presented in separate units for a simultaneous but separate assay.
  • saliva it is also preferable to capture IgG and/or IgM.
  • anti-IgG is preferably used, optionally in combination with IgM and/or IgA.
  • assay of the present invention For those forms of assay of the present invention which utilise the two-component bead/tube format, it is possible to increase even further the adaptability of the assay of the present invention by immobilising one or two classes of anti- immunoglobulins on the inner surface of a plastics tube and immobilizing one or two different classes of anti-immunoglobulins on beads, the beads to be used in the tube.
  • anti- IgG may be coated on one of the two components, and anti-IgM on the other.
  • a combination bead/tube format may be used and, after contacting the tube and bead with the sample, the bead may be removed and contacted with one antigen and the tube contacted with another antigen, enabling two antibody assays to be carried out on a single sample.
  • the assays of the present invention may be carried in a conven ⁇ tional manner see, for example, "ELISA and Other Solid Phase Immunoassays Theoretical and Practical Aspect", Eds. Kemeny D.M. & Challacombe S.J.
  • Immunoglobulins may be immobilized on a solid phase, for example, by contacting the solid phase with a solution of the immunoglobulins at an appropriate concentration and pH, for example, at a pH within the range of from 7 to 11, especially from 9 to 10.
  • a buffer is preferably used, for example, a sodium carbonate/sodium bicarbonate buffer.
  • suitable immunoglobulin preparations are available commercially, and a suitable dilution of a commercial antibody solution is, for example, 1:200 to 1:4000 v/v.
  • a labelled antigen reagent may be produced by any of a variety of methods see for example, Kemeny & Challacombe, loc cit. Antigen-enzyme conjugates are generally used.
  • Samples may be diluted, for example, blood or plasma samples may be diluted 1 in 1 v/v, for example, 50 ⁇ l of sample is added to 50 ⁇ l of diluent in a microwell. It should be noted that any sample diluent used should not contain human immunoglobulins of the class or classes that are to be captured.
  • the sample is then generally incubated with the solid phase. The temperature and time of incubation are interdependent, a longer time being required at a lower temperature. Typical incubation conditions are one hour at 37 * c. After incubation, the plate or beads should be washed thoroughly prior to incubation with the antigen reagent (conjugate).
  • Typical incubation conditions for the conjugate stage are 30 minutes at 37°C After incubation with the conjugate, there is a further washing step, followed by incubation with the substrate for the enzyme in the case of an ELISA. Again, 30 minutes at 37°C are typical incubation conditions. A stop solution is generally added at the end of the incubation. In the case of an ELISA, results are generally obtained by reading the absorbence of each unit in a spectrophotometer. If another labelling system is used, the method is modified accordingly, for example, in the case of a radioimmunoassay or a fluorescence assay, results are obtained after incubation with a radiolabelled or fluorescent antigen conjugate. In the case of coloured particles, it may be possible to determine positive and negative results by eye.
  • coloured particles are particularly useful as label, since a positive or negative result can be determined by eye with a minimum number of reaction steps.
  • an ELISA system may be used for a more sensitive assay.
  • the present invention also provides a kit comprising
  • a solid phase especially plastics beads or a microtitre plate, carrying immobilized antibodies to one or more classes of immunoglobulin, especially a mixture of anti-IgG and anti-IgM antibodies,
  • two or more antigen reagents each capable of binding selectively to an antibody specific for one of the pathogens under investigation, and each antigen being provided with means capable directly or indirectly of providing a detectable signal, and optionally, one or more of the following:
  • the kit may be provided as such, or the antibody-coated component and the antigen reagent component may be provided separately.
  • the present invention also provides a solid phase suitable for use in an immunoassay, on which is immobilized a mixture of anti- IgG and anti-IgM antibodies.
  • the mixture of antibodies may additionally comprise anti-IgA antibodies.
  • the antibodies are especially anti-human antibodies.
  • the solid phase is, for example, any of the solid phases described above, for example, microtitre plates and beads, and includes those suitable for use in assay devices.
  • the use of the immunoglobulin capture format for the determination of antibodies to a plurality of pathogens in different samples (“combination assay”) or for the simultaneous but separate determination of a plurality of different antibodies in aliquots of the same sample (“simultaneous assay”) gives advantages both in time and cost.
  • the present invention is not limited to the detection of pathogens.
  • the present invention may be applied to the detection of any antibody of interest, whatever the nature of the antigen that gives rise to the anti ⁇ body, for example, non-pathogen-associated antibodies.
  • autoimmune diseases and allergies for example, non-organ-specific autoimmune diseases, for example, rheumatoid arthritis, lupus erythematosus and rheumatic fever, and organ-specific autoimmune diseases and diseases considered to have some autoimmune involvement, for example, autoimmune diseases of the thyroid, myasthenia gravis, autoimmune haemolytic anaemias, multiple disseminated sclerosis, aphthous ulcer, pernicious anaemia and ulcerative colitis. All that is required is an entity capable of binding specifically to any antibody of interest. Accordingly, it is to be understood that the teachings of present specification are as relevant to the detection of non-pathogen related antibodies as they are to antibodies to pathogens, and that the present invention includes all such embodiments.
  • the present invention may be used in the detection of determination of human or animal pathogens, and finds both human and veterinary applications, including applications in the meat trade.
  • Solid phase 96-well microtitre plate (Nunc) coated with a mixture of polyclonal anti-human IgG (DAKO) and polyclonal anti- human IgM (DAKO). (Nunc products are available from Life
  • HIV-l recombinant protein comprising core and envelope antigens from the CBL-1 isolate of HIV-l (Sattentau Q.J. et al. (1986) Science 234 1120) conjugated to HRP (horse-radish peroxidase) ("HIV-l conjugate").
  • HIV-2 peptide comprising gp36 envelope antigen, conjugated to HRP ("HIV-2 conjugate").
  • Positive samples a) HIV-l, internal reference No. 4034, diluted in HIV-l negative serum b) HIV-2, internal reference No. 91/174 diluted in HIV-2 negative serum c) anti-HBc core positive samples diluted in HBc negative serum 6.
  • Negative samples normal human serum obtained from donated blood.
  • HIV-l samples each containing one species of antibody of interest (anti-HIV-l, anti-HIV-2 and anti-HBc) were diluted and tested according to the above protocol using the HIV- 1, HIV-2 and HBc conjugates described above, either alone or in various combinations as shown in the Tables below.
  • the HIV-l sample was serially diluted 1/40; 1/80; 1/160 and 1/320.
  • the HIV-2 sample was diluted 1/32; 1/64; 1/128 and 1/256.
  • a number of different HBc-positive samples were diluted 1/2.
  • HIV-l positive samples at increasing dilutions were tested each with the following conjugates: HIV-l; HIV-l+HIV-2; HIV- 1+HBc; HIV-1+HIV-2+ HBc. The results are presented in Table 1.
  • HIV-2-positive samples at increasing dilutions were each tested with the following conjugates: HIV-2; HIV-2+HIV-1; HIV- 2+HBc; HIV-l+HIV-2+HBc. The results are presented in Table 2.
  • results obtained show that specificity is not compromised when antibodies to two different pathogens are detected, and also show that the sensitivity of the combination assay is remarkably high.

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Abstract

Procédé servant à doser des anticorps de deux ou plusieurs pathogènes différents dans un spécimen liquide de contrôle et comprenant la capture des anticorps sur une phase solide sur laquelle sont immobilisés des anticorps d'une ou plusieurs catégories d'immunoglobulines, particulièrement un mélange d'anticorps anti-IgG et anti-IgM, ainsi que le dosage des anticorps capturés. Le procédé peut s'appliquer au dosage d'autres anticorps ne présentant pas de réaction croisée, ainsi qu'au dosage simultané mais séparé d'anticorps de différents pathogènes.
PCT/GB1994/000788 1993-04-14 1994-04-14 Dosage immunologique WO1994024560A1 (fr)

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SK1535-94A SK153594A3 (en) 1993-04-14 1994-04-14 Determination method of antibodies to two or bigger number of different patogens in tested sample
BR9404967A BR9404967A (pt) 1993-04-14 1994-04-14 Imunoensaio
AU65086/94A AU6508694A (en) 1993-04-14 1994-04-14 Immunoassay
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WO1999015898A1 (fr) * 1997-09-22 1999-04-01 Chiron Corporation Procede pour detecter des anticorps dans un echantillon
ES2129358A1 (es) * 1997-04-09 1999-06-01 Vamobi S A Procedimiento de deteccion en una sola prueba de anticuerpos anti-virus de la hepatitis c, de la inmunodeficiencia humano y antigeno de superficie del virus de la hepatitis b".
US6261764B1 (en) 1997-09-22 2001-07-17 Chiron Corporation Buffers for stabilizing antigens
WO2002073204A2 (fr) * 2001-03-12 2002-09-19 Monogen, Inc Détection et différenciation d'états pathologiques par échantillons cellulaires
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WO2020119669A1 (fr) * 2018-12-10 2020-06-18 Kei International Limited Feuille de nitrocellulose comprenant des immunoglobulines immobilisées et des antigènes à base de lipides et son utilisation
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EP0696640A3 (fr) * 1994-08-12 1997-12-29 Boehringer Mannheim Gmbh Antigènes de recombinaison venant de la région NS3 du virus de l'hépatite C
US5801064A (en) * 1995-12-04 1998-09-01 Foresman; Mark D. Assay methods and reagents for detecting autoantibodies
WO1998023956A1 (fr) * 1996-11-28 1998-06-04 University College London Test d'immunocapture
ES2129358A1 (es) * 1997-04-09 1999-06-01 Vamobi S A Procedimiento de deteccion en una sola prueba de anticuerpos anti-virus de la hepatitis c, de la inmunodeficiencia humano y antigeno de superficie del virus de la hepatitis b".
WO1999015898A1 (fr) * 1997-09-22 1999-04-01 Chiron Corporation Procede pour detecter des anticorps dans un echantillon
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SK153594A3 (en) 1995-07-11
EP0646241A1 (fr) 1995-04-05
CA2137786A1 (fr) 1994-10-27
CN1105181A (zh) 1995-07-12
JPH07508102A (ja) 1995-09-07
BR9404967A (pt) 1999-06-15
CZ315094A3 (en) 1996-01-17
FI945857A0 (fi) 1994-12-13
FI945857A (fi) 1994-12-13
AU6508694A (en) 1994-11-08

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