WO1992017781A1 - Dosage par agglutination - Google Patents

Dosage par agglutination Download PDF

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Publication number
WO1992017781A1
WO1992017781A1 PCT/GB1992/000615 GB9200615W WO9217781A1 WO 1992017781 A1 WO1992017781 A1 WO 1992017781A1 GB 9200615 W GB9200615 W GB 9200615W WO 9217781 A1 WO9217781 A1 WO 9217781A1
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WIPO (PCT)
Prior art keywords
particles
ligand
agglutination
sample
antigen
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PCT/GB1992/000615
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English (en)
Inventor
Peter David Weston
James Francis Kelly
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International Murex Technologies Corporation
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Publication of WO1992017781A1 publication Critical patent/WO1992017781A1/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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction

Definitions

  • the present invention relates to an agglutination test for the detection of a ligand.
  • US-A-4419453 describes a latex agglutination test in which the test reagent comprises antigen- or antibody- coated latex particles of one colour and a water-soluble non-latex polymer particle absorbing dye of a different colour.
  • the test reagent comprises antigen- or antibody- coated latex particles of one colour and a water-soluble non-latex polymer particle absorbing dye of a different colour.
  • EP-A-0174195 discloses agglutination assays, particularly latex agglutination assays, for simultaneous testing for a multiplicity of ligands.
  • the reagents for use in the assays comprise two or more insoluble coloured substances, each substance being adapted to form a distinctively coloured agglutinate in the presence of a specific ligand or specific group of ligands.
  • US-A-4115535 describes an assay in which magnetic particles are used to facilitate separation of agglutinated particles from solution and a second kind of particles is present to facilitate detection of agglutination. Both types of particles are coated with a protein capable of inter- acting specifically with a protein whose presence it is wished to determine. Agglutination occurs when the latter protein is present in a test sample and is detected by observing, for example, a fluorescent label on the second kind of particles. Similar assays are disclosed in JP-A-177265/60 and JP-A-128168/61.
  • agglutination assay Two types of agglutination assay are direct assays and indirect assays.
  • a direct agglutination assay for example, as described in US-A-3088875, particles are coated with a substance that binds to the ligand under investigation.
  • ligand present in the sample combines with the binding substance coated on the surface of the particles, and agglutination occurs.
  • the particles are coated with the ligand under investigation, or with an analogue of the ligand, or with a conjugate comprising the ligand or an analogue of the ligand, generally with an inert protein.
  • the coated particles, together with a substance that binds with the ligand under investigation, are incubated with the sample under investigation.
  • the binding substance is not bound to particles as in a direct assay, but is free in the incubation mixture.
  • Indirect assays are generally inhibition assays or competition assays.
  • ligand present in a sample under investigation combines with binding sites on the binding substance in the incubation mixture, so that fewer sites are available to combine with the particle-bound ligand, and so fewer particles are agglutinated. Accordingly, in contrast to a direct assay, in an indirect assay agglutination indicates the absence of ligand and lack of agglutination indicates the presence of ligand.
  • the substance that binds to the ligand is preferably a specific binding substance, especially an antibody to the ligand.
  • Direct assays may be used to investigate, for example, ligands that are antibodies or antigens, or ligands that are receptors or their target molecules. Direct assays are particularly useful in the case where a ligand is multi-epitopic, for example, in the case of antigens that present several different epitopes, or that present repeating epitopes.
  • Indirect assays may be used, for example, when the ligand is small and presents a single epitope, for example, when the ligand is a hapten.
  • Examples of ligands that may be investigated using an indirect assay are therapeutic drugs, drugs of abuse and certain hormones.
  • EP-A-0296883 describes an indirect agglutination assay for haptens.
  • particles are suitable for use in an agglutination assay.
  • coated particle is used to denote a particle that is coated with a substance that binds to the ligand under investigation (for use in a direct agglutination assay), or a particle that is coated with the ligand itself, an analogue of the ligand, or a conjugate comprising the ligand or an analogue of the ligand (for use in an indirect agglutination assay).
  • An improved assay has now been devised which employs a single type of particle and which permits enhanced detection of a ligand, for example, an antigen.
  • Coated particles (and free binding substance for the ligand, where appropriate), are incubated with a liquid sample under investigation. After incubation, the particles are retrieved. Liquid is removed, preferably so that, at most, just enough liquid remains to suspend the particles, and the retrieved particles are suspended in the remaining liquid. Alternatively, the liquid is removed and the particles are suspended in a second liquid. After further treatment, agglutination is observed by eye. This assay simplifies previous assays and it is surprising that agglutination can still be observed after separation of the bulk of the test sample liquid.
  • the present invention provides a method of determining a ligand in a liquid sample, which method comprises:
  • the particles used in the present invention may be magnetic particles.
  • the particles may be retrieved by applying a magnetic field to the dispersion. For example, using a permanent magnet or an electromagnet, the particles are pulled to one surface of the container in which the sample and the particles have been incubated.
  • the particles may be retrieved in step (b) by centrifugation, for example in the case of non-magnetic particles, for example coloured and white particles.
  • ligand (or binding partner for the ligand in the case of an indirect assay) is thus rapidly and effectively concentrated from the bulk of the dispersion onto the surface of the particles.
  • most of the liquid is removed and discarded, and in step (c), the particles are then suspended in the small volume of liquid that remains.
  • the particles may be suspended in a second liquid medium.
  • Step (d) may be carried out according to conventional procedures, for example, a sample of the suspension from step (c) may be placed on a suitable support, for example, a card or a piece of glass, tile or plastics material.
  • the suspension is generally spread out in a thin layer, and the support may be moved in a horizontal plane or rocked to encourage agglutination.
  • a direct assay in the absence of ligand in the original sample, the mixture remains unagglutinated after step (d) and presents a smooth appearance in step (e) .
  • ligand In the presence of ligand, agglutinates form and the sample has a granular appearance.
  • the presence of ligand is indicated by lack of agglutination.
  • the retrieval step (b) in the method of the invention enables a ligand to be detected sensitively in samples in which the ligand is present at a low concentration.
  • Agglutination requires an initial incubation of coated particles with ligand, and it is generally considered to be necessary that free ligand is present during the agglutination. It has now been found, surprisingly, that after the initial incubation, the particles can be washed yet will still agglutinate. Hence it appears that, using the method of the present invention, the presence of free ligand is not required during the agglutination process itself.
  • a further surprising observation is that, if a suspension of magnetic particles resulting from step (c) above is subjected to the influence of an applied magnetic field during the agglutination step (d), agglutination occurs more rapidly than it does in the absence of an applied magnetic field.
  • a dispersion of magnetic particles resulting from step (c) above is transferred to a suitable support, for example, to a card or piece of glass or tile, and spread into a thin layer, and is then subjected to the influence of a magnetic field, preferably from beneath the support, for example, a permanent magnet rotated or held under the support, or an electromagnet beneath the support, the mixture will agglutinate more rapidly than it would without the applied magnetic field.
  • This unexpected observation may also allow detection of ligands to a greater sensitivity than hitherto, using manipulations which are simple, rapid and reliable.
  • steps (b) and (c) may be omitted and a magnetic field may be applied to the dispersion resulting from step (a) above to accelerate agglutination.
  • the present invention also provides a method of determining a ligand in a liquid sample, which method comprises:
  • step (iii) thereafter determining agglutination by eye.
  • the mixture of the sample under investigation and the appropriately coated particles may be incubated in a container and then a portion of the mixture may be removed for step (ii).
  • the sample under investigation and the coated magnetic particles may be admixed directly on the support to be used for agglutination. In both cases a magnetic field is applied to cause the agglutination to occur more rapidly.
  • the present methods are concerned with determining a ligand in a sample.
  • the ligand may be any substance to be investigated for which there is a specific binding substance.
  • the ligand under investigation is an antigen, for example, a protein antigen or a poly- saccharide antigen.
  • the ligand is typically a multivalent immuno-reactive species such as a multi-epitope antigen.
  • the antigen may be derived from a human or animal pathogen, for example, it may be an antigen associated with, or characteristic of, a bacterium, virus, fungus or parasite.
  • the method may therefore be used to detect a target antigen derived from bacteria, for example, selected from Streptococcus spp. , Streptococus pneumoniae, Haemophilus influenza b, Neisseria meninqitidis spp.
  • Neisseria gonorrhea and Gardnarella vaqinalis a viral antigen, for example, derived from a human immunodeficiency virus, for example, HIV-1 or HIV- 2, or from a hepatitis virus, for example, from hepatitis A virus, hepatitis B virus or hepatitis C virus; derived from a fungal species for example, Candida albicans; derived from a parasite for example, Chla ydia spp.
  • the antigen may be a human or animal host-derived protein, for example, human chorionic gonadotrophin, luteinizing hormone, prostatic acid phosphatase, carcinoembryonic antigen or creatine kinase.
  • the ligand may be an antibody, for example, an antibody to an infectious agent, for example, to a human immunodeficiency virus, for example, Hiv-i or HIV-2, or to a hepatitis virus, for example hepatitis A virus, hepatitis B virus, or hepatitis C virus. If an antibody is to be detected, the particles will be coated with an appropriate antigen.
  • ligands that are haptens for example, therapeutic drugs, drugs of abuse and certain hormones, may be investigated according to the methods of the present invention. It may be desired to detect the presence of such a substance in a sample of body fluid from a human or animal, for example, in the case of a drug of abuse, for example, cocaine, heroin or morphine, or in the case of an antibiotic, for example, in the milk of a cow being treated for an infection.
  • a drug of abuse for example, cocaine, heroin or morphine
  • an antibiotic for example, in the milk of a cow being treated for an infection.
  • a substance for example, a therapeutic drug, particularly in the case of drugs the level of which should be maintained within relatively narrow therapeutic limits to obtain the most advantageous therapeutic effects and/or to minimise adverse reactions, for example, theophylline and phenytoin.
  • a substance for example, a therapeutic drug, particularly in the case of drugs the level of which should be maintained within relatively narrow therapeutic limits to obtain the most advantageous therapeutic effects and/or to minimise adverse reactions, for example, theophylline and phenytoin.
  • Such substances may be assayed according to the present invention using an indirect assay.
  • the ligand under investigation may be obtained directly in the form of a liquid sample, or a solid or semi-solid sample comprising the ligand may be dissolved, suspended or extracted into a suitable liquid, generally an aqueous liquid.
  • the sample may be a biological sample derived from a human or animal. Examples of liquid samples are blood, serum, plasma, sputum, milk, urine or cerebrospinal fluid.
  • the sample may be a suspension of whole organisms or an extract of organisms originating from a human or animal source such as a suspension of bacteria obtained from a culture plate or an extract of a swab taken from a site of suspected infection.
  • the sample may be diluted with or extracted into a buffer so that the liquid medium incubated in step (a) has a pH which is appropriate, for example from pH 4 to pH 9 and preferably about neutral pH.
  • the particles that are dispersed in the first liquid medium in step (a) are, for example, polystyrene latex particles, styrene-glycidyl methacrylate latex particles - or other polymer latexes-, for example, those described in US-A-4419453.
  • the particles may be magnetic particles or non-magnetic, and may be white or coloured, for example red, blue or green.
  • Non-magnetic particles are available commercially in a range of colours. Particles of different colours may be employed in the same test, the different colours identifying the specificity of the respective particles. Mixed coloured magnetic particles may be used therefore in which the different coloured particles are coated with substances able to bind to different ligands respectively.
  • the size of particles used in agglutination assays is typically less than 2 ⁇ m in diameter, for example from 0.1 to l.O ⁇ rn diameter, and smaller particles smaller than O.l ⁇ m in diameter may be used. Particles are available commercially in a wide range of suitable sizes.
  • the size may be selected so that, in assessing the results of an assay, an agglutinated positive can easily be distinguished by eye from an unagglutinated negative.
  • the size of the particles preferably therefore is selected so that a negative has a smooth appearance or at least an appearance which is not too granular.
  • the particles are coated with the ligand or with a substance capable of binding to the ligand it is wished to detect. Adsorption, chemical coupling or any other appropriate method may be used to link the coating substance to the particles.
  • the sole particles in the medium in step (a) may be the coated particles.
  • the binding substance may be an antibody, antigen or any other substance that binds, preferably specifically, to a particular target ligand.
  • An antibody used for coating may be a monoclonal antibody or a polyclonal antibody and may be of any class, for example, IgA, IgM or IgG.
  • An antibody may therefore be employed which is capable of binding to an antigen characteristic of one of the organisms listed above or from other organisms of .interest or to host-derived antigens such as those listed above.
  • the specific binding substance may be an antigen derived for example from a virus, bacterium, fungus or parasite.
  • the antigen may be obtained using the techniques of molecular biology or it may be an extract or purified extract from the organism in question.
  • the sample under investigation is contacted with a dispersion of the appropriately coated particles.
  • the mixture also contains a substance that binds to the ligand. under investigation.
  • the first liquid medium generally an aqueous medium, comprising the sample together with the particles and any free binding substance required, is incubated in step (a) preferably for from 30 seconds to 10 minutes, for example from 1 to 3 minutes. Incubation may take place at room temperature or at an elevated temperature, for example up to 50°C.
  • the particles are retrieved in step (b) .
  • the particles are concentrated in this step. Where the particles are magnetic, that may be achieved by applying a magnetic field.
  • step (a) The container in which step (a) has been carried out is typically placed alongside a magnet to which the particles are attracted. After a short period of time, for example 1 or 2 minutes, substantially all the particles have accumulated at the side of the container adjacent to the magnet. Liquid free of particles is removed and discarded. Where the particles are not magnetic particles, retrieval may be achieved by centrifugation and supernatant removed and discarded.
  • the volume of liquid left is preferably about the minimum required for suspension of the particles such that any subsequent agglutination can be observed.
  • the retrieved particles may be suspended in a second liquid medium in step (c).
  • the particles are suspended in a sufficient volume of the second liquid medium to permit observation of any subsequent agglutination.
  • the second liquid medium is typically an aqueous medium and may be an aqueous buffer medium.
  • the retrieved particles may be washed after step (b) .
  • the particles are magnetic particles
  • washing solution is added and, after the particles have been dispersed therein, a magnetic field may be reapplied.
  • the particles are attracted to the magnet and washing solution is removed and discarded.
  • they are suspended in the washing solution and retrieved by centrifugation.
  • the supernatant consisting of the used washing solution is removed and discarded.
  • the particles are generally suspended in a second liquid medium but they may be suspended in a portion of the first medium that had been removed before washing and retained for further use. Washing is an optional step, however, and is not often required.
  • step (d) the suspension, or a sample of the suspension, is allowed to agglutinate or is treated to encourage agglutination.
  • the treatment generally comprises spreading an aliquot of the suspension of particles on a suitable support, for example, a card or a piece of glass, and manually rotating and tipping the support or agitating the support in a single plane. Spreading the suspension may be done first, or may be accomplished by the rotating or shaking.
  • the suspension from step (c) is transferred onto a card suitable for agglutination testing and the suspension is spread, typically with a small wooden or plastic spatula up to the edge of a ring printed on the card.
  • the card may be rotated to allow the formation of aggregates which are then observed by eye.
  • a piece of glass, tile or plastics material may be used instead of a piece of card.
  • the suspended particles are simply observed by eye in step (e) to see if agglutination of the particles has taken place. Initially, the dispersion has a smooth appearance. If agglutination occurs, aggregates form and the dispersion becomes more granular.
  • An advantage of the assay of the present invention is that it does not depend upon assessing agglutination indirectly by detecting a label carried by the particles, such as a fluorescent label. As indicated above, it has surprisingly been found that, when magnetic particles are employed, the rapidity of agglutination can be increased by applying a magnetic field to a sample of the particles after incubation with the sample.
  • the particles may be in a dispersion from step (a), a suspension from step (c), or the liquid comprising the coated particles and the sample comprising the ligand may be admixed in situ on the support on which agglutination is to occur.
  • the agglutination is generally carried out on a suitable support, for example, a card, a piece of glass or tile, or a similar flat support.
  • the dispersion or suspension is preferably spread in a layer on the support.
  • the support may be placed over a magnet, either a permanent magnet or an electromagnet. If desired, the support and/or the magnet may be moved relative to one another, or pulses of a magnetic field may be applied.
  • Application of a magnetic field may be for from 5 to 20 seconds, depending upon the strength of the field. After removal of the magnetic field, agglutination may be observed.
  • the applied magnetic field may serve, in effect, to pull the particles down onto the support and to aid agglutination.
  • all steps of an assay involving the application of a magnetic field to accelerate agglutination may be carried out in situ on the support on which agglutination is to be observed.
  • the use of an applied magnetic field to accelerate agglutination may be carried out independently of or in conjunction with a particle retrieval step assisted by the application of a magnetic field.
  • Assays according to the invention have wide applicability. They may be qualitative or semi- quantitative. They can be used to detect the presence or otherwise of a ligand in a sample. In cases where the ligand is present at only low concentrations where conventional agglutination tests would have failed to show agglutination, they can detect the ligand successfully. This allows detection and identification of an infectious agent in cases where the infection level is low or of antibody where the level of antibodies is low at the time of sampling.
  • the assays of the invention also enable detection and/or monitoring of levels of haptens, for example, therapeutic drugs, drugs of abuse and certain hormones.
  • haptens for example, therapeutic drugs, drugs of abuse and certain hormones.
  • therapeutic agents for example phenytoin, the level of which should be maintained within narrow limits.
  • An indirect assay of the invention may be used to monitor the level of such agents.
  • Each of the assays according to the invention is typically run with a positive control, in which there is used a sample known to contain the ligand it is wished to determine.
  • a positive control is used in order to ensure that the assay is functioning correctly and that the test components have not deteriorated during storage.
  • the agglutination may be determined by eye. It will be appreciated, however, that the methods of the present invention are not limited to detection of agglutination by eye, and that they may also be used in an agglutination assay where the particles carry a detectable label, and the label is detected by the appropriate method for that label.
  • Suitable labels are well known, and include fluorescent, enzyme and radio- isotope labels. Detection of agglutination may be carried out by other known methods, for example, measurement of turbidity. The following Examples illustrate the invention.
  • Rhone-Poulenc supplies polystyrene particles (called polystyrene latex) containing magnetic pigment and five preparations have been used in this work.
  • polystyrene latex polystyrene particles
  • latex is often used to denote particles suitable for carrying out an agglutination assay or, particularly, a dispersion or suspension of such particles in a liquid medium.
  • latex will be used in the sense of a suspension of particles in the following Examples.
  • glycine/saline buffer which was 0.1M glycine in 0.15M saline with 0.05% v/v Bronidox and 0.01% w/v sodium dodecyl sulphate.
  • the particles were separated from the buffer by attracting them to the side of the vessel using a strong magnet as supplied by Dynal U.K. (Magnetic Particle Concentrator MPC-1).
  • the buffer was then removed using a disposable pipette and the latex suspended in fresh buffer to a final concentration of 1% w/v.
  • Example 1 Absorption of antibody to magnetic latex
  • the latex was sensitised with antibody (i.e., the particles were coated with antibody) by methods commonly employed.
  • Purified immunoglobulin from rabbit anti- streptococcus B was mixed with the latex suspension at two levels, 300 ⁇ g IgG/ml latex and 450 ⁇ g IgG/ml latex. The mixture was heated at 56°C for 30 mins. and then bovine serum albumin solution added at the rate of 33 ⁇ l, 30% per ml latex suspension. After standing for 1 hour the latex was washed once by attraction to a magnet, removal of liquid and suspension to the same volume in glycine saline buffer.
  • a pronase extract of a suspension of Streptococcus B organisms was diluted in glycine saline buffer containing 0.1% v/v bovine serum albumin and this provided the antigen to be detected.
  • An aliquot of the antigen solution (1ml) was mixed with the magnetic latex with antibody attached (50 ⁇ l) and left to stand (10 mins.).
  • the test tube containing the suspension was inserted in the magnetic particle concentrator and the brown coloured latex migrated to the side of the tube. After 1 min. all the liquid was removed using a disposable pipette and the tube taken out of the magnetic device.
  • the magnetic latex was suspended by gentle agitation in fresh buffer (200 ⁇ l) and then an aliquot of the suspension (50 ⁇ l) transferred to one of the printed rings (2.5 cm diameter) .on a white card used for latex agglutination testing. The drop was spread on the card to cover the area within the ring using a wooden cocktail stick. The card was then rocked manually or rotated at 150 rpm for 2 minutes on a horizontal flat bed rotator. The card was then allowed to stand 1 minute before the pattern of the latex particles was observed. Agglutination of the brown particles was readily visible and was scored on a 1+ to 3+ scale, see Table 1 below. TABLE 1: DILUTIONS OF STREPTOCOCCUS B ANTIGEN DETECTED WITH WHITE LATEXES AND MAGNETIC LATEX
  • Example 2 Extension of the method to other antigen/- antibody systems Magnetic latex was sensitised with purified immunoglobulin with a high titre against meningo A,C,Y and W135 antigens using 680 ⁇ g IgG/ml latex, heating at 56°C, 40 mins. then adding bovine serum albumin 33 ⁇ l, 30% per ml latex. After standing 35 mins. the particles were washed twice with a large volume of glycine buffered saline and then taken up to give a suspension of 1% w/v latex in buffer.
  • the magnetic latex suspension was used in the same way as previously described mixing an aliquot (lml) of dilutions of the meningo A,C,Y and W135 positive control antigen in buffer with 0.1% BSA together with an aliquot (50 ⁇ l) of the 1% magnetic latex antibody suspension. The mixture was stood for 2 to 3 minutes then the tubes put into the magnetic particle concentrator. After standing (1 min.) the liquid was removed and the solids suspended in buffer (200 ⁇ l); an aliquot taken onto a latex card spread and then rocked for 2 mins. The card was then stood for 1-2 mins. before the agglutination was observed and the result recorded, see Table 2 below. TABLE 2: MENINGO A.C ⁇ , W135 ANTIGEN DETECTED WITH WHITE
  • White latex K8362 used the same preparation of antibody as was used to sensitise the magnetic latex. Also see Notes to Table 1 for a description of the methods used for comparative testing.
  • Example 3 Use of magnetic latex for the detection of antigens from meningo B, streptococcus pneumoniae and haemophilus influenzae B
  • Magnetic latex D at 1% w/v was coupled to immunoglobulin preparations from sera specific to Neisseria meningitidis group B, Streptococcus pneumoniae and Haemophilus influenzae B.
  • Antibody was offered at 450 ⁇ g/ml latex and heated at 56"C for 30 minutes in the presence of 0.1M glycine buffered saline pH 8.2.
  • Bovine serum albumen 30% (33 ⁇ l/ml) was added and the mixture stood 30 min.; the particles were washed twice by attraction to the side of the vessel by magnet, removal of the liquid and suspension in glycine buffered saline containing bovine serum albumen 0.1%.
  • Example 4 Use of a 3 colour latex product to give sensitive assays, concentrating by centrifugation
  • the salmonella latex product Wellcolex (Trade Mark) containing red, blue and green coloured latexes sensitised individually with three different antibodies, was used to discover whether concentration by centri ⁇ fugation would give a sensitive test. All three of the antigens were diluted in 2 fold dilutions using glycine buffered saline containing bovine serum albumen 0.1%. For direct agglutination, product latex (1 drop, 30 ⁇ l) was added to dilutions of the antigens (40 ⁇ l) on the white card printed with circles (2.5 cm diameter). The pairs of drops were mixed with a wooden cocktail stick, the mixture was spread to the edge of the printed circle and the card rotated in a horizontal plane by machine at 150rpm for 2 min.
  • Agglutination was scored on a 1+ to 3+ scale.
  • latex (1 or 2 drops, 30 or 60 ⁇ l) was added to dilutions of antigen (lml) distributed in Sarsted Centrifugable Reagent tubes; Cat No. 72.690/478 and mixed thoroughly. After standing (1 min) the tubes were centrifuged at 6,500 rpm for 2.5 min. in an MSE Micro Centaur centrifuge.
  • Example 5 Identification of bacterial antigen in a clinical sample with increased sensitivity using centri ⁇ fugation of white latex
  • CSF cerebrospinal fluid
  • the new method was employed.
  • the boiled sample (200 ⁇ l) was mixed with Streptococcus pneumoniae latex (30 ⁇ l) and left 10 min., centrifuged 6,500 rp for 2.5 min in a MSE Micro Centaur Centrifuge and the supernatant taken on for a further manipulation adding Haemophilus influenzae latex (30 ⁇ l) waiting 10 min. then centrifuging and removing the supernatant.
  • the latex pellets were suspended in saline (60 ⁇ l), transferred to a card and spread with a wooden cocktail stick. The card was hand rotated for 3 min. then the agglutination pattern was scored as follows:- 1. S. Pneumoniae tr/l+
  • the unboiled sample was tested for non-specific agglutination with a control latex before and after boiling.
  • the agglutination patterns suggested that the child's meningitis was associated with the presence of antigen from Streptococcus pneumoniae.
  • the efficacy of the new procedure was amply demonstrated when previously undetectable antigen was concentrated and detected in a clinical sample.

Abstract

Dosages améliorés par agglutination active et passive, dans lesquels on récupère par centrifugation des particules enrobées après l'incubation de celles-ci avec l'échantillon. Dans le cas des particules magnétiques, la récupération se fait par l'application d'un champ magnétique. L'étape de récupération augmente la sensibilité du dosage. A titre d'amélioration supplémentaire, on peut accélérer l'agglutination par l'utilisation de particules magnétiques et par l'application à l'échantillon d'un champ magnétique pendant l'agglutination.
PCT/GB1992/000615 1991-04-04 1992-04-06 Dosage par agglutination WO1992017781A1 (fr)

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GB9107030.0 1991-04-04
GB919107030A GB9107030D0 (en) 1991-04-04 1991-04-04 Assay

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DE102004033811A1 (de) * 2004-07-12 2006-02-02 Salama, Abdulgabar, Prof. Dr. Verfahren zum einfachen und schnellen Nachweis von Zellen und Biomolekülen mit Hilfe paramagnetischer Partikel
US7723100B2 (en) 2006-01-13 2010-05-25 Becton, Dickinson And Company Polymer coated SERS nanotag
WO2012010654A2 (fr) 2010-07-21 2012-01-26 Diagast Complexe immunomagnétique et son utilisation dans le groupage/phénotypage des hématies
WO2012010666A1 (fr) 2010-07-21 2012-01-26 Diagast Méthodes d'immunodiagnostic magnétique et nécessaires révélant la présence de complexes anticorps/antigènes dans le cadre du groupage et du phénotypage du sang érythrocytaire
US8409863B2 (en) 2005-12-14 2013-04-02 Becton, Dickinson And Company Nanoparticulate chemical sensors using SERS
US8497131B2 (en) 1999-10-06 2013-07-30 Becton, Dickinson And Company Surface enhanced spectroscopy-active composite nanoparticles comprising Raman-active reporter molecules
US9201013B2 (en) 1999-10-06 2015-12-01 Becton, Dickinson And Company Method for tagging material with surface-enhanced spectroscopy (SES)-active composite nanoparticles
US9297766B2 (en) 2001-01-26 2016-03-29 Becton, Dickinson And Company Method of tagging materials with surface-enhanced spectroscopy-active sandwich particles
JP2016520846A (ja) * 2013-06-06 2016-07-14 コーニンクレッカ フィリップス エヌ ヴェＫｏｎｉｎｋｌｉｊｋｅ Ｐｈｉｌｉｐｓ Ｎ．Ｖ． 多量体の標的分子の検出に対する粒子ベースの試験において凝集を防ぐ試薬、方法及び装置

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WO1989001161A1 (fr) * 1987-07-28 1989-02-09 International Institute Of Cellular & Molecular Pa Dosage turbimetrique
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Cited By (12)

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US8497131B2 (en) 1999-10-06 2013-07-30 Becton, Dickinson And Company Surface enhanced spectroscopy-active composite nanoparticles comprising Raman-active reporter molecules
US8918161B2 (en) 1999-10-06 2014-12-23 Becton, Dickinson And Company Methods of use for surface enhanced spectroscopy-active composite nanoparticles
US9201013B2 (en) 1999-10-06 2015-12-01 Becton, Dickinson And Company Method for tagging material with surface-enhanced spectroscopy (SES)-active composite nanoparticles
US9297766B2 (en) 2001-01-26 2016-03-29 Becton, Dickinson And Company Method of tagging materials with surface-enhanced spectroscopy-active sandwich particles
DE102004033811A1 (de) * 2004-07-12 2006-02-02 Salama, Abdulgabar, Prof. Dr. Verfahren zum einfachen und schnellen Nachweis von Zellen und Biomolekülen mit Hilfe paramagnetischer Partikel
US8409863B2 (en) 2005-12-14 2013-04-02 Becton, Dickinson And Company Nanoparticulate chemical sensors using SERS
US7723100B2 (en) 2006-01-13 2010-05-25 Becton, Dickinson And Company Polymer coated SERS nanotag
WO2012010654A2 (fr) 2010-07-21 2012-01-26 Diagast Complexe immunomagnétique et son utilisation dans le groupage/phénotypage des hématies
WO2012010666A1 (fr) 2010-07-21 2012-01-26 Diagast Méthodes d'immunodiagnostic magnétique et nécessaires révélant la présence de complexes anticorps/antigènes dans le cadre du groupage et du phénotypage du sang érythrocytaire
US9618518B2 (en) 2010-07-21 2017-04-11 Diagast Magnetic immunodiagnostic methods and kit for the demonstration of antibody/antigen complexes in erythrocyte blood grouping and phenotyping
JP2016520846A (ja) * 2013-06-06 2016-07-14 コーニンクレッカ フィリップス エヌ ヴェＫｏｎｉｎｋｌｉｊｋｅ Ｐｈｉｌｉｐｓ Ｎ．Ｖ． 多量体の標的分子の検出に対する粒子ベースの試験において凝集を防ぐ試薬、方法及び装置
JP2019060873A (ja) * 2013-06-06 2019-04-18 ミニケア ビー．ブイ． 多量体の標的分子の検出に対する粒子ベースの試験において凝集を防ぐ試薬、方法及び装置

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IE921089A1 (en) 1992-10-07
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