EP0835449A1 - Procede de detection simultanee de differents anticorps et/ou antigenes - Google Patents

Procede de detection simultanee de differents anticorps et/ou antigenes

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Publication number
EP0835449A1
EP0835449A1 EP96920750A EP96920750A EP0835449A1 EP 0835449 A1 EP0835449 A1 EP 0835449A1 EP 96920750 A EP96920750 A EP 96920750A EP 96920750 A EP96920750 A EP 96920750A EP 0835449 A1 EP0835449 A1 EP 0835449A1
Authority
EP
European Patent Office
Prior art keywords
antibodies
antibody
antigens
antigen complexes
substance
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP96920750A
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German (de)
English (en)
Inventor
Henrik Stender
Per Chr. Grauballe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dako Denmark ApS
Original Assignee
Dako AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dako AS filed Critical Dako AS
Publication of EP0835449A1 publication Critical patent/EP0835449A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/536—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a

Definitions

  • the present invention relates to a method for the simultaneous detection of more than one antibody and/or more than one class of antibodies or more than one anti- gen, or of one or more antibodies and/or one or more classes of antibodies and one or more antigens present in a test sample, use of the method for the diagnosis of or screening for disorders or diseases caused by viruses, bacteria, fungi, parasites, toxins or allergens as well as to kits for performing the method.
  • complexes formed between antibodies in the sample to be tested and selected optionally labelled antigens (antibody-antigen complexes), and/or between antigens in the sample to be tested and selected op ⁇ tionally labelled antibodies (antibody-antigen complexes) are captured by means of a substance having ability to bind antibody-antigen complexes, which substance is or will be immobilised onto a solid support.
  • the captured labelled antibody-antigen complexes is observed or measured on the solid support and the observation or measurement is related to the presence of antibodies and/or antigens in said test sample.
  • Substances having ability to bind antibody-antigen complexes are substances that have the property of binding to antibodies that are part of complexes formed be ⁇ tween antibodies and antigens, but that essentially do not bind to non-complexed antibodies.
  • Complexes of antibodies and antigens also called immune complexes
  • Well-known examples of substances that have the property of binding to antibody-antigen complexes are complement 1q (C1q) and rheumatoid factor (RF).
  • C1q is one of the components of the complement system and is known specifically to bind to antibody-antigen complexes. C1q initiates the classical pathway of comple ⁇ ment activation and mediates the solubilisation and clearance of immune complexes from circulation.
  • C1q is a heat-labile 11S protein which is composed of two non-identical and non ⁇ covalently bound subunits made up of three polypeptide chains.
  • the native C1q has six distinct globular heads projecting from a stem-like tail region which contains col ⁇ lagen-like amino acid and carbohydrate sequences. It has been shown that C1q through the heads binds only to antibodies which are part of immune complexes, but essentially do not to monomeric antibodies.
  • RF is an antiglobulin antibody, i.e. an antibody binding to complexes of immuno ⁇ globulins, e.g. antibody-antigen complexes.
  • RF is found in sera from persons suffer- ing from autoimmune diseases such as rheumatoid arthritis but may also be pro ⁇ quizzed experimentally by immunisation of animals with their own immunoglobulins in complexed form.
  • fragments of C1q, synthetic peptides and methods for the detec- tion or quantification of immune complexes present in a sample are disclosed.
  • the method described comprises contacting the immune complexes with a fragment as claimed in this document whereby the immune complexes bind to the fragment, and detecting or quantitating the immune complexes. It has been suggested that this removal of immune complexes may be applied in the treatment or the diagnosis of various diseases.
  • a method for the detection and determination of a com ⁇ plement binding antibody which method is characterised in that a binding partner (i.e. an antigen) to the complement binding antibody is immobilised, the immobilised antigen is reacted with the complement binding antibody and with serum comple ⁇ ment, and the reaction product is reacted with a coupling product and a label.
  • a binding partner i.e. an antigen
  • This method is only useful for antigens that are not adversely influenced by the immo ⁇ bilisation onto a solid support.
  • the methods disclosed herein comprise as an essential feature the formation of agglomerates of the formed antibody-antigen complexes.
  • US 4 143 124 and US 4283 383 a method for the detection of an antibody or an antigen is described, whereby an appropriate antigen or antibody is added to the sample, and subsequently brought into contact with C1q or RF.
  • this method is carried out as a competitive method, i.e. antibody-anti- gen complexes originally in the sample compete for the binding to C1q or RF with antibody-antigen complexes formed by adding the appropriate antigen or antibody which further may be labelled.
  • a method for the quantitative determination of substances present in a fluid whereby an immunosensor having C1q coated thereon as capture substance is brought in contact with a solution containing the antibody-anti ⁇ gen complexes to be determined, and whereby the immunosensor is brought in contact with a reagent that causes the dissociation of C1q and the complexes to be determined.
  • two antibodies may be used in the detection of one antigen. At least one of the antibodies may be labelled.
  • IgM and IgG antibodies are used for detecting heat denatured alkaline phosphatase.
  • a large number of immunological methods have been developed for the detection of various antigens or antibodies. Such methods have to be rapid, reliable and prefera ⁇ bly inexpensive and easy-performed. Especially, in the screening of a large number of samples, e.g. donor blood, it is of utmost importance that screening assays can be performed rapidly, and, furthermore, it will often be advantageous to be able to simultaneously detect the presence of more than one antigen or antibody in a test sample.
  • a standard procedure for the detection of more than one antigen or antibody or different classes of one antibody is to perform individual tests. This is e.g. the case when testing for HIV or Hepatitis C virus. Another example is when testing for IgG and IgM antibodies.
  • IgG antibodies are traditionally determined using an indirect assay whereas IgM antibodies are traditionally detected using an antibody capture assay.
  • the antibody or antigen must be immobi ⁇ lised onto a solid support.
  • Another standard procedure for the detection of more than one antigen or antibody or classes of one antibody comprises immobilisation of more than one antibody or antigen onto the same solid support.
  • competition between antibodies or antigens to be immobilised may be a disadvan ⁇ tage.
  • the present invention relates to a method for the detection of different antibodies and/or antigens simultaneously. It has su ⁇ risingly been found that this can be achieved without loss of sensitivity or specificity.
  • the present invention relates to a method for the detection of more than one antibody and/or more than one class of antibodies or more than one antigen, or of one or more anti ⁇ bodies and/or one or more classes of antibodies and one or more antigens present in a test sample, which method comprises
  • test sample (1) contacting said test sample with one or more selected antigens which are or will be identically or differently labelled and/or one or more selected antibodies which are or will be identically or differently labelled so as in solution to form antibody-antigen complexes of
  • This simultaneous detection can be accomplished without having to reduce the amount of the individual test antigens and/or antibodies in the test assay which may result in loss of sensitivity, since, in the present method, there is no competition between the individual test antibodies or antigens in contrast to traditional methods.
  • This is an advantage as compared to indirect antibody assays wherein all individual test antigens must, in competition, be immobilised onto the solid support prior to the binding of the antibodies in the sample.
  • This is also an advantage as compared to antigen sandwich assays wherein all individual test antibodies, in competition, must be immobilised onto the solid support prior of the binding to antigens in the sample.
  • This is further advantageous as compared to antibody capture assays wherein both specific and non-specific antibodies, in competition, are captured prior to the binding to test antigens.
  • antibodies and/or antigens present in the sample react to form complexes in solution prior to immobilisation onto the solid support. Thus, the antibody-antigen reactions resemble in vivo conditions.
  • P/N ratio positive-to-negative ratio
  • Another advantage of the present method is that it is possible, and economical fea- sible, to screen for antibodies and/or antigens for which the population shows ex- tremely low prevalence since in such cases, an additional antigen or antibody may simply be included in the screening assay.
  • the present invention relates to a method for the simultane ⁇ ous detection of more than one antibody and/or class of antibodies or more than one antigen, or of one or more antibodies and/or classes of antibodies and one or more antigens present in a test sample, which method comprises
  • the present invention relates to a method comprising
  • the present invention relates to a method for the simultaneous detection of antibodies and/or classes of antibodies, wherein said antibodies to be detected are more than one antibody and/or belong to more than one class of anti- bodies.
  • the present invention relates to a simultaneous method for the detection of antigens, wherein said antigens to be detected are more than one antigen.
  • the present invention relates to a method for the simultaneous detection of antibodies and/or classes of antibodies and antigens, wherein said anti ⁇ bodies and antigens to be detected are one or more antibodies and/or one or more classes of antibodies and one or more antigens.
  • the immobilisation onto the solid support of the substance having ability to bind anti- body-antigen complexes may be achieved by immobilisation directly onto the solid support of the substance having ability to bind antibody-antigen complexes.
  • im ⁇ mobilisation may be effected by covalent or non-covalent binding.
  • covalent or non-covalent binding onto a solid support may be performed by methods and by use of agents well known in the art.
  • the immobilisation of the substance having ability to bind antibody-anti ⁇ gen complexes may be achieved by means of a binding partner which binding partner is immobilised onto the solid support.
  • the immobilisation may be effected using an an- tibody to the substance having ability to bind antibody-antigen complexes or using a streptavidin or avidin coated solid support and a biotinylated substance having ability to bind antibody-antigen complexes.
  • Coating of a solid support with streptavidin or avidin may be performed by coating directly with streptavidin or avidin, or by use of macromolecules as described in, e.g., EP 269 092 B1.
  • Examples of substances having ability to bind antibody-antigen complexes are C1q, RF, a portion or fragment thereof, and a peptide which is capable of binding to the formed antibody-antigen complexes.
  • a preferred substance having ability to bind an ⁇ tibody-antigen complexes is C1q.
  • the test sample, the selected antigens and/or selected antibodies, and the substance having ability to bind antibody-antigen complexes may be brought into contact immediately without any preincubation. It is to be understood that the substance having ability to bind antibody-antigen com- plexes may already be present in the test sample as a naturally-occurring compo ⁇ nent of the sample, or it may be provided. The substance having ability to bind anti ⁇ body-antigen complexes may be provided in immobilised form or not in immobilised form.
  • the sample may also be brought into contact with the selected antigens and/or antibodies (preincubation), and thereafter be contacted with the optionally immobi- lised substance having ability to bind antibody-antigen complexes. It is an option to bring the test sample in contact with the optionally immobilised substance having ability to bind antibody-antigen complexes, before the selected antigens and/or anti ⁇ bodies are introduced. Likewise, it is an option firstly to bring the selected antigens and/or antibodies into contact with the optionally immobilised substance having abil- ity to bind antibody-antigen complexes, before providing the test sample.
  • heating of samples to be tested is often carried out as a standard procedure in order to eliminate the risk of transmission of various patho ⁇ gens.
  • such heating procedure may also be carried out so as to inactivate any substance having ability to bind antibody-antigen complexes such as C1q optionally present in the test sample.
  • the heating procedure is carried out at a temperature and for a period of time sufficient to inactivate such substances such as at a temperature of 56°C for 30 minutes.
  • C1q as well as RF may also be removed from the sample by adsorption to aggregated immunoglobulins.
  • the invention relates to a method for the detection of more than one antibody and/or class of antibodies or more than one antigen, or of one or more antibodies and/or classes of antibodies and one or more antigens pres ⁇ ent in a test sample, which method further comprises pretreating the test sample under conditions by which any substance having ability to bind antibody-antigen complexes which substance optionally is present in said test sample, such as, e.g., C1q, is inactivated or removed.
  • any substance having ability to bind antibody-antigen complexes pre- sent in the sample may be used to capture the formed antibody-antigen complexes.
  • additional substances having ability to bind antibody-antigen com ⁇ plexes may optionally be added.
  • C1q of the sample is used, additional C1q may optionally be added.
  • C1q of the sample is used without additional C1q, the stability of this substance should be taken into consideration.
  • the term "selected antigens” is intended to comprise native, syn ⁇ thetic and recombinant proteins or peptides capable of forming antibody-antigen complexes with the antibody to be detected.
  • synthetic proteins or peptides comprises proteins or peptides which are modified naturally-occurring proteins or peptides or which are non-naturally-occurring proteins or peptides.
  • modified naturally-occurring proteins or peptides is intended to comprise proteins or peptides with alterations such as deletions, additions or substitutions. Synthetic proteins or peptides may be synthesised in vitro or in vivo.
  • selected antibodies is intended to comprise complement fixing monoclonal, polyclonal and recombinant antibodies capable of forming anti ⁇ body-antigen complexes with the antigen to be detected.
  • different antibodies is intended to comprise antibodies with different specificities and/or classes of antibodies with the same specificity.
  • the expression "which are or will be labelled" is intended to comprise both a directly and an indirectly labelling of the selected antibodies and the selected antigens.
  • the selected antigens and/or antibodies may, if convenient, be labelled with identical or different labels. In some cases, it may be advantageous to label the antigens and/or antibodies with different labels, allowing observation or measurement of the individ ⁇ ual antigen or antibody associated with that particular label.
  • the "substance having ability to bind antibody-antigen com- plexes” comprises any substance being able to bind to antibodies that are part of complexes formed between antibodies and antigens, but that essentially do not bind to non-complexed antibodies.
  • Such substances may be isolated from humans or animals such as sheep, goats, rabbits or guinea pigs, or may be prepared syntheti ⁇ cally.
  • Such substances comprise C1q, a portion or fragment thereof, RF, a portion or fragment thereof as well as a peptide having said ability. Fragments of C1q and pep ⁇ tides prepared synthetically are described in WO 92/07267.
  • a substance having ability to bind anti ⁇ body-antigen complexes is intended to include one or more substances, each hav- ing such binding ability.
  • An example is C1q and a fragment thereof.
  • test sample or “sample” is intended to include, but is not limited to, serum, saliva, tears, semen, urine, cerebrospinal fluid, milk, excre ⁇ tions, nasopharyngeal secretions (NPS), bronchial alveolar lavage (BAL), swabs, faecal samples, aspirates and the like.
  • samples may be analysed immediately or stored under suitable conditions. The samples may be used undiluted (neat), diluted or concentrated.
  • binding partner is a compound by which the substance having ability to bind antibody-antigen complexes is immobilised.
  • the term is intended to comprise, but is not limited to, antibodies to the substance having ability to bind antibody-antigen complexes.
  • An example of a useful binding partner may be rabbit anti-human C1q antibody, rabbit anti-guinea pig C1q antibody, streptavidin, and avidin. When streptavidin or avidin is used as binding partner, the substance having ability to bind antibody-antigen complexes must be biotinylated. Examples of monoclonal antibodies which are able to react with human C1q-containing com ⁇ plexes are described in WO 85/02261.
  • the expression "is or will be immobilised” is intended to encompass that the sub- stance having ability to bind antibody-antigen complexes is immobilised prior to contact with the test sample and the selected antigens and/or antibodies, as well as immobilisation after contact with the test sample.
  • the substance having ability to bind antibody-antigen complexes must be immobilised by means of a bind ⁇ ing partner.
  • the solid support capture system may take a wide variety of forms well known in the art, such as e.g. a plate, a microtiter plate having one or more wells, a microscope slide, a filter, a membrane, a tube, a dip stick, a strip, beads such as paramagnetic beads, beads made of polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides and agaroses.
  • a filter, a membrane, a strip or beads is (are) used as the solid support, it (they) may, if conveniently be incorporated into a single-use device.
  • Another attractive solid sup ⁇ port is a multi-well microtiter plate which is very attractive, since such system is fea ⁇ sible for automatization of the analysis.
  • the label which is or will be attached to the selected antigen or selected antibody may suitably be an enzyme label, a fluorophore, a hapten, a radioisotope label, a peptide label, a coenzyme label, a dye, a donor or acceptor for electron transfer or a chemiluminiscent label.
  • Useful enzyme labels include, but are not limited to, peroxidases such as horserad ⁇ ish peroxidase (HRP), alkaline phosphatase (AP), alcohol dehydrogenases and glu ⁇ cose oxidase.
  • Useful coenzyme labels include, but are not limited to, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide and flavin adenine dinucleotide phosphate.
  • Useful hapten labels include biotin, dinitrobenzoic acid and digoxigenin.
  • Useful dye labels are, e.g., cyanine dyes such as Cy2, Cy3 and Cy5, methylene blue and colloidal gold.
  • fluorophore is intended to mean a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable range such as 5- (and 6)-carboxyfluorescein, 5-carboxyfluorescein, 6-carboxyfluorescein, fluorescein isothiocyanate (FITC), rhodamine, dansyl, umbeiiferone, tetramethylrhodamine, cyanine dyes such as Cy2, Cy3 and Cy5, coumarin, R-phycoerythrin (RPE), allophy- coerythrin, Texas Red and Princeston Red as well as conjugates of R-phycoerythrin and e.g. Cy5 and Texas Red.
  • FITC fluorescein isothiocyanate
  • RPE R-phycoerythrin
  • RPE allophy- coerythrin
  • Texas Red and Princeston Red as well as conjugates of R-phycoerythrin and e.g. Cy5 and Texas Red.
  • Particularly useful labels are HRP, AP, biotin, digoxigenin, 5-(and 6)-carboxyfluore- scein, FITC, streptavidin, avidin, dinitro benzoic acid, rhodamine, Cy5, R-phyco ⁇ erythrin (RPE), RPE-Cy5, RPE-Texas Red and colloidal gold.
  • the label When the selected antibody or selected antigen is directly labelled, the label is cou ⁇ pled to such antibody or antigen.
  • the label When the selected antibody or antigen is indirectly labelled, the label may, e.g., be introduced by using a labelled antibody or a frag ⁇ ment thereof which is bound to or is able to bind to the selected antibody or antigen.
  • the observing or measuring of the captured labelled antibody-antigen complexes may be performed using techniques generally known in the art. Such techniques include, i.a., the application of enzyme immunoassays (EIA), fluorescence immuno ⁇ assays and chemiluminiscence immunoassays.
  • EIA enzyme immunoassays
  • fluorescence immuno ⁇ assays fluorescence immuno ⁇ assays
  • chemiluminiscence immunoassays i.a., the application of enzyme immunoassays (EIA), fluorescence immuno ⁇ assays and chemiluminiscence immunoassays.
  • the selected antigens or the selected antibodies used in the present method are antigens derived from or antibodies against virus, bacteria, fungi, parasites, toxins or allergens.
  • selected antigens may be derived from or selected antibodies may be raised against virus such as RSV (Respiratory Syncytial Virus), Astrovirus, Adenovirus, Rotavirus, SRSV (small round structured viruses), Parvovirus B19, Measles, Mumps, Rubella, CMV (Cytomegalovirus), EBV (Epstein- Barr Virus), Herpes Simplex Virus (HSV), hepatitis viruses, HIV (human immunode- ficiency viruses), HTLV (human T cell lymphotropic viruses), Influenza Virus, Para ⁇ influenza Virus, Papillomavirus, Enterovirus and the like.
  • virus such as RSV (Respiratory Syncytial Virus), Astrovirus, Adenovirus, Rotavirus, SRSV (small round structured viruses), Parvovirus B19, Measles, Mumps, Rubella, CMV (Cytomegalovirus), EBV (Epstein- Barr Virus), Herpes Simplex Virus (HSV), hepati
  • selected antigens may be derived from or selected antibodies may be raised against bacteria such as Chlamydia spp., Borrelia spp., Rickettsia spp., Leptospira spp., Yersinia spp., Treponema spp., Mycobacte ⁇ rium spp., Mycoplasma spp., Neisseria spp., Streptococcus spp., Helicobacter spp., Staphylococcus spp., Salmonella spp., Legionella spp. and the like.
  • bacteria such as Chlamydia spp., Borrelia spp., Rickettsia spp., Leptospira spp., Yersinia spp., Treponema spp., Mycobacte ⁇ rium spp., Mycoplasma spp., Neisseria
  • selected antigens may be derived from or selected antibodies may be raised against fungi such as Candida spp., Cryptococcus spp. and the like.
  • selected antigens may be derived from or selected antibodies may be raised against parasites such as Toxoplasma spp. and the like.
  • selected antigens may be derived from or selected antibodies may be raised against toxins such as Clostridium toxins, Pas ⁇ teurella multocida toxin and the like.
  • the present invention relates to the use of the method described herein for the diagnosis of or screening for disorders or diseases caused by virus, bacteria, fungi, parasites, toxins or allergens.
  • the method of the invention may be used to detect the presence of Astrovirus, Adenovirus and Rotavirus in faecal sam- pies by applying the relevant selected antibodies.
  • the method ofthe invention may further be used to detect classes of antibodies to Borrelia burgdorferi, or to Tre ⁇ ponema pallidum by applying the respective selected antigen.
  • the method of the invention may also be used to detect antibodies to HIV-l, HIV-l I, HTLV-I, HTLV-II and Hepatitis C virus by applying five selected antigens.
  • the method of the invention may be used to detect Hepatitis B virus and antibodies to Hepatitis C virus by applying the relevant selected antibody and antigen.
  • kit for performing the method described above also constitutes part of the present invention, which kit comprises one or more selected antigens and/or one or more selected antibodies, and one or more reagents for labelling the antibody-antigen complexes or constituents of such complexes, and a solid support capture system with a substance having ability to bind antibody-antigen complexes immobilised thereon, or a solid support and one or more reagents for such immobilisation.
  • such kit comprises one or more selected labelled antigens and/or one or more selected labelled antibodies, and a solid support capture system with a substance having ability to bind antibody-antigen complexes immobilised thereon, or a solid support and reagents for such immobilisation.
  • the substance having ability to bind antibody-antigen complexes is preferably C1q or a fragment thereof.
  • C1q may suitably be immobilised onto the solid support by means of a binding partner, which binding partner may preferably be an antibody against C1q such as rabbit anti-human C1q antibody.
  • the kit may further comprise a detection system for visualising any labels such as enzyme labels that may not be observed directly.
  • the detection system may suitably comprise enzyme substrates as well as reagents for stopping the enzyme reaction.
  • the kit may further comprise a signal amplifying system.
  • the different selected antigens and/or antibodies may be label with different labels (non-identical labels), thereby relating different observed signals to the presence of particular antigens and/or antibodies.
  • AP and HRP may suitably be used.
  • the enzyme activity may then be visualised using substrates consecutively, whereby the solid support capture system is separated from the first substrate before the second substrate is intro ⁇ quizled. The activity may then be observed at different wavelengths. More than two labels may be used.
  • FITC and RPE may suitably be used.
  • Flow cytometry is another technique which is very suitable for analysing samples and which is, furthermore, suitable for automated analysis.
  • the flow cytometer may utilise a single light source or more than one separate light source may be used to excite each different type of fluorochrome or immunofluorescent labels.
  • Useful labels are FITC, RPE, RPE-Cy5, RPE-Texas Red and allophycocyanine.
  • Beads for use in flow cytometry may preferably be spherical particles with a diameter of from 1 to 30 ⁇ m, preferably from 1 to 20 ⁇ m, more preferably from 2 to 15 ⁇ m and most preferably from 2 to 10 ⁇ m. Particles having a diameter of from 5 to 10 ⁇ m are particular convenient since this is approximately the size of biological cells, and, thus, the parameters of the flow cytometer need not to be changed.
  • the size of the particles should, of cause, be chosen so as to be suitable for the desired assay per- formance.
  • the invention is further illustrated by the following examples.
  • EXAMPLE 1A 43 serum samples from patients suffering from Lyme Borreliosis as well as 144 serum samples from presumed healthy blood donors from an endemic area were tested as described below for the presence of antibodies to B. burgdorfe .
  • the results obtained by use of the method according to the invention for the detection of IgM and IgG antibodies to B. burgdorferi are compared with the results obtained by use of commercially available test kits for the separate detection of IgG and IgM an ⁇ tibodies to B. burgdorferi (IDEIATM Borrelia burgdorferi IgG and IDEIATM Boirelia burgdorferi IgM, DAKO).
  • Microwells (MaxiSorp ® , NUNC) were precoated with 100 ⁇ l, 5 ⁇ g/ml rabbit anti- human C1q antibody (DAKO) and were stored at 4°C prior to use.
  • the serum samples were diluted 1 to 100 in tris buffered saline with pH 7.2 (TBS).
  • TBS tris buffered saline with pH 7.2
  • 150 ⁇ l of the diluted sample were mixed with volumes of 150 ⁇ l flagellum conjugate (biotinylated ⁇ . burgdo/ e ⁇ flagellum complexed with strept- avidin labelled with HRP) which are components of the IDEIATM Boirelia burgdorferi IgM test kit) and were preincubated 1 hour at room temperature.
  • flagellum conjugate biotinylated ⁇ . burgdo/ e ⁇ flagellum complexed with strept- avidin labelled with HRP
  • the microwells were washed four times with 300 ⁇ l TBS, and 100 ⁇ l TMB (tetra- methylbenzidine) substrate was added to the wells.
  • Bound HRP was visualised by the development of a blue colour.
  • the enzyme reaction was stopped after 10 min ⁇ utes by addition of 100 ⁇ l 0.5 M sulphuric acid which changed the colour from blue to yellow.
  • the intensity of the colour corresponded to the amount of the captured labelled antibody-antigen complexes.
  • Table 1 and 2 are the number of positive and nega ⁇ tive responses, respectively, for the 43 serum samples from patients suffering from Lyme Borreliosis when tested with the IDEIATM Borreiia burgdorferi IgM and IDEIATM Boirelia burgdorferi IgG kits.
  • Table 3 the results from Table 1 and 2 are summarised.
  • the number of serum samples indicated as being positive corresponds to the number of serum samples containing either IgM or IgG antibodies to B. burgdorferi as well as both IgM and IgG antibodies to B. burgdorferi.
  • IDEIATM IgM IDEIATM Borreiia burgdorferi IgM
  • MAI Method according to the invention for detection of IgM and IgG antibodies to
  • Table 3 summarises the results from Table 1 and 2. It can be seen that the diagnos ⁇ tic sensitivity of the present method was similar to the diagnostic sensitivity of the combined use of the IDEIATM Borreiia burgdorferi IgM and IgG kits since all positive serum samples (i.e. serum samples containing either IgM or IgG antibodies as well as serum samples containing both IgM and IgG antibodies when tested in the com ⁇ flashally available kits) were positive when tested by use of the present method.
  • Table 4 and 5 show the number of positive or negative responses for the 144 serum samples from blood donors (with a low prevalence of antibodies to B. burgdorferi) when tested with the IDEIATM IgM and IgG kit.
  • Table 6 the results obtained when the same serum samples were tested by use of the present method are shown.
  • each microwells were firstly coated with 100 ⁇ l, 5 ⁇ g/ml rabbit anti-human C1q antibody, washed 4 times in 300 ⁇ l TBS, and subsequently coating with 100 ⁇ l, 2.5 ⁇ g/ml human C1q (double-coating) and were stored at 4°C prior to use. Thus, no preincubation with human C1q is necessary.
  • the test results so ob ⁇ tained were similar to the above-described.
  • serum samples diluted 1:100 in TBS were incubated for two hours with flagellum conjugate (biotinylated B. burgdorferi flagellum complexed with streptavidin labelled with horseradish peroxidase (HRP)) directly in microwells dou ⁇ ble-coated with a rabbit anti-human C1q antibody-human C1q.
  • flagellum conjugate biotinylated B. burgdorferi flagellum complexed with streptavidin labelled with horseradish peroxidase (HRP)
  • HRP horseradish peroxidase
  • Serum samples from three patients suffering from Lyme Borreliosis (samples de ⁇ noted numbers 9, 12 and 15, respectively) and serum samples from five healthy blood donors (samples denoted numbers 343, 348, 318, 347 and 302, respectively) were tested using the method according to the invention.
  • the detection of antibodies to ⁇ . burgdorferi and/or to Parvovirus B19 was carried out as described below.
  • the serum samples were selected using the following commercially available test kits: IDEIATM Parvovirus B19 IgG, IDEIATM Parvovirus B19 IgM, IDEIATM Borreiia burgdorferi IgG and IDEIATM Borreiia burgdorferi IgM kits (all kits from DAKO).
  • the samples were chosen so as to represent all combinations of IgG antibodies to B. burgdorferi and Parvovirus B19, respectively, i.e. both B. burgdorferi and Parvovirus B19 positive or negative, B. burgdorferi positive and Parvovirus B19 negative, and ⁇ . burgdorferi negative and Parvovirus B19 positive.
  • the samples were further selected so as to be negative with respect to IgM antibodies to both ⁇ . burgdorferi and Par- vovirus B19.
  • Microwells (MaxiSorp ® , NUNC) were precoated with human C1q (100 ⁇ l, 1 ⁇ g/ml human C1q) and were stored at 4°C prior to use.
  • Serum samples were diluted 1:100 in tris buffered saiine with pH 7.2 (TBS) and were heated at 56°C for 30 minutes in order to inactivate any C1q present in the serum samples.
  • TBS tris buffered saiine with pH 7.2
  • volumes of diluted sample were mixed with an equal volume of diluted biotinylated recombinant Parvovirus B19 capsids, an equal volume of bio- tinylated Borreiia burgdorferi flagellum, or an equal volume of a mixture of biotiny ⁇ lated recombinant Parvovirus B19 capsids and biotinylated Borreiia burgdorferi fla ⁇ gellum, respectively.
  • Biotinylated recombinant Parvovirus B19 capsids and biotiny ⁇ lated ⁇ . burgdorferi flagellum were components of the IDEIATM Parvovirus B19 IgM kit and the IDEIATM Borreiia burgdorferi IgM kit, respectively.
  • the sample mixtures were preincubated one hour at room temperature. After incubation, 100 ⁇ l of each sample mixture was added in duplicate to microwells coated with human C1q. The microwells were incubated for one hour at room temperature on an orbital shaker.
  • Table 7 shows the results obtained by use of the IDEIATM kits as well as the results obtained by the present method according to the invention for detection of IgM and/or IgG antibodies to Parvovirus B19, to Borreiia burgdorferi and simultaneously to Parvovirus B19 and ⁇ . burgdorferi, respectively.
  • IDEIATM Parvo IgG IDEIATM Parvovirus B19 IgG IDEIATM Parvo IgM: IDEIATM Parvovirus B19 IgM IDEIATM Borreiia IgG: IDEIATM Borreiia burgdorferi IgG IDEIATM Borreiia IgM: IDEIATM Borreiia burgdorferi IgM MAI Parvo: Method according to the invention for the detection of antibodies to
  • Parvovirus B19 MAI Borreiia Method according to the invention for the detection of antibodies to B. burgdorferi
  • a serum sample containing only IgG antibodies to B. burgdorferi was selected for this experiment and was diluted in a serum sample that did not contain IgM nor IgG antibodies to ⁇ . burgdorferi in order to imitate serum samples containing different concentrations of specific IgG antibodies.
  • These serum samples were analysed by the IDEIATM Borreiia burgdorferi IgG kit and by use of the method according to the invention for the detection of antibodies to ⁇ . burgdorferi. The detection was carried out as described in Example 1A.
  • IgG ⁇ DE ⁇ ATM Borreiia burgdorferi ⁇ gG
  • MAI Method according to the invention for the detection of IgM and IgG antibodies to
  • the P/N ratios obtained with the indirect IDEIATM Borreiia burgdorferi IgG are shown.
  • the OD-values obtained by use of the method according to the invention for the detection of IgM and IgG antibodies are also shown.
  • the P/N ratio is defined as the OD-values of each sample divided by the OD-value of the sample represent ⁇ ing a dilution of 256 ⁇ .
  • the cut-off of the commercially available IgG kit is between dilutions of 32 ⁇ and 64 ⁇ (not shown).
  • C1q present in human serum samples was used as substance with ability to bind antibody-antigen complexes.
  • Rabbit anti-human C1q antibody was used as binding partner for immobilising C1q onto the solid support.
  • microwells precoated with 100 ⁇ l, 0.05 ⁇ g/ml rabbit anti-human C1q antibody
  • 5 ⁇ l of the serum samples was mixed with 50 ⁇ l TBS and 50 ⁇ l Treponema flagellum conjugate (biotinylated Treponema flagellum (Scand. J. Immunol. 15, 341-348 (1982)) complexed with HRP labelled streptavidin) and incubated two hours at room temperature.
  • Treponema flagellum conjugate biotinylated Treponema flagellum (Scand. J. Immunol. 15, 341-348 (1982)) complexed with HRP labelled streptavidin
  • HRP labelled streptavidin HRP labelled streptavidin
  • MIA - are the results obtained with microwells coated only with rabbit anti-human C1q antibody
  • MIA + are the results obtained with the rabbit anti-human C1q antibody-human C1q double-coated microwells (for reference).
  • the following serum samples were tested by the present method: One serum sam ⁇ ple being positive with respect to antibodies against Borreiia burgdorferi (Borreiia positive) and negative with respect to antibodies against Treponema pallidum (Syphilis negative), one serum sample being Borreiia negative and Syphilis positive, and one serum sample being Borreiia negative and Syphilis negative.
  • the serum samples were confirmed positive or negative with the commercially available kit IDEIATM Borreiia burgdorferi IgG (DAKO), and with a prototype Syphilis IgG (made by DAKO).
  • Sulphate polystyrene latex beads (0.1% w/v) having a diameter of 5 ⁇ m (Interfacial Dynamics Corporation) were coated with human C1q (5 ⁇ g/ml C1q in PBS two hours at room temperature). C1q coated beads were washed three times in TBS and were mixed in a tube with serum sample diluted 1:100 in TBS, and FITC labelled antigens (biotinylated Borreiia burgdorferi flagellum mixed with FITC labelled streptavidin (DAKO) or biotinylated Treponema flagellum mixed with FITC labelled streptavidin (DAKO)). The mixture was incubated without further treatment in the dark for two hours at room temperature. The beads were then analysed by flow cytometry (Becton Dickinson FACScan flow cytometer).
  • MAI Borrelia/FITC Method for the simultaneous detection of MAI Syphilis/FITC antibodies to B. burgdorferi and T. pallidum
  • Microwells were double-coated with rabbit anti-human C1q antibody-human C1q as described in Example 1 B and stored at 4°C prior to use.
  • Biotinylated rabbit anti-human IgA antibodies (DAKO), biotinylated rabbit anti-human IgM antibodies (DAKO) and biotinylated rabbit anti-human IgG antibodies (DAKO), each diluted 1:10 in TBS, were each preincubated for one hour in test tubes with streptavidin labelled with HRP (DAKO) diluted 1:5000 in buffer TBS.
  • the HRP labelled antibodies were used separately or in combination (see Table 11).
  • the labelled antibodies were mixed 1:1 with 2.5 ⁇ g/ml human IgA (DAKO), 5 ⁇ g/ml human IgM (DAKO) or 2.5 ⁇ g/ml human IgG (DAKO) either separately or in combi ⁇ nation. All mixtures were additionally tested against buffer as a control. Incubation was performed for one hour at room temperature.
  • the signal is positive if OD > 3 x OD Buff ⁇ r
  • the signal is negative if OD ⁇ 3 x OD Buff ⁇ r

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Abstract

L'invention porte sur un procédé de détection simultanée de plus d'un anticorps et/ou de plus d'une classe d'anticorps ou de plus d'un antigène, ou d'un ou plusieurs anticorps et/ou d'une ou plusieurs classes d'anticorps et d'un ou plusieurs antigènes présents dans un échantillon à tester, sur l'utilisation de procédés de diagnostic ou de dépistage de troubles ou de maladies causées par des virus, des bactéries, des champignons, des parasites, des toxines ou des allergènes, ainsi que sur des trousses de mise en oeuvre de ce procédé. Selon ledit procédé, les complexes formés entre les anticorps de l'échantillon à tester et les antigènes facultativement marqués sélectionnés (complexes anticorps-antigène), et/ou entre les antigènes de l'échantillon à tester et les anticorps facultativement marqués sélectionnés (complexes anticorps-antigène) sont capturés à l'aide d'une substance capable de se fixer à des complexes anticorps/antigène, ladite substance étant ou devant être immobilisée sur un support solide. Le complexe anticorps-antigène marqué capturé s'observe et se mesure sur le support solide, l'observation et la mesure étant en relation avec la présence d'anticorps et/ou d'antigènes dans l'échantillon.
EP96920750A 1995-06-29 1996-06-27 Procede de detection simultanee de differents anticorps et/ou antigenes Withdrawn EP0835449A1 (fr)

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DK75395 1995-06-29
PCT/DK1996/000288 WO1997001758A1 (fr) 1995-06-29 1996-06-27 Procede de detection simultanee de differents anticorps et/ou antigenes

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GB9624750D0 (en) * 1996-11-28 1997-01-15 Univ London Capture assays
US6379909B1 (en) 1998-06-24 2002-04-30 Alk-Abello A/S Method of detecting an antibody in a liquid sample
WO1999067642A2 (fr) * 1998-06-24 1999-12-29 Alk-Abelló A/S Procede de detection d'un anticorps dans un echantillon liquide
US6673614B2 (en) 2000-06-27 2004-01-06 Cell Genesys, Inc. Rapid methods and kits for detection and semi-quantitation of anti-adenovirus antibody
GB0025245D0 (en) 2000-10-14 2000-11-29 Lee Helen Multiple target detection
US7101683B2 (en) 2001-06-26 2006-09-05 Abbott Laboratories Methods for the simultaneous detection of HCV antigens and HCV antibodies
WO2003002749A2 (fr) 2001-06-26 2003-01-09 Abbott Laboratories Procedes servant a detecter simultanement des antigenes de hcv et des anticorps anti-hcv
US20050048545A1 (en) * 2003-07-10 2005-03-03 Cull Millard Gambrell Universal detection of binding
US20050186642A1 (en) 2004-02-24 2005-08-25 Biocare Medical, Inc. Immunoassay reagents and methods of use thereof
CN111521804B (zh) * 2019-02-01 2024-01-19 科美诊断技术股份有限公司 一种处理剂及其应用
CN112816701B (zh) * 2020-12-28 2023-12-26 军事科学院军事医学研究院环境医学与作业医学研究所 胶体金侧流层析法快速检测蓖麻毒素的方法及胶体金侧流层析试剂盒
CN115792248A (zh) * 2023-02-13 2023-03-14 江西赛基生物技术有限公司 食物特异性IgG抗体检测试剂盒及其使用方法

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US4283383A (en) * 1974-05-20 1981-08-11 Technicon Instruments Corporation Analysis of biological fluids
US5364930A (en) * 1990-10-16 1994-11-15 Northwestern University Synthetic C1q peptide fragments
DE4214589C2 (de) * 1992-05-04 2002-06-20 Imtec Immundiagnostika Gmbh Immunosensor und Meßverfahren zur quantitativen Bestimmung von Bestandteilen in Flüssigkeiten
WO1994017410A1 (fr) * 1993-01-22 1994-08-04 Imre Corporation Dosage de complexe immun ameliore

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