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WO2019053328A1 - Procédé de dosage de la présence d'un anticorps dans un échantillon et kit associé - Google Patents

Procédé de dosage de la présence d'un anticorps dans un échantillon et kit associé Download PDF

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Publication number
WO2019053328A1
WO2019053328A1 PCT/FI2018/050652 FI2018050652W WO2019053328A1 WO 2019053328 A1 WO2019053328 A1 WO 2019053328A1 FI 2018050652 W FI2018050652 W FI 2018050652W WO 2019053328 A1 WO2019053328 A1 WO 2019053328A1
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Prior art keywords
donor
acceptor
particle
antibody
kit
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PCT/FI2018/050652
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English (en)
Inventor
Visa NURMI
Original Assignee
Helsingin Yliopisto
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Publication of WO2019053328A1 publication Critical patent/WO2019053328A1/fr

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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
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • This invention relates to a method and a kit for qualitatively and/or quantita- tively determining the presence of an antibody in a sample of body fluid.
  • This invention particularly relates to a homogenous method for medical diagnostic purposes and a kit for use in such a method.
  • FRET Forster resonance energy transfer
  • Saraheimo S. et al . (PLOS ONE 2013, 8(5), e62739) describe an assay based on measuring TR-FRET between donor-labelled antigen and acceptor-labelled antigen bridged by a specific IgG molecule.
  • the assay disclosed relies on labelling the antigen of interest, in two different reactions, with both acceptor and donor fluoro- chrome. Since the two labelling reactions are commonly done utilizing different methods for activation, the labelling of antigen with other fluorophores may result in "masking" of the antigens epitopes thus preventing the recognition of the antigen by antibodies.
  • the simultaneous binding of donor- and acceptor- labelled antigens to an IgG molecule can be measured by TR-FRET, therefore enabling detection of antibodies specific to the given antigen.
  • the FRET-bridge assay was set up utilizing labelled streptavidin (SA) as the antigen.
  • SA streptavidin
  • the FRET-bridge assay also requires the antigen to be separately labelled with two fluorophores, which is rather challenging. The labelling chemistries of donor and acceptor are usually different, which may result in the antigens being labelled at different sites.
  • WO2015/128548 discloses an assay setup using a fragment antigen-binding (Fab) binding moiety (FBM) bearing one fluorophore (donor or acceptor) and antigen bearing the other fluorophore (acceptor or donor, respectively).
  • FBM fragment antigen-binding binding moiety
  • donor or acceptor fluorophore
  • antigen bearing the other fluorophore acceptor or donor
  • Oxygen channelling assay especially luminescent oxygen channelling (LOC) assay, represents an energy transfer approach in which the donor label is in donor beads and consists of a photosensitizer able to generate oxygen radicals upon excitation (Ullman EF et al, Clin Chem 1996; 42: 1518-26). The excited oxygen when in proximity ( ⁇ 200 nm) with acceptor beads, triggers reactions producing detectable emission.
  • LOC luminescent oxygen channelling
  • Nanobead-embedded donors and acceptors suitable for this application are commercially available from PerkinElmer (AlphaScreen ® beads, PerkinElmer, Wall- tham, MA, USA).
  • PerkinElmer AlphaScreen ® beads, PerkinElmer, Wall- tham, MA, USA.
  • the publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
  • the present invention relates to a method for determining qualitatively and/or quantitatively the presence of an antibody in a sample comprising a body fluid of an animal based on the inhibition of a signal formed by energy transfer between an energy donor and an energy acceptor.
  • the present invention relates to a method for determining the presence of an antibody in a sample wherein said method employs:
  • the present invention relates to a method for determining the presence of an antibody in a sample wherein said method employs:
  • a particle comprising an antigen of said antibody and a ligand molecule, b) a donor coupled to a molecule able to bind to the ligand, forming a donor- ligand binding molecule complex, and
  • the present invention relates to a method for determining the presence of an antibody in a sample wherein said method employs:
  • a particle comprising an antigen of said antibody and a ligand molecule, b) a donor embedded in the particle, and
  • the present invention relates to a method for determining the presence of an antibody in a sample wherein said method employs:
  • a particle comprising an antigen of said antibody and a ligand molecule, b) a donor coupled to a molecule able to bind to the ligand, forming a donor-lig- and binding molecule complex, and
  • the present invention relates thus to a method for determining qualitatively and/or quantitatively the presence of an antibody in a sample wherein said method comprises the steps of:
  • the present invention relates also to a kit for determining qualitatively and/or quantitatively the presence of an antibody in a sample
  • the kit comprises: a) a particle comprising an antigen of said antibody and a ligand molecule, b) a first reagent comprising a donor,
  • said donor and said acceptor form an energy transfer pair capable of forming a detectable signal.
  • FIG. 1 schematically illustrates the assay principle of the present invention as implemented in example 1 .
  • donor and acceptor labelled streptavidins SA
  • VLP biotinylated virus like particle
  • the present invention is based on a finding that the determination of the presence of antibodies specific for a given antigen can be carried out by employing said antigen attached to a particle in combination with a ligand molecule, a donor (of an energy transfer pair) attached to a molecule able to bind to the ligand and an acceptor (of the energy transfer pair) attached to a molecule able to bind to the ligand.
  • the antibod- ies When the sample comprises antibodies against the said antigen, the antibod- ies will bind to the particle through the formation of the antibody-antigen complex thus hindering the binding of the donor-ligand binding molecule complex and/or the acceptor-ligand binding molecule complex to the particle. This means that the distance between the donor and the acceptor stays too long for enabling energy transfer between the donor and acceptor and thus the formation of the signal is prevented or diminished compared to a situation where no antibodies are present. The more antibodies of said specificity the sample contains, the lower is the detected signal.
  • antibody refers, as would be apparent to a person skilled in the art, to various antibody structures, including but not limited to monoclonal antibodies. Since the present invention is particularly directed to serological determination for diagnostic purposes, the term “antibody” refers to a soluble antibody.
  • the term "antigen" refers, as would be apparent to a person skilled in the art, to a structure/moiety/epitope to which an antibody binds.
  • body fluid refers to any fluid within or excreted from the body of an animal, including human.
  • Body fluid refers thus to e.g. blood, plasma, serum, cerebrospinal fluid, saliva, urine, nasopharyngeal and bron- choalveolar secretion, aspirate or lavage fluid, ocular fluids, pleural fluid, exudate and ascites, semen, bile and effusions (e.g. from middle ear or paranasal sinuses).
  • body fluid refers to blood, plasma, serum and cerebrospinal fluid.
  • the terms "ligand/ligand molecule” and "ligand binding molecule” refer to molecules that is able to bind to each other and form a pair. Such molecule pairs are known to a person skilled in the art.
  • the ligand-ligand binding molecule pair is formed from a peptide or an amino acid and an antibody recognising the peptide or the amino acid in question.
  • the ligand is biotin and the ligand binding molecule is selected from the group consisting of avidin, a modified avidin and streptavidin.
  • the ligand is a nucleotide sequence and the ligand binding molecule is its complementary sequence.
  • label refers to a detectable label molecule, which may comprise a fluorescent compound, suitable metallic element, such as, lanthanide, or chelates thereof, fluorescent or energy accepting. It may also be a nanobead embedded with fluorescent compounds, or a photosensitizer, fluorescent nanodots, chelate-embedded beads, quantum dots, upconverting crystal or nanobeads containing those.
  • the labels, described above can be directly, covalently or non-covalently coupled to a ligand binding molecule or they may be attached by indirect means generally known to a person skilled in the art.
  • the term "energy transfer” refers a signal generation wherein the donor, upon excitation by electromagnetic radiation is able to transport energy to the acceptor which accept the energy and generates a detectable signal.
  • the term "donor” refers to a compound able to absorb energy and in close proximity to a suitable acceptor donates the energy to the acceptor.
  • the term “acceptor” refers to a compound able to accept the energy donated by the donor and to generate a detectable signal.
  • the acceptor can be coloured, fluorescent or non-fluorescent organic compound, for example.
  • Quantum dots are small nanocrystals, which have unique photophysical properties and are able for energy transfer. Thus, quantum dots can serve as energy donors or energy accep- tors in FRET or LOC assays, for example.
  • chromophore/fluorophore refers to a moiety that contains a region that adsorbs certain wavelengths of light and interacts with suitable region of another chromophore so as to be useful, for example, for Forster resonance energy transfer (FRET) or Luminescent oxygen channelling (LOC) assay.
  • FRET Forster resonance energy transfer
  • LOC Luminescent oxygen channelling
  • the detection of the presence of antibodies in question is based on the energy transfer between a donor and an acceptor.
  • the detection of the presence of antibodies in question is based on the principle of Forster resonance energy transfer (FRET).
  • FRET the pair of two light-sensitive molecules (fluorophores/chromophores), a donor and an acceptor, generates a specific and measurable signal only when they are in close proximity i.e., when their mutual distance is ⁇ 10 nm.
  • the detection of the presence of antibodies in question is based on the principle of luminescent oxygen channelling (LOC) assay.
  • LOC luminescent oxygen channelling
  • the method and the kit of the present invention for determining the presence of an antibody in a sample employ:
  • method and the kit of the present invention for determining the presence of an antibody in a sample employ:
  • method and the kit of the present invention for determining the presence of an antibody in a sample employ:
  • a particle comprising an antigen of said antibody and a ligand molecule, b) a donor embedded in the particle, and
  • the particle comprises the antigen of said antibody, the ligand molecule, and the donor.
  • method and the kit of the present invention for determin- ing the presence of an antibody in a sample employ:
  • a particle comprising an antigen of said antibody and a ligand molecule, b) a donor coupled/attached to a molecule able to bind to the ligand, forming an acceptor-ligand binding molecule complex, and
  • the particle comprises the antigen of said antibody, the ligand molecule, and the acceptor.
  • the present invention relates to a method for determining qualitatively and/or quantitatively the presence of an antibody in a sample wherein said method comprises the steps of:
  • the donor is coupled/attached to a molecule able to bind to the ligand, forming a donor-ligand binding molecule complex or the donor is embedded in the particle.
  • the donor is coupled/attached to a molecule able to bind to the ligand, forming a donor-ligand binding molecule complex.
  • the donor is embedded in the particle.
  • the acceptor is coupled/attached to a molecule able to bind to the ligand forming a donor-ligand binding molecule complex or the acceptor is embedded in the particle.
  • the acceptor is coupled/attached to a molecule able to bind to the ligand forming a donor-ligand binding molecule complex.
  • the acceptor is embedded in the particle.
  • the present invention relates to a method for determining qualitatively and/or quantitatively the presence of an antibody in a sample wherein said method comprises the steps of:
  • the present invention relates to a method for determining qualitatively and/or quantitatively the presence of an antibody in a sample wherein said method comprises the steps of:
  • the present invention relates to a method for determining qualitatively and/or quantitatively the presence of an antibody in a sample wherein said method comprises the steps of:
  • the present invention relates also to a kit for determining the presence of an antibody in a sample wherein said kit comprises:
  • a first reagent comprising a donor, and/or
  • said donor and said acceptor form an energy transfer pair capable of forming a detectable signal.
  • the donor and/or the acceptor can be coupled/attached to a molecule able to bind to the ligand, thus forming a donor-ligand binding molecule complex and/or an acceptor-ligand binding molecule complex.
  • the present invention relates to a kit for determining the presence of an antibody in a sample wherein said kit comprises:
  • a particle comprising an antigen of said antibody and a ligand molecule
  • a first reagent comprising the donor in the form of a donor-ligand binding molecule complex
  • a second reagent comprising the acceptor in the form of an acceptor-lig- and binding molecule complex
  • said donor and said acceptor form an energy transfer pair capable of forming a detectable signal.
  • the donor or the acceptor can be/is embedded in the particle comprising also the antigen and the ligand molecule.
  • the present invention relates to a kit for determining the presence of an antibody in a sample wherein said kit comprises:
  • the present invention relates to a kit for determining the presence of an antibody in a sample wherein said kit comprises:
  • a particle comprising an antigen of said antibody and a ligand molecule, b) the donor in the form of a donor-ligand binding molecule complex, and c) the acceptor embedded to the particle,
  • said donor and said acceptor form an energy transfer pair capable of forming a detectable signal.
  • the sample, the particle, the donor-ligand binding molecule complex and/or the acceptor-ligand binding molecule complex are sequentially combined together.
  • the sample and the particle are combined together first and after that the other compo- nents are sequentially added thereto.
  • the particle of the present invention can be any particle suitable for carrying the antigen, the ligand molecule and optionally also the donor or the acceptor.
  • a particle is a nanoparticle, a virus, a virus like particle, for example.
  • Such particles are known to a person skilled in the art and they are also com- worthally available.
  • the particle is a virus like particle (VLP).
  • the virus like particles typically resemble structurally authentic viruscapsids. Methods for producing VLPs are described by Kaikkonen, L, et al., (Journal of Clinical Microbiology, 1999. 37(12): p. 3952-3956) and Yliharsila, M., et al.
  • the particle is a quantum dot particle.
  • the particle carrying the antigen and the ligand molecule act as an immunogenic platform, whereto the antibodies as well as the donors and acceptors coupled with a ligand binding molecule (SA) are able to bind. This way, the donor and acceptor get close to each other for transferring energy and thus developing a signal, which can be measured.
  • SA ligand binding molecule
  • the antibody molecules cover also the ligand molecules that are present on the particles hindering thus the binding of the ligand binding molecules, wherein the signal formed from the energy transfer between the donor and the acceptor is not developed or the development is diminished.
  • the sample contains antibodies against the antigen in question, the lower is the measurable signal.
  • the particle comprises also the donor or the acceptor.
  • the particle is a quantum dot particle.
  • the particle carries the donor or the acceptor as well as the antigen and the ligand molecule.
  • the antibodies are attached to the particle via antigen-antibody binding. Due to their big size, the antibody molecules cover also the ligand molecules that are present on the particles hindering thus the binding of the ligand binding molecules carrying the acceptor or the donor, respectively, wherein the signal formed due to the energy transfer between the donor and the acceptor is not developed or the development is diminished. The more the sample contains antibodies against the antigen in question, the lower is the measurable signal.
  • the ligand molecule is biotin. In one embodiment, the ligand binding molecule is selected from streptavidin, avidin or modified avidins. In one embodiment, the ligand binding molecule is streptavidin. In one embodiment, the ligand molecule is biotin and the ligand binding molecule is streptavidin.
  • the donor is a compound able to absorb energy, e.g. by excitation radiation, and in close proximity to a suitable acceptor, will donate the energy to the acceptor by a suitable mechanism such as FRET or LOC.
  • Pairs of donor-acceptor-labels are commercially available and thus known to a person skilled in the art.
  • Preferred pairs of donor-acceptor-labels consists of fluorescent Eu chelates (see e.g. Hemmila I and Mukkala V-M; Crit Rev Clin Lab Sci 2001 ; 38; 441 -519, Mathis G and Bazin H, Lanthanide Luminescence, Springer, Heidelberg, pp 47-88) together with suitable acceptors, such as allophycocyanin, Cy-5, some rhodamines, AlexaFluor 647 or similar product from other sources (HiLight, DyLight, Ulight etc) (Hemmila I and Laitinen V, Lanthanide Luminescence, Springer, Heidelberg, pp.361 -280).
  • a label pair consists of fluorescent terbium (Tb) chelates and sets of acceptors, such as rhodamine, fluorescein, Cy-3.
  • the donor is europium (Eu) chelate.
  • the acceptor is AlexaFluor 647 (AF647).
  • the donor is europium (Eu) chelate and the acceptor is AlexaFluor 647 (AF647).
  • the energy transfer between the energy transfer pair of the donor and the acceptor is detected by measuring Forster resonance energy transfer (FRET). In one embodiment, the energy transfer between the donor and the acceptor is detected using a time-resolved FRET (TR-FRET). In one embodiment, the energy transfer between the donor and the acceptor is detected using a two- photon excitation FRET. In one embodiment, the energy transfer between the energy transfer pair of the donor (D) and the acceptor (A) is detected using lumines- cent oxygen channelling (LOC) assay.
  • FRET Forster resonance energy transfer
  • TR-FRET time-resolved FRET
  • TR-FRET time-resolved FRET
  • the energy transfer between the donor and the acceptor is detected using a two- photon excitation FRET.
  • the energy transfer between the energy transfer pair of the donor (D) and the acceptor (A) is detected using lumines- cent oxygen channelling (LOC) assay.
  • LOC lumines- cent oxygen channelling
  • Labelling of a ligand binding molecule with a donor and/or an acceptor for the assay according to the invention depends on the system of choice.
  • FRET type assays the most efficient way technically is to covalently label the ligand binding molecule with respective sets of donors and acceptors.
  • the method of the invention comprises addition of auxiliary reagents in different steps of the method.
  • the method of the invention comprises addition of a denaturing, kosmotropic, chaotropic, precipitating, and/or outsalting reagent such as, but not limited to, sodium dodecyl sulphate (SDS), ammonium sulphate, polyethylene glycol (PEG), organic solvent, salt, urea, thiocyanate, guanidine, diethylamine, acid and/or base.
  • SDS sodium dodecyl sulphate
  • PEG polyethylene glycol
  • organic solvent salt
  • salt salt
  • urea urea
  • thiocyanate guanidine
  • diethylamine acid and/or base.
  • the body fluid is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, saliva, urine, nasopharyn- geal secretion, bronchoalveolar secretion, aspirate fluid, lavage fluid, ocular fluid, pleural fluid, exudate, ascites, semen, bile, effusions and any combination thereof.
  • the body fluid is selected from plasma, serum and/or cerebrospinal fluid.
  • the antibody is a human antibody. In one embodiment, the antibody is an animal, excluding human, antibody. In one embodiment, the antibody is soluble antibody. In one embodiment, the antibody is a soluble human antibody. In one embodiment, the antibody is an animal, excluding human, soluble antibody.
  • the antibody is selected from the group consisting of human immunoglobulins A, D, E, G (including subclasses 1 , 2, 3 and 4), and M; and corresponding immunoglobulins of other species (e.g. IgY is considered the avian and reptilian equivalent of IgG).
  • the antibody is selected from the group consisting of human immunoglobulins A, E, G (including subclasses 1 , 2, 3 and 4), and M.
  • the method and the kit of the present invention are suitable for detecting antibodies of the IgG-glass.
  • the antibody is selected from the group consisting of antibodies against antigens selected from the groups consisting of microbial antigens, including bacteriological, parasitic, fungal and viral antigens; exogenous antigens; autoantigens; allergens; and tumour antigens.
  • microbial antigens including bacteriological, parasitic, fungal and viral antigens; exogenous antigens; autoantigens; allergens; and tumour antigens.
  • the antibody is selected from the group consisting of antibodies against viral antigens. In one embodiment of the invention, the antibody is selected from the group consisting of antibodies against parvoviruses), Epstein-Barr virus, Varicella-zoster virus, cytomegalovirus, bocavirus(es), or polyomavirus(es). In one embodiment, the antibody is selected from antibodies against parvovirus(es). In one embodiment, the antibody is selected from antibodies against polyomavirus(es).
  • the present invention is directed to a method and a kit for assessing the immunity status of an individual. In one embodiment, the method and the kit of the present invention are directed for assessing the lifelong infection history of an individual. In one embodiment, the method and the kit of the present invention are directed for detecting past immunity or latency.
  • the method of the present invention is homogenous (wash free) e.g. provides direct measurement after combining the reagents.
  • the method and the kit of the present invention are suitable for point-of-care (POC) diagnostic.
  • POC point-of-care
  • the method of the present invention is rapid, giving results in less than 60 minutes, preferably in less than 30 minutes. In one embodiment, method of the present invention is able to give results in 30 to 60 minutes. In one embodiment, method of the present invention is able to give results in 15 to 30 minutes. In one embodiment, method of the present invention is able to give results in less than 15 minutes.
  • the invention relates to a method and a kit wherein the presence of an antibody in a sample of a body fluid, such as plasma or serum, of an animal is qualitatively and/or quantitatively determined.
  • the invention relates to a method and a kit wherein the presence of an antibody in a sample is qualitatively determined.
  • the invention relates to a method and a kit wherein the presence of an antibody in a sample is quantitatively determined.
  • the assay was developed for parvovirus B19, using B19V IgG and IgM EIAs as reference.
  • the B19V immunity status was assessed with 186 well-characterized IgG-positive and IgM-negative sera from tonsillectomy patients.
  • the performance in IgM detection was investigated with 18 IgG-negative and IgM-positive sera from individuals with acute B19V infection.
  • the analyzed samples consisted of two groups of sera as follows:
  • Group 1 consisted of 186 sera from tonsillectomy patients (Toppinen, M., et al., Journal of Virological Methods, 2015. 218: p. 40-45). The tonsillectomies had been done due to chronic tonsillitis or tonsillar hypertrophy. Of these sera 109 (58.6 %) were B19V seronegative and 77 (41 .4 %) were B19V-lgG positive, B19V-lgM-neg- ative representing long term B19V immunity.
  • Group 2 consisted of B19V-lgG-negative, B19V-lgM-positive sera from 18 individuals with acute B19V infection.
  • Biotinylated B19V-VP2-VPLs (3480 kDa) were prepared as described in Kaikkonen L. et al. Biotinylation efficiency was not quantitated.
  • Donor fluorophore labelled streptavidin was purchased from Perkin Elmer (Eu- W1024 labelled streptavidin). Acceptor fluorophore labelled streptavidin was pur- chased from Thermo Fisher Scientific (streptavidin, Alexa Fluor 647 conjugate), respectively.
  • Tris-HCI buffer 50 mM, pH 7.4 containing 9 g/l NaCI and 2 mg/ml BSA.
  • the assay protocol for the TR-FRET assays was: 5 ⁇ of biotinylated VLPs (0.3 ng, final concentration 60 pg/ ⁇ ) was combined with 10 ⁇ of undiluted sample (final dilution 1 :2). VLPs and sample were incubated for 1 h at RT before addition of 5 ⁇ of the labelled SAs (final concentration 1 nM Eu-SA, 1 nM AF-SA). The final reaction volume (20 ⁇ /well) was pipetted onto a 384-well microplate (ProxiPlate-384 Plus F, Black 384-shallow well Microplate from PerkinElmer).
  • the final concentrations of each component were initially optimized by cross titration of VLP from 550 pM to 9 pM at twofold steps, SA from 9 nM to 1 nM and sample dilutions 1 :20 and 1 :200. Further cross titration was done with VLP concen- trations 120, 60 and 30 pg/ ⁇ , SA concentrations 9, 3 and 1 nM and sample dilutions 1 :2, 1 :20 and 1 :200.
  • AF647N AF647-k * Eu
  • AF647N normalized AF647 fluorescent counts
  • AF647 unnormalized A647 counts (at 655 nm)
  • k Eu emission at 665 nm/Eu emission at 615 nm
  • Eu Eu fluorescent counts (at 615 nm).
  • the constant k was found to be independent of Eu-SA concentration whereby a value of 0.001342 (average of AF647- to Eu-counts in Eu-SA dilutions of 1 :1000 to 1 :8000) was used in subsequent calculations. With reactions having low AF647 counts and high Eu counts, the normalization would occasionally result in negative value; in these cases the buffer background, typically 3 to 10 counts, was used as the final value.
  • Cutoff between positive and negative samples was acquired by adding two standard deviations (SD) to average Rl of seronegative samples. Alternatively, separate cutoffs were used for positive and negative samples average Rl + 3 SD and +1 SD, respectively. Samples with Rl between the cutoffs were classified as borderline. Cutoffs were calculated for each consecutive measurement. The re- suits are present in tables 1 and 2 below. Table 1 . Results of FRET B19V antibody assay with a single cutoff. B19V EIA was used as reference.
  • the assay was developed for JC-polyomavirus (JCV), using HUSLAB (Helsinki University Hospital Laboratory) JCV IgG EIA [www.huslab.fi/ohjekirja, assay no. 6329, S -JCVAb] as reference.
  • JCV immunity status was assessed with 8 JCV seronegative and 10 JCV IgG-positive sera from 18 healthy pregnant women. Subjects were tested for JCV IgG throughout trimesters l-lll and were either JCV seronegative in all trimesters or showed JCV IgG-positivity in all trimesters.
  • the sera were acquired from Finnish Maternity Cohort (FMC) serum bank (Oulu, Finland) and are further described by Saraste M. et al., in Mult Scler Relat Disord. 21 : 1 1 -18, 2018.
  • Biotinylated JCV-VP1 -VPLs (14.3 MDa) were prepared in Helsinki as described by Kardas, P. et al., in Clin Vaccine Immunol. 21 (1 1 ): 1581-1588, 2014. Biotinylation efficiency was not quantitated.
  • Donor fluorophore labelled streptavidin was purchased from Perkin Elmer (Eu-W1024 labelled streptavidin).
  • Acceptor fluorophore labelled streptavidin was purchased from Thermo Fisher Scientific (streptavidin, Alexa Fluor 647 conjugate), respectively. Reagents and samples were diluted in Tris-HCI buffer (50 mM, pH 7.4) containing 9 g/l NaCI and 2 mg/ml BSA.
  • the assay protocol for JCV TR-FRET assays was: 5 ⁇ of biotinylated JCV
  • VLPs (10 ng, final concentration 500 pg/ ⁇ ) was combined with 10 ⁇ of undiluted sample (final dilution 1 :2). VLPs and sample were incubated for 15 min at RT before addition of 5 ⁇ of the labelled SAs (final concentration 4 nM Eu-SA, 4 nM AF-SA). The final reaction volume (20 ⁇ /well) was pipetted onto a 384-well microplate (Prox- iPlate-384 Plus F, Black 384-shallow well Microplate from PerkinElmer). Fluorescence was then measured after 15 min incubation at room temperature (RT) as described in Example 1 . Each sample was analyzed in triplicates and average FRET-signal was used as a final result. FRET-signal is reduced in presence of antibodies against the VLP. Cutoff between positive and negative samples was acquired by subtracting one standard deviation (SD) from average FRET-signal of seronegative samples. The results are presented in Table 3.
  • SD standard deviation

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Abstract

La présente invention concerne un procédé et un kit pour déterminer qualitativement et/ou quantitativement la présence d'un anticorps dans un échantillon de fluide corporel. La présente invention concerne en particulier un procédé homogène mis en œuvre à des fins de diagnostic médical et un kit destiné à être utilisé dans un tel procédé.
PCT/FI2018/050652 2017-09-12 2018-09-12 Procédé de dosage de la présence d'un anticorps dans un échantillon et kit associé WO2019053328A1 (fr)

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CN113125419B (zh) * 2019-12-31 2023-06-23 科美博阳诊断技术(上海)有限公司 一种供体试剂及其应用

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