WO2018065417A1 - Procédé de mesure de l'avidité ou de l'affinité fonctionnelle - Google Patents

Procédé de mesure de l'avidité ou de l'affinité fonctionnelle Download PDF

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Publication number
WO2018065417A1
WO2018065417A1 PCT/EP2017/075083 EP2017075083W WO2018065417A1 WO 2018065417 A1 WO2018065417 A1 WO 2018065417A1 EP 2017075083 W EP2017075083 W EP 2017075083W WO 2018065417 A1 WO2018065417 A1 WO 2018065417A1
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biological sample
ligand
microchannel
molecule
microcarriers
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PCT/EP2017/075083
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English (en)
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François TOPIN
Wouter LAORY
Simon Morling
Joris SCHUURMANS
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Mycartis N.V.
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Publication of WO2018065417A1 publication Critical patent/WO2018065417A1/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
    • G01N33/557—Immunoassay; Biospecific binding assay; Materials therefor using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction

Definitions

  • the invention relates to the field of biological and chemical assays performed using micro fluidics.
  • the invention provides methods to measure avidity, also called functional affinity, of non-covalent binding interactions.
  • the invention further relates to methods of determining antibody avidity, for example, IgG or IgE antibody avidity for an antigen.
  • polyclonal antibodies such as those found in the serum consequently to an infection, a vaccination or the development of an allergy, can bind to one specific binding partner.
  • Polyclonal antibodies are heterogeneous and will contain a mixture of antibodies of different affinities, each one possibly recognizing a different epitope.
  • each individual binding event increases the likelihood of other interactions to occur (i.e. increase the local concentration of each binding partner in proximity to the binding site).
  • the notion of functional affinity was introduced to apply to the binding strength of a bi- or polyvalent ligand to binding partners that present more than one binding site.
  • Avidity refers specifically to the strengthening of binding of one specific ligand to one specific binding partner through more than one point of interaction. This effect can be quantified as the ratio of the dissociation constant (Ka) for the intrinsic affinity (Ka of only one interaction) over the Ka for the functional affinity of said specific ligand to the binding partner. Since the association constant Ka corresponds to 1/ Ka, the avidity as strictly defined can also be quantified as the ratio of the K a for the functional affinity of said specific ligand to the binding partner over the Ka for the intrinsic affinity.
  • avidity is commonly used in a looser sense as a synonym for functional affinity, as in the terms “avidity assay” and “avidity index”, largely used in the field. It is thus classically used to measure the global apparent affinity of antibodies to an antigen in a complex medium such as serum, and thus the polyclonal response to an antigen.
  • avidity is to be understood as a synonym of functional affinity, and refers to the apparent affinity of polyvalent ligands for their binding partner, that is to say the global Ka measured for the binding of the ligand(s) to the binding partner.
  • the terms "avidity, strictly defined” or “strict avidity” of a specific ligand with respect to a defined binding partner in contrast herein refers to the ratio of Ka for the intrinsic affinity over Ka for the functional affinity.
  • the main method used to determine avidity is the chaotrope-based avidity assay. Briefly, this assay involves the treatment of bound antibody in an enzyme-linked immunosorbent assay such as ELISA with a chaotropic ion such as thiocyanate and the subsequent measurement of the effect on the binding titer.
  • an enzyme-linked immunosorbent assay such as ELISA with a chaotropic ion such as thiocyanate
  • Avidity is a thus a parameter which enables assessing the maturation of the humoral immune response and has been used to characterize allergic individuals (El-Khouly F. et al, Pediatr Allergy Immunol; 18(7):607-13; 2007), or distinguish between ongoing or recent infections from those in the more distant past, for example to determine infectiousness.
  • the determination of avidity of Toxoplasma-specific IgGs is classically used in pregnant women, to determine if infection during pregnancy has occurred.
  • Avidity assays have also been used successfully in the context of multiple virus infections. Chaotrope-based avidity indices differentiate between current and past infections with tick-borne encephalitis virus, West Nile virus, hantavirus, parotitis virus, morbilli virus, rubella virus, hepatitis C virus, parvovirus, human herpes virus 6, cytomegalovirus, and human and simian immunodeficiency virus (SIV).
  • the chaotrope-based avidity assay is time consuming and cannot be used in high- throughput assays or automated instruments, as chaotropic reagents can be hazardous or corrosive to an automated immunoassay instrument platform and may also cross-contaminate other assays (thereby causing aberrant results).
  • SPR Surface Plasmon Resonance
  • Biacore Surface Plasmon Resonance
  • the inventors have discovered that it is possible to obtain very precise data regarding the kinetics of both association and dissociation of molecules of interest with specific ligands in a complex medium, such as serum for instance, using microfluidics based on pressure driven laminar flow and functionalized microcarriers.
  • the inventors have thus set up a method for the determination of the avidity of a ligand to at least part of a molecule of interest, in a biological sample, characterized in that it comprises the steps of:
  • the method of the invention does not comprise a step of purification of the ligand from the biological sample.
  • ligand it is herein referred to a molecule or a class of molecule capable of binding specifically to the at least part of a molecule of interest.
  • a “class of molecules” refers to molecules which structures have a common motif, sequence, or structure.
  • the ligand may be a nucleotide or a group of nucleotides comprising a common motive or sequence, an antibody or a class of antibodies, such as for example any of the IgG, IgE, IgA, IgM classes of antibodies, or a protein of a class of proteins harboring a common peptide sequence.
  • the ligand is preferably an antibody or a class of antibodies, such as for example any of the IgG, IgE, IgA, IgM classes of antibodies.
  • the ligand is a class of antibodies, preferably chosen in the list consisting in the IgG, IgE, IgA and IgM classes of antibodies.
  • a molecule of interest By “at least part of a molecule of interest” it is herein referred to at least a fragment of any molecule such as an antigen, an antibody, a protein, a peptide, a nucleic acid, a chemical compound, a sugar or carbohydrate, a lipid, an hormone, a toxin, or any other molecule of interest.
  • binding partners i.e. a ligand and a molecule of interest
  • binding partners are protein/protein, antigen/antibody or carbohydrates/antibody.
  • biological sample it is herein referred to a sample obtained from a biological subject, including sample of biological tissue or fluid origin.
  • the biological sample is a fluid.
  • the biological sample is a body fluid, preferably chosen in the list consisting of blood, plasma, serum, or urine.
  • Other biological samples may be contemplated, such as organs, tissues, fractions, and cells isolated from mammals including, humans.
  • the sample comprises cells or tissues, it is preferably treated so as to eliminate those biological structures prior to implementing the method, for instance using homogenization followed by filtration.
  • the biological sample comprises at least two different ligands binding to the same or to different molecules of interest.
  • the method of the invention comprises a step of treating the biological sample in order to specifically label said ligand.
  • specifically label the ligand it should be understood that the step of treating the biological sample leads to the specific labelling of said ligand. It should be understood that the labelling is specific of the ligand.
  • following this step in the labelled biological sample, only the ligand is labelled. As a result, the other molecules, different from the ligand, which may be present in the biological sample, are not labelled and will thus not be detected by the method of the invention. This step insures that the data collected is specific to the binding reaction which is considered.
  • the step of treating the biological sample corresponds to the specific labelling of said protein or antibody present in the sample.
  • the step of treating the biological sample corresponds to the specific labelling of IgG antibodies present in the sample.
  • the step of treating the biological sample corresponds to the specific labelling of IgE antibodies present in the sample.
  • Methods for specific labelling of a molecule are known in the art, and typically involve indirect labelling, wherein a molecule capable of binding specifically to the ligand and conjugated to a label, is used.
  • the biological sample which comprises the ligand
  • a molecule capable of binding specifically to the ligand herein called binding partner
  • binding partner a molecule capable of binding specifically to the ligand conjugated to labeling means.
  • the binding partner may for instance be a chemical compound, a protein such as a receptor of fragment thereof, or a specific antibody or fragment thereof.
  • a protein such as a receptor of fragment thereof
  • a specific antibody or fragment thereof for example, when the ligand considered is a known protein or family of proteins.
  • a secondary antibody specific of the IgG class and conjugated to labeling means may be used.
  • those antibodies are known in the art as "secondary antibody", and are typically capable of binding both heavy and light chains of the IgG antibodies.
  • the binding agent may be coupled to any type of labeling means.
  • labeling means it is herein referred to any species that can be used for detection and/or quantitation. Labelling means are well known in the art, and need not be thoroughly detailed herein.
  • the labelling means is preferably a luminophore. Examples of luminophores that can be used include fluorophores, phosphorescent compounds, or luminescent compounds. Luminophores appropriate in the context of the invention have for instance been disclosed in WO2002033419.
  • the means used in this step is a fluorophore.
  • the step of treating the biological sample comprises or consists in incubating the biological sample with a labelled antibody specific of the ligand.
  • said labelled antibody specific for the ligand is coupled to a luminophore, preferably a fluorophore.
  • the step of treating the biological sample comprises or consists in incubating the biological sample with a labelled antibody specific of said ligand, i.e. of said protein or antibody, preferably coupled to a luminophore, yet preferably a fluorophore.
  • the step of treating the biological sample comprises or consists in incubating the biological sample with a labelled antibody specific for IgG antibodies, preferably coupled to a luminophore, yet preferably a fluorophore.
  • the step of treating the biological sample comprises or consists in incubating the biological sample with a labelled antibody specific for IgE antibodies, preferably coupled to a luminophore yet preferably a fluorophore.
  • the method further includes a step of measuring the data relative to the binding of said ligand to said at least part of the molecule of interest while flowing the labelled biological sample in at least one microfluidic channel comprising at least a set of microcarriers, wherein the microcarriers are functionalized with said at least part of the molecule of interest.
  • the binding between the ligand in the biological sample, and the molecule of interest or its fragment thereof, attached to the surface of the microcarrier, occurs relatively fast, and the initial interaction are likely to occur as soon as the biological sample contacts the microcarriers in the microchannel. This step therefore ensures that the data relative to the binding of said ligand to said at least part of the molecule of interest is collected as the reaction occurs.
  • microchannel and microcarriers are designed to use the properties of microfluidics, in particular the advantages of the laminar flow that is created by flowing the biological sample herein.
  • One microchannel comprises at least one set of microcarriers, advantageously several microcarriers, thereby enabling average calculations and thus accuracy of the data.
  • this system may be used for multiplexing, for instance in embodiments where the microchannel comprises several sets of identical microcarriers.
  • the microchannel comprises a plurality of sets of microcarriers.
  • the microcarriers are preferably encoded, and advantageously each set of microcarriers harbors a different code.
  • the microchannel comprises at least two distinct sets of microcarriers, each set of microcarriers being functionalized with a distinct molecule of interest.
  • microchannels may be used in the method of the invention.
  • the labelled biological sample is flown in a plurality of microchannels, each microchannel comprising at least a set of microcarriers.
  • each microchannel may comprise a plurality of sets of microcarriers. It is not necessary that all microchannels comprise the same number of either microcarriers nor sets of microcarriers, and various arrangements may be used by the person skilled in the art.
  • the specific features of the microchannel and microcarrier are as follows.
  • microchannel or “microfluidic channel” it is herein referred to a hollow structure appropriate for the passage of fluids, i.e. an enclosed passage, having sub-millimeter dimensions.
  • the at least one microchannel according to the invention has a cross-section microscopic in size, i.e. with the largest dimension (of the cross-section) being typically from 1 to 500 micrometers, preferably 10 to 500 micrometers, more preferably from 20 to 400 micrometers, even more preferably from 30 to 400 micrometers.
  • a microchannel has a longitudinal direction, which is not necessarily a straight line, and that corresponds to the direction in which the fluids are directed within the microchannel, i.e. essentially to the direction corresponding to the vector addition of the speed vectors of a fluid passing in the microchannel, assuming a laminar flow regime.
  • a microchannel has, at one end, an entry and, at the other end, an exit, which are openings in the microchannel that e.g. let the fluids enter into the microchannel, respectively leave the microchannel.
  • the length of the microchannel of the invention can vary depending on its cross-section and on the desired footprint, generally to fit into the microfluidic chip it is typically comprised in.
  • the width and height of the microchannel is preferably from 500 nm to 300 micrometers.
  • the microchannel has preferably a cross-section that is rectangular or close to rectangular, trapezoid or like a parallelogram.
  • the microchannel has typically two lateral walls, a base and a cover.
  • the microcarriers are restricted from exiting said at least one microchannel.
  • the microchannel is thus preferably designed to enable the flowing of fluids inside the at least one microchannel without allowing the microcarriers to exit the microchannel.
  • the at least one microchannel of the microfluidic cartridge comprises restriction means, preferably restriction means configured to restrict the longitudinal movement of said microcarrier in said microchannel while still letting fluids flow.
  • restriction means include a grid, a wire, a mesh filter, a weir construct, one or more pillars, a reduction of the section of the microchannel.
  • the entry of the microchannel is typically the extremity by which the biological sample is flown.
  • the microcarriers of the microchannel are responsive to magnetic, electrostatic, or dielectrophoretic forces.
  • the microcarriers preferably have magnetic, electrostatic, or dielectrophoretic properties. Movements of the microcarriers may then be restricted using a magnetic or electronic field, preferably operated from the microfluidic device.
  • the microchannel is transparent on at least one side.
  • the microcarriers can be readily observed via means for optical inspection, such as a microscope.
  • This particular configuration of the microchannel allows for an easy identification of the sets of microcarriers when they are placed within the microchannel and determination of the chemical or biological readout by conventional techniques based on optical response used in the art for that determination.
  • the microchannel is made of or comprises silicon, thermoplastic polymers, quartz, glass or plateable metals such as nickel, silver or gold, most preferably of transparent polymers. Most preferably, the microchannel is made of Cyclic Olefin (Co)polymer.
  • the microchannel can be manufactured using conventional photolithography and/or stamping and/or injection molding techniques that are extensively described in the literature (Fundamentals of microfabrication, Madou M., CRC Press, 2002, and Fundamentals and Applications ofMicrofluidics, Nguyen and Wereley, Artech House, 2002).
  • the microchannel and microcarriers may be designed to facilitate mass transfer of the fluids and/or the microcarriers within the microchannels, so as to guaranty accuracy of the data. Ways to design such microchannel and microcarriers have been described in WO 2010/072011.
  • the shape and size of the microcarriers relative to the cross-section of the at least one microchannel allows to have, over the entire length of the microchannel, at least two of any of the microcarriers arranged side by side without touching each other and without touching the perimeter of the microchannel when travelling in the longitudinal direction of the microchannel.
  • microcarrier it is herein referred to any type of particle microscopic in size, typically with the largest dimension being from 100 nm to 300 micrometers, preferably from 1 ⁇ to 200 ⁇ .
  • microcarrier functionalized with at least part of the molecule of interest it is herein referred to a microcarrier having said part of the molecule attached to its surface.
  • the molecule of interest or fragment thereof is typically attached to the microcarrier by means conventionally used for attaching molecules to microcarriers in general, including by means of a covalent bound and through direct attachment or attachment through a linker.
  • the microcarrier of the invention may be made from or comprise any material routinely used in high-throughput screening technology and diagnostics.
  • the microcarriers are made of silicon.
  • the microcarrier may be of any shape.
  • the microcarrier has preferably a spherical shape or the form of a wafer which means that their height is notably smaller (e.g. by at least a factor of two) than both their width and their length and that they have two essentially parallel and essentially flat surfaces (front faces) at the top and at the bottom.
  • the microcarrier has a disk-like shape with the front face in form of a circle.
  • the microfabrication techniques described in relation to the microchannel can also be used to produce microcarriers, for example for producing silicon microcarriers on a wafer as described in EP 1 276 555.
  • the microcarrier may further be encoded, to facilitate their identification.
  • the microcarrier of the invention is encoded in such a way that its function, i.e. the type of molecule(s) attached to its surface, can be determined by reading the code, preferably using optical means.
  • Codes and method for encoding microcarriers are known in the art, and have been disclosed for instance in EP 1 276 555 and EP 1 346 224.
  • a "set of microcarriers" herein refers to one or more microcarriers with the same functionalization, i.e. with the same part of the molecule of interest attached to their surface.
  • a set may be only one microcarrier or a plurality of microcarriers.
  • the set of microcarriers is thus defined by the part of the molecule of interest attached to the microcarriers.
  • sets of microcarriers are said to be different (i.e. from one another) when said at least part of the molecule of interest differs.
  • the microcarriers When the microcarriers are encoded, the microcarriers of a same set usually harbor the same code. When several sets of microcarriers are present within a same microchannel, each set of microcarrier harbors a different code. Flowing of the labelled biological sample into the microchannel will produce a laminar flow which enable accurate binding and dissociation data, as it minimizes the effects of diffusion.
  • the labelled biological sample if flown in the at least a microchannel using an active flow.
  • an active flow implies a reaction limited regime where binding rate is maximal, and mostly becomes independent of flow rate.
  • Such active flow may be obtained by using a differential pressure between the entry and the exit of the at least one microchannel.
  • Such active flow may be obtained using micropumps, preferably located at the entry of the microchannel.
  • a first micropump is used near or at the entry of the microchannel and a second micropump is used near or at the exit of the microchannel.
  • the active flow is set so as to enable reaction limited regime. Indeed, it is known that the flow rate may can positively impact the conditions of the assay, in that above a flow rate threshold, the reaction is most likely to occur in reaction limited regime.
  • Appropriate flow rates for enabling reaction limited regime vary according to the samples and reagents used, and the person skilled in the art may adapt the flow rate according to the molecule of interest and the ligand using common knowledge.
  • Many microfluidics platforms have been developed using pressure-driven laminar flow, and any of them may be used to implement the method of the invention.
  • a platform appropriate for implementing the method of the invention is the Evalution® platform.
  • the viscosity of the product used in the platform may need to be adjusted, to facilitate implementation of the method.
  • the labelled biological sample may for instance be diluted prior to being flown in the microchannel in order to facilitate data acquisition.
  • the labelled biological sample may be diluted with conventional buffers such as TRIS buffer, HEPES buffer or PBS buffer.
  • the buffer used does not contain molecules susceptible to specifically bind the molecule of interest.
  • the method is very versatile as it enables the use of a plurality of microchannels, which may for instance be useful when various dilutions of the same labelled biological sample need to be tested. Alternatively, this set up enables the concurrent assay of several different labelled biological samples, each one being for instance flown in a different microchannel.
  • the labelled biological sample if flown in a plurality of microchannels, each microchannel comprising at least a set of microcarriers.
  • association and dissociation rates K on and Koff can be derived from kinetic data
  • the binding rate Kon can be influenced by the concentration of ligand in a binding reaction, while the dissociation rate K 0 ff is not.
  • This technical issue is classically overcome in the field by either measuring the concentration of the labelled ligand, for instance using routine techniques such as ELISA, or by measuring the data on serial dilutions of the labelled biological sample.
  • the concentration of labelled ligand may be determined using routine techniques such as ELISA.
  • the method of the invention further comprises a step of measuring the concentration of the labelled ligand present in the labelled biological sample flown in the at least one microchannel. This step may be performed prior, after, or in parallel of the other steps of the method of the invention.
  • dilutions of the labelled biological sample in a buffer are prepared prior to the biological sample being flown in the at least one microchannel, and said dilutions are flown in a plurality of microchannels comprising at least a set of microcarriers, wherein each dilution is flown in a distinct microchannel.
  • the data relative to the binding of said ligand to said at least part of the molecule of interest is measured while the labelled biological sample is flown in the at least one microchannel. It should be understood that if a plurality of microchannels are used, the data should be measured for each of said microchannels.
  • this step may further comprise a step of reading the code, preferably using optical means.
  • the microcarriers are preferably encoded, and advantageously each set of microcarriers harbors a different code. Because each code usually correspond to a specific functionalization, i.e. a specific part of a molecule of interest, the data relative to the binding of the ligand may thus be attributed to a specific at least part of a molecule of interest, even when the microchannel comprises a plurality of sets of microcarriers. This aspect is particularly useful in multiplexing set-ups.
  • the ligand which is labelled, will bind to the at least part of the molecule of interest attached to the surface of the microcarrier.
  • the local accumulation of the label for instance the accumulation of fluorescence, compared to the background noise of the label present in the labelled biological sample, is therefore indicative of a binding reaction.
  • Measurement of the signal emitted by the label can be used as data relative to the binding of said ligand to said at least part of the molecule of interest.
  • This set up ensures that the data which is measured is specific of the binding interaction of interest.
  • the data relative to the binding is measured at defined time-points.
  • the data relative to the binding is measured immediately when the labelled biological sample is being flown, and then on further defined time points.
  • the frequency of the defined time points may easily be adapted by the person skilled in the art, in order to adapt the method to the molecule of interest, and the biological sample.
  • the defined time points are more frequent within the first 5 minutes after the labelled biological sample is being flown in the microchannel.
  • the person skilled in the art may decide to modify the frequency, for instance to lower the frequency of the measurement.
  • the data relative to the binding is measured immediately when the labelled biological sample is being flown, and then every 2 to 20 seconds, preferably every 5 to 15 seconds, yet preferably every 10 seconds, during at least 1 minute, preferably at least 2 minutes.
  • the data relative to the binding is measured up until the binding reaction reaches an equilibrium state. This can be estimated visually, by observing the binding curve reaching a plateau.
  • the person skilled in the art may then proceed to the next step of the method, i.e. measuring the data relative to the dissociation of said ligand from said at least part of the molecule of interest while flowing a buffer in said microfluidic channel.
  • the dissociation between the ligand and the molecule of interest or its fragment thereof also occurs extremely fast, and the initial reaction of dissociation are likely to occur as soon as the buffer is flown in the microchannel and diluted the labelled biological sample, and thus the ligand.
  • This step therefore ensures that the data relative to the dissociation of said ligand to said at least part of the molecule of interest is measured as the reaction of dissociation occurs.
  • this step may further comprise a step of reading the code, preferably using optical means.
  • the buffer used in this step does not require specific compounds, and the person skilled in the art may use conventional buffers such as TRIS buffer, HEPES buffer or PBS buffer.
  • the buffer used does not contain molecules susceptible to specifically bind the molecule of interest.
  • the data relative to the binding is collected at defined time-points.
  • the data relative to the dissociation is measured immediately when the buffer is being flown, and then on defined time points.
  • the frequency of the defined time points may easily be adapted by the person skilled in the art, in order to adapt the method to the molecule of interest, and the biological sample.
  • the defined time points are more frequent within the first 2 minutes after the buffer is being flown in the microchannel.
  • the data relative to the dissociation is measured immediately when the buffer is being flown, and then every 2 to 20 seconds, preferably every 5 to 15 seconds, yet preferably every 10 seconds, during at least 1 minute, preferably at least 2 minutes.
  • the data relative to the binding is measured for about 2, about 5, about 10 minutes.
  • the person skilled in the art will be able to adapt the measuring step according to the ligand and molecule of interest used, and of the data collected.
  • the person skilled in the art may proceed to determining the avidity of said ligand for said at least part of the molecule of interest.
  • the data may first be used to draw the curve of the kinetic of the binding and dissociation reaction. Those curves are well known.
  • immediate processing by the Evalution® software is obtained so that a binding/dissociation curve is readily available for analysis.
  • the binding rate Kon can typically be derived from the data, in particular by the shape of the binding curve for a specific concentration of labelled ligand (taking into consideration the earliest points, before reaching equilibrium).
  • the dissociation rate Koff corresponds to the slope of the dissociation data (taking into consideration the earliest points, before reaching equilibrium), and is not influenced by the concentration of labelled ligand.
  • the K on may thus be derived easily.
  • the functional affinity i.e. avidity
  • Ka is the functional affinity, i.e. the avidity.
  • the method of the invention can advantageously be used for determining antibodies avidity, for example, IgGs or IgEs avidity for an antigen, and thus for assessing the maturation of the humoral immune response to an antigen, thereby be applicable to:
  • the invention further pertains to a method to diagnose and/or characterize allergy to a specific antigen in a subject, comprising determining the avidity of IgEs to said specific antigen from a biological sample of the subject using the method of the invention.
  • the method is used to diagnose and/or characterize allergy to more than two specific antigens in a subject, comprising determining the avidity of IgEs to each of said specific antigens from a biological sample of the subject using the method of the invention.
  • the method further comprises comparing the avidity obtained from a biological sample of the subject to a reference value, preferably corresponding to the avidity obtained using the method of the invention with a non-specific molecule as the molecule of interest.
  • bovine serum albumin may be used as a control molecule for such purpose.
  • Figure 1 Measurements of the binding and dissociation kinetics of IgEs to common allergens using the method of the invention.
  • Example 1 application of the invention to the field of allergy detection The method of the invention was used to measure the avidity of IgEs for common allergens (deriving from peanut) in the serum of a human subject.
  • microcarriers functionalized with common allergens Different sets of microcarriers were prepared, each set being functionalized with one of the following molecules of interests, related to peanut allergy in the literature:
  • Ara h 1 which is a protein from Arachis hypogaea (peanut);
  • Ara h 2 which is a protein from Arachis hypogaea (peanut);
  • Ara h 3 which is a protein from Arachis hypogaea (peanut);
  • Ara h 9 which is a protein from Arachis hypogaea (peanut);
  • bovin serum albumin (BSA) Proteins (allergens and controls) were immobilized on COOH-microparticles (Evalution®), using established 2-step EDC/NHS chemistry. To facilitate data treatment, each set of microcarriers was associated with a different code, written on the microcarriers. The 7 sets of microcarriers were then mixed together to create a multiplex population.
  • BSA bovin serum albumin
  • the multiplex population is loaded into the microchannels of an assay cartridge (Evalution®) to a height of 1 mm (height is dependent on the multiplex level and is not specific for this type of assay).
  • the cartridge is inserted into the Evalution® instrument.
  • Serum samples from human subjects were treated by pre-incubation of the sample with phycoerythrin-labeled anti-human IgE antibodies.
  • the serum sample(s) containing specific anti-peanut allergen IgE antibodies are diluted in LowCross-Buffer® (CANDOR Bioscience) and spin-filtered using 0.45 ⁇ filter at 16.1xl000g for 2 min at 4°C.
  • the phycoerythrin-labeled anti-human IgE antibodies are diluted in LowCross-Buffer® and centrifuged at 16.1xl000g for 2 min at 4°C.
  • a reaction mix is prepared by mixing the diluted sample(s) with the diluted antibodies.
  • thermomixer 1100 rpm
  • the treated serum samples can be used in the Evalution® instrument (i.e. flown in the microchannels comprising the microcarriers).
  • the treated serum samples were introduced in the channels of an assay cartridge loaded with the functionalized microparticles with immobilized peanut allergens and controls (cf. step 1).
  • Serum and secondary antibody were flown constantly through the microchannels through acquisition of binding data. Binding was monitored live by measuring fluorescence buildup at regular time-points and immediate processing by the Evalution® software.
  • Dissociation data is recorded first every 10 seconds for 2 min and then every 30 seconds for 3 minutes or longer.
  • Association data and dissociation data were exported from the Evalution Explorer Software and processed using a software allowing curve fitting (in the case of the experiment: GraphPad®).
  • the association rate (Kon, typically measured as a number of association events per unit of time) anddissociation rate (K 0 ff, typically measured as a number of dissociation events per unit of time) were determined.
  • the functional affinity (i.e. avidity) was calculated using the well-known ratio:
  • Ka is the affinity constant (in this case the functional affinity).

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Abstract

Cette invention concerne un procédé de détermination de l'avidité d'un ligand spécifique pour au moins une partie d'une molécule d'intérêt, dans un échantillon biologique, caractérisé en ce qu'il comprend les étapes suivantes : a) traitement de l'échantillon biologique en vue de marquer spécifiquement le ligand, pour obtenir ainsi un échantillon biologique marqué, b) mesure des données relatives à la liaison dudit ligand à ladite au moins partie de molécule d'intérêt pendant la circulation de l'échantillon biologique marqué dans au moins un canal microfluidique comprenant au moins un ensemble de microsupports, où les microsupports sont fonctionnalisés avec ladite au moins partie de molécule d'intérêt, c) mesure des données relatives à la dissociation dudit ligand de ladite au moins partie de molécule d'intérêt pendant la circulation d'un tampon dans ledit canal microfluidique ; et d) détermination de l'avidité dudit ligand pour ladite au moins partie de molécule d'intérêt.
PCT/EP2017/075083 2016-10-03 2017-10-03 Procédé de mesure de l'avidité ou de l'affinité fonctionnelle WO2018065417A1 (fr)

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