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EP4295155A1 - Kit et méthode d'analyse d'un état d'immunisation chez un sujet par rapport à un micro-organisme pathogène - Google Patents

Kit et méthode d'analyse d'un état d'immunisation chez un sujet par rapport à un micro-organisme pathogène

Info

Publication number
EP4295155A1
EP4295155A1 EP22705903.7A EP22705903A EP4295155A1 EP 4295155 A1 EP4295155 A1 EP 4295155A1 EP 22705903 A EP22705903 A EP 22705903A EP 4295155 A1 EP4295155 A1 EP 4295155A1
Authority
EP
European Patent Office
Prior art keywords
ligand
labelled
test
antibody
test strip
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.)
Pending
Application number
EP22705903.7A
Other languages
German (de)
English (en)
Inventor
Giovanni Melioli
Marco Pizzi
Valentina GALLO
Michela SALVI
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.)
Eltek SpA
Original Assignee
Eltek SpA
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 Eltek SpA filed Critical Eltek SpA
Publication of EP4295155A1 publication Critical patent/EP4295155A1/fr
Pending legal-status Critical Current

Links

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
    • G01N33/68—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
    • G01N2470/04—Sandwich assay format

Definitions

  • the COVID-19 pandemic is caused by the SARS-CoV-2 virus, which belongs to the Coronavirus family, i.e., a family of viruses known to cause the onset of respiratory tract diseases ranging from common cold to Severe Acute Respiratory Syndrome (SARS).
  • Coronaviruses are viruses of rounded morphology and comprise, among their various constituent elements, the Glycoprotein S, or Spike , which determines the specificity of the virus for the epithelial cells of the respiratory tract.
  • the onset of the COVID-19 pandemic has made it necessary, among other things, to test whether a subject affected by the virus has been able to recover naturally, in particular in the case of subjects defined as "asymptomatic", or as a result of specific therapeutic treatments, and if he/her has consequently developed a reaction such as to make him immune at the time of a related test.
  • Recent availability of vaccines aimed at countering the spread of the aforementioned pandemic has also made it necessary to verify whether a subject that underwent vaccination has really acquired the ability to defend himself/herself from the virus, already starting from a relatively short time after the administration of the vaccine. There is currently no knowledge about the duration of the immunization, so a corresponding test would also be useful to understand if it is necessary to repeat the vaccination.
  • WO 2020/046857 A1 discloses antibodies binding to and neutralizing norovirus and methods for use thereof.
  • a method is provided for detecting a norovirus infection in a subject, by exploiting a strip-based competitive assay, wherein the antibodies of the sample compete with a labelled antibody specific to the norovirus, to bind to the antigen fixed on the strip.
  • US 2012/178105 A1 discloses a competitive assay for the detection of the GL3 protein in human samples using a sandwich-based immunoassay, in which a pair of GL3 specific monoclonal antibodies are used, one for capture and one for detection, to create an antibody “sandwich” around the GL3 ligand.
  • the assay has been modified by complexing the capture antibody with GL3 before adding the sample or detector antibody, providing an inhibition-based assay.
  • WO 2005/069002 A1 discloses devices and methods suitable for rapid detection of endogenous urine antibodies, particularly antibodies directed against HIV viral coat proteins.
  • an aim of the present invention is to indicate tools and methods for the execution of a quick functional test, designed to verify a state of immunization of a subject with respect to a pathogenic microorganism, in particular to the SARS-CoV-2 virus, both for the case in which such immunization should result from the administration of a vaccine, and the case in which such immunization could have been acquired in a natural way by an infected subject (i.e., in the absence of specific therapeutic treatments) or else following a therapeutic treatment other than vaccination.
  • Another aim of the invention is to indicate tools and methods that allow to quickly and easily assess whether the subject in question has acquired the ability to defend himself from the virus or not.
  • Another aim of the invention is to indicate tools and methods that allow to evaluate the presence of antibodies, in particular antibodies directed against the Spike protein (known as "Anti-Spike”), capable of counteracting or stopping the progress of viral replication.
  • Anti-Spike antibodies directed against the Spike protein
  • At least one of the above aims is achieved, according to the present invention, by a kit and a process for testing the state of immunization to a pathogenic microorganism, in particular the SARS-CoV-2 virus, as described below.
  • a kit and a process for testing the state of immunization to a pathogenic microorganism, in particular the SARS-CoV-2 virus as described below.
  • Preferential embodiments of the invention are indicated in the attached claims. The claims form an integral part of the technical teaching provided herein in relation to the invention.
  • the invention relates to a rapid test (assay) based on detection of a viral protein, in particular the Spike protein, supplied in lyophilized or dried form in a first container, that is preferably an integral part of a diagnostic kit.
  • a buffer solution is provided, which is mixed with the contents of the first container containing the lyophilized or dried viral proteins, to solubilize them.
  • a biological sample of the subject to be examined, the viral protein of the first container and the buffer solution of the second container are mixed together to form a test sample, a fraction of which is then fed into a rapid test device of the diagnostic kit, of a disposable type: detection of the possible presence of viral proteins in the test sample on the test device takes place by antigen-antibody binding, i.e., viral protein-antibody, and provides indications as to the immunization status of the subject.
  • antigen-antibody binding i.e., viral protein-antibody
  • test device or the diagnostic kit that includes it, is preferably designed to allow the use, as a starting biological sample, of a saliva sample of the subject: this possibility makes the carrying out of the test quicker and less invasive, compared to the case of a biological sample consisting of blood obtained from a finger-pricking device or from a classic sampling, which, however, is not excluded from the scope of the invention.
  • the test device of the diagnostic kit is prearranged for performing a lateral flow chromatographic immunological test, also known in literature as LFIA (. Lateral Flow Immuno Assay) or LFD (. Lateral Flow Device) or LFA (. Lateral Flow Assay) or LFT (. Lateral Flow Test ): this allows to obtain the results concerning possible immunization already after a few minutes from the execution of the test, in particular through direct visibility (with the naked eye) of the outcome, possibly exploiting chemiluminescence or fluorescence effects.
  • LFIA Lateral Flow Immuno Assay
  • LFD Lateral Flow Device
  • LFA Lateral Flow Assay
  • LFT Lateral Flow Test
  • the test device of the diagnostic kit is conceived to allow the carrying out of a quick test of POC type ⁇ Point Of Care) ⁇ in addition to being minimally invasive, such a type of test has the advantage of being able to be performed even by non-expert operators, that is, operators not necessarily having the technical skills normally required for a classic laboratory test, for example, an ELISA test.
  • the antibody for detection in the visible is associated with coloured nano-particles, for example latex nano-spheres (blue colour) or gold nano-spheres (red/purple colour), which - when concentrated in one and the same area of the test device - are visible with a classic detection line, for example a pink/purple line.
  • coloured nano-particles for example latex nano-spheres (blue colour) or gold nano-spheres (red/purple colour)
  • the coloured detection line will be visible thanks to a chemiluminescence reaction, with a suitably labelled detection antibody, for example by means of a catalyst enzyme of the HRP type ⁇ Horseradish peroxidase ), and a suitable development liquid, for example containing luminol, capable of causing the reaction that will emit blue light, more easily visible in the dark.
  • a suitably labelled detection antibody for example by means of a catalyst enzyme of the HRP type ⁇ Horseradish peroxidase
  • a suitable development liquid for example containing luminol
  • the methods and tools proposed according to the invention allow to evaluate presence of antibodies, particularly Anti-Spike antibodies, necessary to stop the progress of the viral replication.
  • the main advantage of the invention consists in that a functional test is carried out on the biological sample, in particular saliva, by evaluating the ability of the subject to block the virus precisely in the areas that may present for him/her the main access points to the organism.
  • the preferential application of the method is to evaluate the effectiveness of a vaccination in a direct way, i.e., to see if the vaccination has worked by evoking an antibody response at the level of the mucous membranes, very relevant in terms of prevention of a respiratory tract infection.
  • test subject of the invention is conceived such that it can be adapted quickly to any variants of the virus that involve sequential or conformational variations of the Spike protein.
  • the version of the same in mutated form can be produced and inserted into the corresponding container in lyophilized or dried form, both in purity to have a specific test on the mutated virus version, and in mixed form with the other versions of Spike protein, to evaluate the subject's ability to cope with the different forms of virus.
  • a related advantage of the invention is that the proposed methods and tools make it possible to determine whether the plasma of a cured subject can be used effectively to treat other subjects severely affected by the virus, in particular by the SARS-CoV-2 virus, that is, subjects requiring urgent and intensive treatment.
  • a peculiarity of the invention compared to the classic rapid antibody serological tests, lies in the fact that the proposed test is functional, i.e., it not only allows to detect the presence of antibodies, such as Anti-Spike antibodies, but also allows to detect the correct functioning in the ability of these antibodies to inhibit the bound between a ligand, for example represented by the Spike protein, and an appropriate anti-ligand, for example represented by the hACE2 protein ⁇ Human Angiotensin Converting Enzyme 2) or other capture antibody, and thus neutralize the viral antigen.
  • a ligand for example represented by the Spike protein
  • an appropriate anti-ligand for example represented by the hACE2 protein ⁇ Human Angiotensin Converting Enzyme 2
  • other capture antibody for example, as mentioned, if a subject has enough antibodies to neutralize the virus, his/her plasma can be used for urgent treatments of severe COVID-19 patients: otherwise, such plasma could not be effectively used.
  • test sample which is the lyophilized or dried viral protein initially placed in a container that is part of the diagnostic kit, and then diluted with a buffer solution and a biological sample of the subject
  • test is positive
  • antibodies are not present in the starting biological sample, or are present in insufficient amount to provide the subject with immunization to the virus, i.e., to neutralize it: in more detail, referring in particular to the SARS-CoV-2 virus, the subject's biological sample does not contain antibodies, preferably IgA antibodies, in sufficient amount to occupy all the epitopes of the Spike protein, and to prevent its binding with a corresponding Anti-Spike capture antibody immobilized in the test device.
  • the invention can also be used with other viruses, by identifying the appropriate anti-receptor present on the capsid or pericapsid of the virus (the Spike protein, in case of Coronavirus) and the appropriate receptor on the membrane of the host cell ( hACE2 , in case of Coronavirus).
  • the capture element on the test line can be both the receptor itself ( hACE2 , in case of Coronavirus) and other appropriate anti-ligand (in the case of Coronavirus, any Anti-Spike antibody).
  • the anti-receptor is supplied in lyophilized form for the immunization test (in the exemplificative case of Coronavirus, it is the Spike protein).
  • FIG. 1 is a schematic perspective view of a test device that can be used in a diagnostic kit according to possible embodiments
  • FIGS. 2 and 3 are schematic views, respectively in perspective and in section, of a first container that can be used in a diagnostic kit according to possible embodiments
  • - Figures 4 and 5 are schematic views, respectively in perspective and in section, of a second container that can be used in a diagnostic kit according to possible embodiments;
  • FIG. 6 is a schematic perspective view of a third container that can be used in a diagnostic kit according to possible embodiments
  • FIG. 7 and 8 are schematic views, respectively in perspective and in section, of a dosing tool that can be used in a diagnostic kit according to possible embodiments;
  • FIG. 12 is a schematic perspective view of a funnel that equips the container of Figures 10-11 in the first configuration of use;
  • FIG. 13 is an exploded schematic view of the container of Figures 10-11 with a corresponding dosing cap that can be used in a second configuration of use;
  • FIG. 14 is an exploded schematic view of a test device that can be used in a diagnostic kit according to possible embodiments
  • FIG. 16 and 17 are schematic representations aimed at exemplifying the mechanism of operation of a diagnostic assay according to possible embodiments, respectively in the case of immunization and non-immunization to a pathogenic microorganism;
  • FIG. 18 is a representation intended to exemplify possible indications detectable by a test device according to possible embodiments
  • FIG. 26 is a schematic perspective view of a test device of a diagnostic kit according to possible variant embodiments.
  • pathogenic microorganism or “pathogen” as used hereinafter refer to any biological agent responsible for the occurrence of a disease condition in a host organism, including viruses, for example viruses belonging to the Coronavirus family, viruses capable of causing the onset of respiratory tract diseases, viruses of rounded morphology and/or viruses including Glycoprotein S ⁇ Spike) such as SARS-CoV-2 virus.
  • viruses for example viruses belonging to the Coronavirus family, viruses capable of causing the onset of respiratory tract diseases, viruses of rounded morphology and/or viruses including Glycoprotein S ⁇ Spike) such as SARS-CoV-2 virus.
  • the indication relating to the verification of "a state of immunization" of a subject must be understood in its broadest sense, i.e., indicative of a condition of complete immunization of the subject, or of his/her condition of non-complete immunization (i.e., of his/her partial immunization).
  • antibody refers to a protein produced by a subject in response to an infection by a pathogenic microorganism, such as an infection caused by a virus.
  • immunoglobulins for example immunoglobulins of type A (IgA), M (IgM) or G (IgG).
  • ligand and “anti-ligand” as used hereinafter refer respectively to any pathogenic protein that is involved in inhibition, neutralization or removal mechanisms, and to any protein (normally antibodies) that is able to operate as a specific recognition system of the ligand.
  • the diagnostic kit comprises a device for carrying out a lateral flow chromatographic immunological test.
  • a test device exemplified in Figure 1 and indicated as a whole with 1
  • a first opening or window la is defined in a body of the device 1, for the introduction of a sample to be tested, and a second opening or window lb, at which the result of the test is visible, as explained below.
  • the body of device 1 is preferably formed of plastic material.
  • a first sealed container is indicated with 2, for example comprising a hermetically sealed body or vial 2a.
  • Vial 2 can be sealed in any known manner.
  • the container 2 has a cap 2b, for example coupled to an external thread 2c defined near the mouth of vial 2a.
  • the cap 2b is preferably a dosing cap or dropper, in particular configured for dosage and dispensing of drops having a substantially predefined volume, for example provided for the purpose of a dripper or spout 2b' to which a corresponding cap 2b" is associated.
  • a second container is indicated with 3, preferably but not necessarily having a structure similar to the container 2.
  • the container 3 is a single-dose container.
  • the container 3 is a multi-dose container, such as a flacon or bottle, from which - preferably by means of a syringe or a calibrated pipettor with disposable tips, or with a calibrated pipette, or with a dosing cap - a plurality of doses for a plurality of tests can be drawn.
  • the container 3 comprises a hermetically sealed body or vial 3a.
  • the container 3a also has a cap 3b, for example coupled to an external thread 3c of the vial 3a.
  • the cap 3b can be a dosing cap or dropper, in particular configured for dosage and dispensing of drops of substantially predefined volume, for example provided for the purpose of a dripper or spout 3b' to which a corresponding cap 3b" is associated.
  • Vial 3a contains a suitable buffer solution 3d, such as a typical aqueous solution that opposes the change in pH as a result of moderate additions of acids or bases.
  • a suitable buffer solution 3d such as a typical aqueous solution that opposes the change in pH as a result of moderate additions of acids or bases.
  • an amount of buffer solution of between 0.05 and 3 millilitres can be provided, preferably between 0.1 and 0.5 millilitres.
  • Vials 2a, 3a and their caps 2b, 3b can be formed with plastic material.
  • vial 2a, 3a can also possibly be provided with a removable sealing film, which initially closes the mouth of the vials themselves, below the corresponding cap; such a film can be metal-based, or plastic-based, or composite.
  • the diagnostic kit comprises a tool for dosing a fraction of the biological sample taken from the subject.
  • this tool is a dosing pipette or dropper, for example of plastic material, such as that exemplified in Figures 7 and 8, designated by 5.
  • Pipette 5 can be used to take a part of the saliva previously collected in the container 4 of Figure 6, and then dose it, as described below.
  • Components 1-5 are preferably packaged in a protected atmosphere and each contained, preferably, in a corresponding sterile protective casing, for example a flexible sachet. However, there is nothing to prevent packaging of several components (e.g., the two containers 2, 3 and the pipette 5) in one and the same sterile envelope.
  • the measuring cap 3b of the container 3 is used to introduce a dosed amount of the buffer solution 3d into the vial 2a of the container 2, after the relevant cap 2b has been removed therefrom.
  • the dosed amount can be equal to three drops (corresponding to about 100 microliters).
  • the vial 2a can be closed using the corresponding cap 2b and shaken moderately, to suspend or solubilize the protein 2d.
  • a dosed amount of a biological sample of the subject to be examined is added to the solution containing the viral proteins, i.e., to the container 2, with reference to the example given.
  • the biological sample is made up of saliva of the subject.
  • the subject expels from his mouth the saliva in the cup 4, and at least a fraction of it (for example about 100 microliters) is taken through the pipette 5.
  • a dosed amount of saliva (corresponding to about 100 microliters) is fed into the vial 2a now containing the protein 2d and the corresponding dosed amount of the buffer solution, to form the test sample; this operation may be done by removing the cap 2b again from the vial 2a, if necessary, or without removing the cap 2b, if the outlet section of the pipette 5 is sufficiently narrow to allow it to be inserted into the spout 2b' of the cap 2b.
  • the container 2 is again shaken moderately. After a waiting time (for example, about fifteen minutes), a dosed amount of the test solution (for example, three drops, corresponding to about 100 microliters) is fed into the test device 1.
  • a dosed amount of the test solution for example, three drops, corresponding to about 100 microliters
  • a fraction e.g., six drops, corresponding to about 200 microliters
  • a fraction e.g., six drops, corresponding to about 200 microliters
  • a dosed amount of the test solution e.g., three drops, corresponding to about 100 microliters
  • the test device 1 is fed into the test device 1, as described below.
  • a further alternative is to provide in the kit a collection container of the biological sample that can be equipped with a funnel element, also supplied in a sterile casing.
  • a collection container can be used as an alternative to the cup 4 and the pipette 5, or to the absorbent stick 6.
  • the container indicated as a whole with 7, includes a vial 7a, which can be of similar construction as the vials 2a and/or 3a.
  • a funnel element is coupled to the vial 7a, designated as a whole with 8, for example formed with plastic material.
  • the funnel element 8 is preferably prearranged for coupling with the vial 7a: for this purpose, in various embodiments, the outlet part of the funnel element 8 is provided with an internal thread 8a, suitable for coupling with an external thread 7c provided near the mouth of the vial 7a, as shown in Figure 12.
  • a dosed amount of the collected saliva for example three drops, corresponding to about 100 microliters
  • the vial 2a can be closed by means of the corresponding cap 2b and shaken moderately.
  • a dosed amount of the test solution for example, three drops, corresponding to about 100 microliters
  • cup 4 of Figure 6 and the pipette 5 of Figures 7-8, or else the use of the container 7, a funnel element 8 and a measuring cap 7b, must be considered as preferential ones, in view of the greater precision of dosage of the biological sample, compared to the case of use of the absorbent stick 6 of Figure 9 (for which the dose of biological sample is dependent on the absorbent capacity of part 6a).
  • the main and secondary antibodies are immobilized preferably in the transverse direction, with respect to the longitudinal extension of portion 23, to form the two lines T and C.
  • the viral protein 2d, the buffer solution 3d and the biological sample here represented by the saliva of the subject, are mixed, in order to form the test sample.
  • the liquid then flows by capillarity for the entire length of the strip 20, up to the collection part 24.
  • the viral protein 2d contained in the test sample TS will be masked by the antibodies AB produced by the subject, i.e., it will be completely covered by said antibodies, as exemplified in part (A) of Figure 16.
  • the unmasked viral protein 2d binds to the antibody AC T with the coloured nano particles NP thereof, forming a corresponding complex: the fluid flow then carries this complex formed by the protein 2d and the labelled antibody AC T therewith.
  • the flow of the fluid in the passage through part 22, also carries the labelled antibody ACc labelled with the relevant NP nanoparticles therewith.
  • the unmasked (or not completely masked) viral protein 2d with the associated labelled antibody AC T concentrates at the capture antibody of the test line T, binding therewith, this concentration being optically detectable thanks to the nano-particles NP of the labelled antibody AC T .
  • the labelled antibody ACc concentrates at the capture antibodies of the control line C, to be optically visible in the corresponding area of the strip 20.
  • test result can be viewed through the window lb of the device, at which an area of part 23 of the strip including the test line T and the control line C is exposed (see for example Figure 18).
  • Figure 18 just exemplifies the different situations that can occur following a test carried out through the device 1, in the manner described above.
  • test line T and the control line C will be clearly identifiable at window lb, thanks to the coloured nano-particles NP associated with the antibodies AC T and ACc.
  • test line T indicates that binding between the viral protein 2d (target ligand) and the capture antibody (anti-ligand) has been inhibited. Inhibition can be caused by IgA, IgM and IgG antibodies, or any other ligand that has specificity for this reaction.
  • Part B of Figure 18 shows a second case of a positive test, i.e., a subject who does not have a sufficient antibody load to neutralize the pathogenic virus.
  • the control line C will be clearly perceptible, thanks to the coloured nano-particles NP associated with the antibodies ACc, while the test line T will be poorly visible, due to the reduced number of coloured nano-particles NP associated with the antibodies AC T that have concentrated at said line T.
  • Part D of Figure 18 shows the case of an invalid test, in which neither the test line T nor the control line C are in any way identifiable at window lb of the device 1; this may be due, for example, to an insufficient amount of the test sample TS into device 1 or, for example, to a malfunction of the fluidics of the device.
  • the described methodology also indicates the presence of such antibodies. It has also been demonstrated that antibodies against structures other than RBD (epitopes in other locations of the Spike molecule) are able to inhibit the infectivity of the virus on target cells. The described methodology is therefore able to identify (although not differentiate) Anti-Spike antibodies capable of inhibiting binding to the target cell. This is an advantage over cases where anti -RBD antibodies are identified that inhibit hACE2 bond, which is a natural ligand of the Spike protein of the SARS-CoV-2 virus.
  • Another advantage of the proposed methodology is that, by using the whole Spike protein, antibodies directed against different epitopes, generated by a polyclonal response, can have a three-dimensional physical hindrance such as to amplify the inhibition of the bond of the capture antibody with the Spike protein.
  • the diagnostic kit may include, in addition to the components previously exemplified, at least a third container for a development liquid, and preferably a fourth container for a washing liquid, indicated with 9a and 9b, respectively, in Figure 19.
  • These containers 9a, 9b can be of similar construction to that of the containers previously indicated with 2 and 3, except, of course, for the different contents.
  • the antibodies that obtain the test and control lines T and C will be conjugated, instead of coloured nano-particles, with an enzyme suitable for obtaining the effect of chemiluminescence (or fluorescence), such as the catalyst enzyme HRP ( Horseradish peroxidase).
  • HRP Horseradish peroxidase
  • the contents of container 9a may be any development liquid suitable to cause the effect of chemiluminescence (or fluorescence), for example a solution of luminol and water, for example in amounts between 50 and 100 microliters, preferably about 66 microliters.
  • the washing liquid of the container 9b may be a mixture of Phosphate Buffered Saline (PBS) with a detergent, such as polysorbate 20 (known commercially as Tween 20).
  • PBS Phosphate Buffered Saline
  • this liquid may include 50 microliters of saline phosphate buffer with the addition of 0,05 % polysorbate 20.
  • the methods of carrying out the test are similar to those described above, but with the difference that, a few minutes after introduction of the test sample TS into the device 1 (for example, about 3 minutes), a cleansing of the strip 20 is carried out, by introducing a few drops (for example, two drops) of the washing liquid into window la of the test device 1, so as to soak the corresponding part 21 of the strip 20. This is followed by a wait of a few minutes (for example, about 10 minutes), so that all the washing liquid also runs through the entire strip 20, from one end to the other.
  • a dose of the development liquid will be introduced, approximately between 40 and 80 microliters (for example 2 drops), which, in contact with the aforementioned catalyst enzyme (for example the aforementioned HRP), will develop the chemiluminescence (or fluorescence) reaction.
  • the aforementioned catalyst enzyme for example the aforementioned HRP
  • the detection in chemiluminescence derives from the reaction of the luminol solution in water with the HRP enzyme, which generates a blue light, which is more clearly perceptible in the dark, and which lasts for a few minutes.
  • suitable equipment may be provided which, in various preferential embodiments, can take advantage of a normal mobile phone, equipped with a special software application and having a camera with adequate shooting definition.
  • such equipment may include, for example, a shooting container, or black box , essentially box-shaped, indicated as a whole with 30, having a body 31 having a housing seat for the test device and a shooting window.
  • a body 31 has a sliding drawer 32 associated thereto, defining the aforementioned seat, and a shooting window 33 is defined at the upper face of the body 31.
  • the body 31 may be made of plastic material that is not transparent to light, with the exception of a possible transparent closure part 33a of window 33, for example made of glass or other light-transparent material.
  • the body 31 is internally provided with guides 34 (only one visible) for the sliding of drawer 32, here a linear sliding, so that the drawer itself can assume a closed position, represented in the figures, and an open position, not represented.
  • Drawer 32 defines a seat or upper cavity 32a, within which the test device 1 can be positioned with precision. The arrangement of parts is such that, when the test device 1 is at seat 32a of the drawer 32, and the drawer itself is in its closed position, window lb of device 1 is aligned axially with window 33 of the shooting container 30.
  • the upper face of the shooting container 30 is configured so that a mobile phone, indicated with TC in Figure 22, can be placed on it with its shooting sensor or camera at window 33.
  • the appropriate software application loaded on the phone TC is conveniently designed to set the camera as a function of the vertical distance between the upper face of the body 30 and the test device 1, in order to ensure the capture of an image of the window lb of the device 1 having a sufficient quality to enable evaluation of the result of the test.
  • the position shown in the drawings for the shooting window 33 is merely illustrative, as it may be otherwise allocated.
  • the upper face of the body 31 could also include a plurality of windows in different positions, corresponding to different typical positions of the shooting sensor or camera of various commercially available mobile phone models.
  • the same drawer 32 could be conformed to enable shooting of the window lb of the device 1 as a function of the shooting window 33 selected according to the phone used (for example by defining a plurality of seats 32a, in which to place the device 1 according to the type of mobile phone used).
  • a possible operating sequence is as follows: i) the mixing between the viral protein 2d and the buffer solution 3d is carried out, as already explained above; ii) the dedicated application on the phone TC is run, and the phone itself is placed on the shooting container 30, with its camera at the shooting aperture 33; obviously phases i) and ii) can be reversed; iii) the test device 1 is positioned at the corresponding seat 32a of the drawer 32, when the drawer is in the open position; iv) the biological sample of the subject is mixed with the buffer solution 3d containing the viral protein 2d, as already explained above, to obtain the test sample; obviously phases iii) and iv) can be reversed; v) in window la of the test device 1 the necessary amount of the test sample is introduced, as already explained above; vi) after the required waiting time, the necessary amount of washing liquid is introduced in window la of the device 1, as already explained above; vii) after the necessary waiting time, the necessary amount of development liquid is introduced in window lb of device 1, as already explained above
  • the dedicated software application loaded on the mobile phone TC can possibly be configured to provide on the display TCd, in addition to, or as an alternative to, the exemplified symbology, a textual indication of the result of the test.
  • the dedicated software application can be prearranged for detecting and interpreting the different intensities of the test line T, as in the case of an intermediate intensity of the type shown in part (A) of Figure 23, in particular to provide corresponding diagnostic indications.
  • the shooting container 30, instead of being equipped with a window 33 for use in combination with a mobile phone or another shooting instrument, could itself be equipped with a camera, such as an optical matrix sensor suitable for detection of luminescence, with a display, possibly of a touch screen type for the input of the required commands, and with a corresponding control electronics.
  • a camera such as an optical matrix sensor suitable for detection of luminescence
  • a display possibly of a touch screen type for the input of the required commands, and with a corresponding control electronics.
  • the target ligand is intended to be mixed with a buffer solution and a dose of a biological sample previously obtained from the subject, to obtain a test sample, which is then introduced into the receiving zone of the first test strip, the latter being capable of causing a flow of the test sample through the conjugation zone and the detection zone.
  • the invention also concerns the corresponding method or process for in vitro testing of a state of immunization in a subject with respect to a pathogenic microorganism.
  • the execution of the diagnostic test according to the preferential way i.e., using saliva as a biological sample, instead of blood
  • saliva as a biological sample, instead of blood
  • this response is most useful - that is, at the oral mucous membranes - for the prevention of an infection of the respiratory tract.
  • - high positive 1 microgram/10 microliters of plasma
  • - medium positive 0.063 micrograms/10 microliters of plasma
  • the amount of Spike protein used in the tests was 150 nanograms.
  • Figure 28a schematizes in graphic form the essay in accordance with the invention, with the corresponding elements, represented by:
  • the test according to the invention is carried out on a strip, in the form of Lateral Flow Assay, and exploits a competitive assay scheme, that is, it determines presence/absence of neutralizing anti-Spike antibodies in the sample by making them to compete with an anti-Spike monoclonal antibody labelled with an optical detection element, in the presence of a known amount of Spike protein dispersed in the test solution. If an optical signal is observed at the section of the strip to which the capture anti-Spike antibody is bound, the sample is negative, i.e., it does not contain neutralizing anti-Spike antibodies, since the labelled antibody forms a complex with the anti-Spike capture antibody and the Spike protein dispersed in the test solution, so developing an optical signal.
  • the first proposed comparative test essentially concerns a first possible combination of the competitive assay schemes known from documents WO 2020/046857 A1 and US 2012/178105 A1 as mentioned in the introductory part of this description.
  • This assay scheme essentially concerns a second possible combination of the competitive assay schemes known from WO 2020/046857 A1 and US 2012/178105 A1 as mentioned in the introductory part of this description.
  • This scheme differs from the previous one in the first phase, i.e., that of the competition of the two antibodies, i.e., the anti-Spike neutralizing antibody present in the sample under analysis and the anti-Spike monoclonal antibody labelled with an optical detection element.
  • the labelled monoclonal antibody was immobilized to the substrate by binding to the Spike protein, in turn bound to the capture antibody, in this case the labelled monoclonal antibody is dispensed at the same time as the neutralizing antibody present in the sample under analysis.
  • the solid phase here contains the Spike protein bound to the capture antibody, as shown graphically in figure 30a. In this case we start from a condition wherein the test line of each strip is not visible.
  • the methodology proposed in accordance with the invention allows to correctly determine and classify the sample under analysis, enabling identification of concentrations of neutralizing antibody even very low within the sample under analysis, unlike what happens in the comparative tests based on the aforementioned prior art documents.
  • an additional test line - exemplified with TA in Figure 24, obtained with antibodies for the capture of A- type immunoglobulins (IgA) - may be added to part 23 of strip 20 of test device 1.
  • suitable labelled antibodies with coloured or fluorescent nano-particles, or with chemiluminescence substance
  • IgA indicated with ACTA
  • the methodologies and tools described are particularly advantageous as they allow execution of the immunization test using the saliva of a subject, and therefore with a non-invasive mode.
  • the aforementioned methodologies and tools are perfectly suitable for use with a biological sample consisting of the blood of the subject subjected to verification, instead of saliva, with the same modalities: the sampling will take place in this case by a finger-pricking device or venous sampling with standard needle and syringe, and the kit will not necessarily comprise the components previously indicated with 4, 6 and 7+8.
  • an additional test line with antibodies for the capture of G-type immunoglobulins (IgG), exemplified with TG in Figure 25, may be conveniently added.
  • IgG G-type immunoglobulins
  • suitable labelled antibodies with coloured or fluorescent nano-particles, or with chemiluminescence substance
  • IgG indicated with ACTG
  • test device of the diagnostic kit with two test strips, equal and parallel to each other, which will be loaded differently.
  • Figure 26 exemplifies the case of a test device designed in this way.
  • the diagnostic kit includes at least one further container, containing lyophilized or dried Spike protein, and preferably a further container containing buffer solution.
  • These two further containers are exemplified in Figure 27, indicated by 2' and 3, and may be similar to those previously indicated with 2 and 3.
  • Container 2' contains an amount of Spike protein similar to, or substantially similar to, that of container 2.
  • Container 3' may contain a volume of buffer solution approximately corresponding to the estimated volume of the test sample (i.e., the mixture formed among the protein 2d of container 2, the dosed amount of the buffer solution 3d taken from container 3, and the dosed amount of the subject's biological sample 7d).
  • the volume of buffer solution of the container 3' can be greater, without prejudice to the possibility of dosing a fraction to be introduced into the container 2'.
  • the buffer solution of containers 3 and 3' is preferably identical.
  • windows la and lb will be used as already described above, in relation to the test sample containing the subject's biological sample (saliva or blood or serum or plasma).
  • the contents of the two additional containers 2' and 3', mixed together in a way similar to what has already been described above in relation to containers 2 and 3, but without the addition of any biological sample will be introduced in window la', constituting the reference liquid, i.e., a liquid that allows maximum intensity of visualization at the test line T (and/or TA and/or TG) in the absence of antibodies.
  • the reference liquid i.e., a liquid that allows maximum intensity of visualization at the test line T (and/or TA and/or TG) in the absence of antibodies.
  • the tools and methods object of the invention can also be used with viruses other than those exemplified above, selecting the appropriate anti receptor present on the capsid or pericapsid of the virus (the Spike protein, in the previous examples) and the appropriate receptor on the host cell membrane (hACE2, in the previous examples).
  • the capture element on the test line can be either the receptor itself (hACE2, in the previous examples) or other appropriate anti-ligand (any Anti-Spike antibody, in the previous examples).
  • the anti-receptor is supplied in lyophilized form for the immunization test.

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Abstract

Un kit d'analyse d'un état d'immunisation chez un sujet par rapport à un micro-organisme pathogène comprend : - un récipient contenant au moins un ligand cible, caractéristique du micro-organisme pathogène, le ligand cible étant sous forme séchée ou lyophilisée ; - un dispositif d'analyse à écoulement latéral doté d'un trajet d'écoulement défini par une bandelette réactive comprenant une zone de réception, une zone de conjugaison et une zone de détection. Au moins un anti-ligand marqué, spécifique du ligand cible, est immobilisé de manière réversible dans la zone de conjugaison de la bandelette réactive, et au moins un anti-ligand de capture est immobilisé de manière irréversible dans la zone de détection de la bandelette réactive. Le ligand cible est destiné à être mélangé à une solution tampon et un échantillon biologique du sujet, afin d'obtenir un échantillon d'analyse, et la bandelette réactive est configurée pour provoquer un écoulement de l'échantillon d'analyse ajouté à la zone de réception en passant par la zone de conjugaison et la zone de détection de telle sorte que : - si le ligand cible est complètement masqué par des anticorps présents dans l'échantillon biologique, pendant le passage de l'échantillon d'analyse à travers la bandelette réactive, le ligand cible ne peut former un complexe ni avec l'anti-ligand marqué, ni avec l'anti-ligand de capture, - si le ligand cible n'est pas complètement masqué par des anticorps présents dans l'échantillon biologique, pendant le passage de l'échantillon d'analyse à travers la bandelette réactive, le ligand cible forme un complexe avec l'anti-ligand marqué, en attirant le ligand marqué en son sein, et forme ensuite un complexe avec l'anti-ligand de capture, et - l'absence ou la présence éventuelle de l'anti-ligand marqué dans la zone de détection, au niveau de l'anti-ligand de capture, est optiquement détectable au niveau de la zone de détection afin de permettre de déduire ainsi des informations concernant un état d'immunisation du sujet.
EP22705903.7A 2021-02-17 2022-02-16 Kit et méthode d'analyse d'un état d'immunisation chez un sujet par rapport à un micro-organisme pathogène Pending EP4295155A1 (fr)

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IT202100003641 2021-02-17
PCT/IB2022/051384 WO2022175842A1 (fr) 2021-02-17 2022-02-16 Kit et méthode d'analyse d'un état d'immunisation chez un sujet par rapport à un micro-organisme pathogène

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CN1609617B (zh) * 2003-09-29 2012-02-15 香港中文大学 诊断与预防严重急性呼吸道综合症（sars）的组合物和方法
WO2005069002A1 (fr) * 2003-12-30 2005-07-28 Calypte Biomedical Corporation Test rapide de detection d'anticorps diriges contre le vih dans les urines
US8956859B1 (en) * 2010-08-13 2015-02-17 Aviex Technologies Llc Compositions and methods for determining successful immunization by one or more vaccines
US20120178105A1 (en) * 2011-01-10 2012-07-12 Genzyme Corporation Detection of globotriaosylceramide (glc) in human urine samples using an antibody sandwich
HUE064603T2 (hu) * 2011-11-21 2024-04-28 Zoetis Services Llc Jelerõsítés laterális áramlási és rokon immunassay-kben
US12339284B2 (en) * 2017-08-08 2025-06-24 Orasure Technologies, Inc. Assay methods for improved analyte detection
US12188053B2 (en) * 2018-08-27 2025-01-07 Vanderbilt University Human monoclonal antibodies that neutralize pandemic GII.4 noroviruses
CA3116909A1 (fr) * 2018-10-24 2020-04-30 Orasure Technologies, Inc. Dosages a ecoulement lateral pour detection differentielle d'isotypes
DE20175031T1 (de) * 2020-05-15 2021-09-09 Euroimmun Medizinische Labordiagnostika Ag Verfahren zur Bestimmung der Wirksamkeit eines SARS-CoV-2 Impfstoffs

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