WO2022175842A1

WO2022175842A1 - Kit and process for testing a state of immunization in a subject with respect to a pathogenic microorganism

Info

Publication number: WO2022175842A1
Application number: PCT/IB2022/051384
Authority: WO
Inventors: Giovanni Melioli; Marco Pizzi; Valentina GALLO; Michela SALVI
Original assignee: Eltek S.P.A.
Priority date: 2021-02-17
Filing date: 2022-02-16
Publication date: 2022-08-25
Also published as: EP4295155A1

Abstract

A kit for testing a state of immunization in a subject with respect to a pathogenic microorganism comprises: - a container containing at least one target ligand, characteristic of the pathogenic microorganism, the target ligand being in dried or lyophilized form; - a lateral flow test device with a flow path defined by a test strip which includes a receiving zone, a conjugation zone and a detection zone. At least one labelled anti-ligand, specific for the target ligand, is reversibly immobilized in the conjugation zone of the test strip, and at least one capture anti-ligand is irreversibly immobilized in the detection zone of the test strip. The target ligand is intended to be mixed with a buffer solution and a biological sample of the subject, to obtain a test sample, and test strip is configured for causing a flow of the test sample adducted to the receiving zone through the conjugation zone and the detection zone in such a way that: - if the target ligand is completely masked by antibodies present in the biological sample, during passage of the test sample through the test strip, the target ligand cannot form a complex neither with the labelled anti-ligand nor with the capture anti-ligand, - if the target ligand is not completely masked by antibodies present in the biological sample, during passage of the test sample through the test strip, the target ligand forms a complex with the labelled anti-ligand, dragging the labelled anti-ligand therewith, and subsequently forms a complex also with the capture anti-ligand, and - possible absence or presence of the labelled anti-ligand in the detection zone, at the capture anti-ligand, is optically detectable at the detection zone to enable to infer thereby information concerning a state of immunization of the subject.

Description

"Kit and process for testing a state of immunization in a subject with respect to a pathogenic microorganism"

DESCRIPTION Field of invention

The present invention refers in general to the field of testing a state of immunization of a subject with respect to a pathogenic microorganism, in particular in order to verify possible presence in that subject of antibodies which are functionally and quantitatively sufficient to make him/her immune to the microorganism concerned. The invention has been developed with particular attention being paid to the detection of a state of immunization to the SARS-CoV- 2 virus.

Background art

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). In particular, 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.

In addition, the possibility of treating patients with viruses through the antibodies of other cured donors, for example through plasma with high levels of antibodies, can be facilitated through rapid tests that enable to identify donors who have said high level of antibodies. This technique is generally little used, but its use cannot be excluded in emergency cases or when more modern and effective methods were available.

The tests of reference for verifying the state of neutralization of a virus are generally tests carried out in a laboratory, which require expert personnel and involve relatively long times, both in terms of analysis and in terms of communication of the corresponding results. Such tests are often invasive for the subject, as they are based on blood samples.

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. To further increase sensitivity, the assay has been modified by complexing the capture antibody with GL3 before adding the sample or detector antibody, providing an inhibition-based assay.

US 2005/112559 A1 discloses compositions and methods for using proteins, peptides and nucleic acids related to the SARS CoV nucleocapsid protein and the spike glycoprotein in the determination of anti-SARS CoV antibodies in a biological sample.

WO 2005/069002 A1 discloses devices and methods suitable for rapid detection of endogenous urine antibodies, particularly antibodies directed against HIV viral coat proteins.

The need for suitable solutions to simplify and speed up the testing activity in question is therefore felt.

Aim and summary of the invention

In its general terms, 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.

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. 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.

In summary, 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. In a second container of the 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. The use of viral proteins in lyophilized or dried form is extremely advantageous, as it allows them to be stored for a longer period of time and at room temperature, compared to the case of supply of viral proteins already in liquid solution.

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.

The 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.

In various preferential embodiments, 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.

In various preferential embodiments, 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.

In various preferential embodiments, 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.

In other embodiments, for chemiluminescence detection, 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. In these embodiments, thanks to the persistence of the reaction for a few minutes, and thanks to the possible integration of the signal emitted with a suitable optical system (for example the camera of a mobile phone), it is possible to have a greater detection sensitivity, that is, the possibility of seeing the viral protein even if it is present in reduced amounts. Alternatively, detection antibodies appropriately labelled for fluorescence use may also be used.

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. In particular, 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.

Another substantial advantage is that the 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. Once the mutation of the Spike protein has been identified, 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. 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.

If, as a result of the proposed test, the presence of the antigen is detected in a 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), and therefore the test is positive, it means that 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.

Otherwise, i.e., if the test is negative, it means that the subject has developed an adequate immunization against the virus, as his/her antibodies have been able to bind to the antigen in lyophilized form added to the liquid supplied, and to neutralize the virus itself making it harmless. In this case, still referring to the SARS-CoV-2 virus, the biological sample of the subject contains enough antibodies, preferably IgA antibodies, such as to occupy all the epitopes present in the RBD (. Receptor Binding Domain ), i.e., the area responsible for the binding between the Spike protein and the natural ligand hACE2 expressed on the target cells: this makes the virus harmless (neutralized) as its ability to enter the cells to replicate is lost.

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).

Brief description of the drawings

Further aims, characteristics and advantages of the invention will be clear from the detailed description that follows, made with reference to the attached drawings, provided by way of non-exhaustive example only, wherein:

- Figure 1 is a schematic perspective view of a test device that can be used in a diagnostic kit according to possible embodiments;

- Figures 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;

- Figure 6 is a schematic perspective view of a third container that can be used in a diagnostic kit according to possible embodiments;

- Figures 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;

- Figure 9 is a schematic perspective view of an absorbent stick that can be used in a diagnostic kit according to other possible embodiments;

- Figures 10 and 11 are schematic views, respectively in perspective and in section, of an additional container, in a first configuration of use, that can be used in a diagnostic kit according to possible other embodiments;

- Figure 12 is a schematic perspective view of a funnel that equips the container of Figures 10-11 in the first configuration of use;

- Figure 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;

- Figure 14 is an exploded schematic view of a test device that can be used in a diagnostic kit according to possible embodiments;

- Figure 15 is a schematic perspective view of a test strip that can be used in the device in Figure 14;

- Figures 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;

- Figure 18 is a representation intended to exemplify possible indications detectable by a test device according to possible embodiments;

- Figure 19 is a schematic perspective view of two additional containers that can be used in a diagnostic kit according to other possible embodiments;

- Figures 20 and 21 are a perspective view and a sectioned perspective view of a shooting container that can be used in combination with a test device in accordance with possible embodiments;

- Figure 22 is a perspective view of the shooting container of Figure 19, in combination with a mobile phone;

- Figure 23 is an example of possible test outcomes that can be depicted on a display of the mobile phone of Figure 22;

- Figures 24 and 25 are schematic perspectives views similar to those of Figure 15, but relating to possible variants embodiments;

- Figure 26 is a schematic perspective view of a test device of a diagnostic kit according to possible variant embodiments;

- Figure 27 is a schematic perspective view of two additional containers that can be used in a diagnostic kit according to other possible embodiments;

- Figures 28a e 28b represent, in graphic and photographic form, respectively, the execution and the result of a first test carried out in accordance with the invention;

- Figures 29a e 29b represent in graphic form the execution of a first comparative test in accordance with a first possible combination of known techniques, whereas Figure 29c represent in photographic form the result of such a test; and

- Figures 30a e 30b represent in graphic form the execution of a second comparative test in accordance with a second possible combination of known techniques, whereas Figure 30c represent in photographic form the result of such a test.

Description of preferred embodiments

Reference to an embodiment within this description indicates that a particular configuration, structure, or characteristic described in relation to the embodiment is included in at least one embodiment. Thus, phrases such as "in one embodiment", "in various embodiments" and the like, possibly present in different places of this description, are not necessarily referred to one and the same embodiment. In addition, particular conformations, structures or characteristics defined within this description may be combined in any appropriate way in one or more embodiments, even different from those depicted. The numerical and spatial references (such as "upper", "lower", "top", "bottom", etc.) as used herein are for convenience only and therefore do not define the scope of the embodiments.

Unless otherwise specified, the terms "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. Unless otherwise specified, or evident from the context, 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).

Unless otherwise specified, the term "antibody" as used hereinafter 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. Examples of antibodies are immunoglobulins, for example immunoglobulins of type A (IgA), M (IgM) or G (IgG).

Unless otherwise specified, the terms "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.

In Figures 1-8 there are represented in a schematic form some components that can be used for the implementation of the invention, according to possible embodiments thereof, preferably supplied in the form of a diagnostic kit. The components shown are disposable ones (possibly with the exception of the container indicated with 3, if this is a multi-dose container).

In various embodiments the diagnostic kit comprises a device for carrying out a lateral flow chromatographic immunological test. Such a test device, exemplified in Figure 1 and indicated as a whole with 1, can have any architecture of LFIA or LFD or LFA or LFT, and be designed to enable execution of a rapid test of the POC type. For this purpose, 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.

The diagnostic kit preferably includes a number of further components, which include pre-packaged containers, that is, in which specific substances are contained.

Referring to Figures 2 and 3, 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. In the case exemplified, 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.

Vial 2a contains a dosed amount of at least one protein that determines a specificity of the virus subject of the test. The viral protein, indicated with 2d in figure 3, is for example the Spike protein, specific for the SARS-CoV-2 virus. According to the invention, the protein 2d is in lyophilized (freeze-dried) or dried form, substantially in powder form, preferably adhered to at least one wall of the container 2, for example adhered to the bottom of the container during the freeze- drying process by means of secondary bonds on the aforementioned bottom. In container 2, an amount of protein 2d between 25 and 250 ng can be provided, preferably between 125 and 150 ng.

Referring to Figures 4 and 5, a second container is indicated with 3, preferably but not necessarily having a structure similar to the container 2. In various preferential embodiments, the container 3 is a single-dose container. In other embodiments, 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.

In various embodiments also the container 3 comprises a hermetically sealed body or vial 3a. In the example, the container 3a also has a cap 3b, for example coupled to an external thread 3c of the vial 3a. Also 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. In the container 3, 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. Note that 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.

In various embodiments, the diagnostic kit comprises a container for the collection of a biological sample, preferably a container for the collection of saliva from a subject to be subjected to immunological testing. Such a container may consist of a cup, for example of paper material, such as that exemplified in Figure 6 and designated by 4.

In various embodiments, the diagnostic kit comprises a tool for dosing a fraction of the biological sample taken from the subject. In preferential embodiments, 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.

In various embodiments, for the purpose of carrying out the test, after the removal of the cap 3b", 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). After dosing, the vial 2a can be closed using the corresponding cap 2b and shaken moderately, to suspend or solubilize the protein 2d.

Subsequently, 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.

As mentioned, in preferential embodiments, the biological sample is made up of saliva of the subject. For this case 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. Through the same 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.

After the vial 2a is reclosed by means of the relevant cap 2b (or the reclosing of the spout 2b' by means of its 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.

Of course, the type of construction of the containers 2 and 3 can be different from the one exemplified, as well as the methods of collecting the sample from the subject, and the number of pipettes used.

For example, according to a possible alternative embodiment, the kit may comprise an absorbent stick, such as the one exemplified in Figure 9, designated with reference 6, also supplied in a sterile casing. In this case, the absorbent part 6a of the stick 6 is immersed in the saliva contained in the collection cup 4, in order to absorb a dose of the saliva itself, or this absorbent part is passed on the oral walls of the subject, to soak up saliva (in which case the collection container 4 is not necessary). Afterwards, after removal of cap 3b, part 6a of the stick 6 is shaken inside the vial 3a containing the buffer solution 3d, for example in an amount of about 300 microliters. Alternatively, at least one piece of the stick including part 6a is inserted into the vial 3a: in this case the vial 3a can be closed with its cap 3b and shaken, to carry out the mixing of the saliva sample with the buffer solution.

Then, using the measuring cap 3b, a fraction (e.g., six drops, corresponding to about 200 microliters) of the contents of container 3 (buffer solution and saliva) is introduced in the container 2 containing the protein 2d, in order to obtain the test solution. After modest shaking and a subsequent waiting time (e.g., about fifteen minutes), a dosed amount of the test solution (e.g., three drops, corresponding to about 100 microliters) 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. Such a collection container can be used as an alternative to the cup 4 and the pipette 5, or to the absorbent stick 6.

Such a case is exemplified in Figures 10-13. In the example, 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. Also in this case, for the purpose of carrying out the test, after the removal of the cap 2b, the measuring cap 3b of the container 3 is used for introducing a dosed amount of the buffer solution 3d into the vial 2a of the container 2 (for example three drops, corresponding to about 100 microliters), after the corresponding cap 2b has been removed therefrom. Subsequently, the vial 2a can be closed using the corresponding cap 2b and shaken moderately, to bring into suspension or solubilize the protein 2d.

The subject can directly expel saliva from his/her mouth to the inside of the funnel element 8, so that this saliva - indicated in Figure 11 with 7d - reaches the inside of the vial 7a. Funnel element 8 is removed and vial 7a is equipped with a measuring cap 7b, as exemplified in Figure 13; this cap can be of similar construction to those previously indicated with 2b and 3b, or equipped with a spout 7b' to which a corresponding cap 7b" is associated (the structure of Figure 13 can therefore be referred to any other of the containers exemplified here, such as those indicated with 2, 3, 9a, 9b, 2', 2").

Subsequently, by using the measuring cap 7b, a dosed amount of the collected saliva (for example three drops, corresponding to about 100 microliters) are placed in the vial 2a containing the dosed amount of buffer solution 3d with the protein 2d already in suspension or solubilized, in order to obtain the test solution. Subsequently, the vial 2a can be closed by means of the corresponding cap 2b and shaken moderately. After a waiting time (for example, about 15 minutes), a dosed amount of the test solution (for example, three drops, corresponding to about 100 microliters) is fed into the test device 1.

The use of the 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).

In Figure 14 there is schematically represented in an exploded view a disposable test device 1, usable in a diagnostic kit according to the invention. Device 1 has a body preferably formed in at least two parts 10 and 11 coupled together, in particular of moulded plastic material. In the non-limiting example, the upper body part 11 defines the openings or windows la and lb, while the lower body part 10 integrally defines elements 10a, 10b for the positioning and support of a test strip, indicated as a whole with 20, as well as elements 10c for coupling to the upper body part 11. Similar positioning and support elements and similar coupling elements may be provided in the non-visible underside of the body part 11, in particular elements having a complementary shape with respect to the shape of the elements 10a, 10b and 10c.

The device 1 may in any case have any known structure in the field of LFIA or LFD or LFA or LFT type devices, or any other structure capable of enabling the carrying out of a rapid test of the POC type.

Test strip 10, visible in more detail in Figure 15, is composed of a plurality of different parts assembled together on a support (" baking "), and conceived in a known manner such that the test sample moves by capillarity from one end of the same strip to the other end. The various parts, designated by 21-24, have essentially different functions.

The end part indicated with 21, known as " sample pad ", obtains a pad to receive the test sample. This part 21 is formed with an absorbent material, for example a cellulose-based material, or a fiberglass-based material; materials that can be used for this purpose are for example those identified by the codes CFSP001700 (cellulose) or GFDX001000 (fiberglass), marketed by Merck KGaA, Darmstadt, Germany, or other similar materials.

The intermediate part indicated by 22, known as "conjugate pad", obtains a pad to which a specific antibody, or anti-ligand, is reversibly associated for a target analyte, or ligand, that is identifiable through the next part of the strip 23, on which an additional anti-ligand is irreversibly immobilized, as explained below. In the example shown, given that the part of the strip 23 includes both a test line (T) and a control line (C), in part 22 corresponding antibodies are provided, exemplified in AC_T and ACc. Part 22 also has the function of ensuring a uniform transfer of the test sample from the end 21 to the next part 23 of the strip 20; also such part 22 can be formed with a fiberglass material, such as the aforementioned GFDX 001000 marketed by Merck KGaA.

The intermediate part indicated with 23 obtains a detection membrane of the strip 20, known as " membrane test " or " detection membrane" , on which one or more lines of antibodies are provided, preferably a test line T and a control line C, as explained below. Part 23 is preferably formed from a nitrocellulose-based material, such as that identified by the code SHF 1800225 marketed by Merck KGaA.

Finally, the end part indicated with 24 obtains an absorption pad, known as an " absorption pad ' or "wicking pad' , which essentially fulfils the functions of final collection of the test sample, and which can be formed with the same material used to form the end part 21.

Parts 21-24 of strip 20 are assembled in sequence, preferably with partial overlap, on a suitable backing, formed e.g., of paper or plastic material.

As mentioned, on the nitrocellulose membrane that obtains the part 23 there is at least one test line T and, preferably, also a control line C.

In various embodiments, the test line T is obtained by immobilizing in an irreversible way on part 23 a main antibody, which represents an anti-ligand, in particular an anti-ligand specific to the viral protein initially contained in container 2 of the kit, this viral protein representing a ligand. The main antibody can be any Anti-Spike antibody, with reference to the SARS-CoV-2 virus, for example the one marketed under the code 40150-D003 or the hACE2 protein marketed under the code 10108-H08H, both from Sino Biological Inc., Beijing, China. The antibody that makes the test line T is diluted so as to maximize the difference between positive signal and background. In various embodiments, the control line C, when provided, is formed by immobilizing in an irreversible way on part 23 a secondary capture antibody, such as Goat Anti Rabbit IgG.

The antibody of the test line T and of the possible control line C are in lyophilized form and, as mentioned, associated with portion 22 in a non-reversible way.

The membrane that obtains part 23, after deposition of the capture antibodies that obtains the test line T and the possible control line C, is saturated with albumin, in order to avoid aspecific bonds. Instead of albumin, other proteins can be used: for example, a 3% powdered milk solution can be used for the same purpose.

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.

As already mentioned, on part 22 of strip 20 there is bound in a reversible way at least one antibody AC_T of the same nature as that provided for the test line T of part 23 (and therefore also representing a ligand) or referred to different epitopes but still capable of binding to the viral protein, which is labelled with coloured nano-particles, for example in gold or latex.

In addition, when the control line C is provided on part 23, a control antibody ACc is also provided on part 22, capable of binding to the control line C, labelled with coloured nano-particles, for example in gold or latex. Referring to the example indicated above, the control antibody can be, for example, the Rabbit-IgG antibody (the arrangement of the two control antibodies could be reversed compared to the example provided, i.e., with Goat Anti Rabbit IgG on portion 22 and Rabbit-IgG on portion 23).

For the purpose of performing the test, as previously indicated in relation to the different examples of kits referred to in Figures 1-13, 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. A few drops of the test sample, for example three drops, are placed on part 21 of the strip 20, one area of which is exposed through the window la of the device 1 (see for example Figure 18). The liquid then flows by capillarity for the entire length of the strip 20, up to the collection part 24.

The mechanism of operation of the test device 1, i.e., of the strip 20 thereof, is exemplified in Figure 16, in relation to the case of an immunized subject.

For such a case, 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.

As exemplified in part (B) of Figure 16, when the fluid passes through part 22, the viral protein 2d masked by the antibodies AB cannot bind to the antibody AC_T labelled with the corresponding coloured nano-particles NP, i.e., a corresponding complex cannot be formed. In the passage through part 22 the flow of the fluid carries therewith the antibody ACc labelled with the corresponding nano-particles NP.

In the subsequent passage through part 23, as exemplified in part (C) of Figure 16, the masked viral protein 2d cannot concentrate at the capture antibody of the test line T - i.e., also in this case a corresponding complex cannot be formed - and then continues up to the terminal part 24 of the strip 20. By contrast, the labelled antibody ACc concentrates at the capture antibody of the control line C, binding therewith, this concentration being optically detectable thanks to the nano particles NP of the labelled antibody ACc.

Figure 17 exemplifies the case of a non-immunized subject. For such a case, the viral protein 2d contained in the test sample TS will not be completely masked by antibodies produced by the subject, i.e., it may be completely exposed or only partially covered by such antibodies; part (A) of Figure 17 exemplifies the total absence of antibodies produced by the subject.

As exemplified in part (B) of Figure 17, when the fluid passes through part 22, 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. As in the case of Figure 16, in the passage through part 22, the flow of the fluid also carries the labelled antibody ACc labelled with the relevant NP nanoparticles therewith.

In the subsequent passage through part 23, as exemplified in part (C) of Figure 17, 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. Similarly, 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.

Within approximately five to twenty minutes from introducing the sample TS into the device 1, the 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.

In part A of Figure 18 a first case of positive test is shown, i.e., of a subject not immunized, as for Figure 17. In this case, the 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.

The non-visibility of the 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. In this case, at window lb 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.

In Part C of Figure 18 the case of a negative test is shown, i.e., of an immunized subject, in which the pathogen has been completely neutralized, as for Figure 16. In this case, only the control line C will be clearly identifiable at window lb, thanks to the coloured nano-particles NP associated with the antibodies ACc. As explained in relation to Figure 16, in this case, in fact, the viral protein 2d (target ligand), which is completely masked by antibodies AB, will not be able to be captured by the antibodies (anti-ligand) at the test line T, and will concentrate in an area of the strip 20 not exposed at window lb of the device 1 (i.e., at the final part 24 of the strip 20).

Finally, 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.

Since it has been shown that one of the mechanisms of inhibition of the binding of the viral proteins to the target cells is mediated by anti-receptor binding domain antibodies, 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.

Another advantage lies in the fact that the steric hindrance determined by the antibodies of the natural polyclonal response also inhibits the binding of the second detection antibody or anti-ligand, i.e., the antibody AC_T labelled with coloured or fluorescent nano-particles, or labelled with substance for chemiluminescence, determining a further inhibition of the signal. The mechanisms just described significantly increase the sensitivity of the proposed method, without affecting specificity. A non-exclusively specific immune response is effective for protection purposes and can only be detected with this type of approach, unlike where Spike fragments corresponding to the epitope that binds to hACE2 are used.

As previously indicated, in possible embodiments, the detection of the outcome of the test can be emphasized by exploiting effects of chemiluminescence (or fluorescence). In such embodiments, 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. In addition, in this case, 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).

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). For example, 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.

Subsequently, in a suitable opening of device 1 (for example the same window lb) 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.

Referring to the examples of used substances, 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.

In order to facilitate detection and make it more sensitive, in terms of possibility of detecting even a minimal presence of the viral protein (for example in the case of a subject who is not sufficiently immune, as in the case of part (B) of Figure 18), or for the purposes of remote diagnostics, 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.

Referring to Figure 20, 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. In the non-limiting example, 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.

As can be seen from Figure 21, in the example, 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.

Of course, 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; viii) the drawer 32 is then closed quickly, so as to bring window lb into alignment with the shooting window 33 of the shooting container 30; ix) on the mobile phone TC the start of the shooting by the dedicated application is controlled; x) after the time required for shooting (approximately 10 seconds), on the display TCd of the phone TC the image related to the test is obtained.

An example of possible graphic representations present on the aforementioned display TCd is visible in schematic form in Figure 23, wherein the parts (A) - (D) correspond, as regards the result of the test, to those of the parts (A) - (D), respectively, of Figure 18.

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.

It will be appreciated that 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.

The characteristics of the present invention are clear from the given description, as are its advantages.

In accordance with the invention, a kit is provided for testing a state of immunization in a subject with respect to a pathogenic microorganism, comprising:

- a container containing at least one target ligand characteristic of the pathogenic micro-organism, the target ligand being in dried or lyophilized form;

- a lateral flow test device with at least one flow path defined by at least one first test strip which includes at least a receiving zone, a conjugation zone and a detection zone, wherein at least one labelled anti-ligand, specific for the target ligand, is reversibly immobilized in the conjugation zone of the first test strip, and wherein at least one capture anti-ligand is irreversibly immobilized in the detection zone of the first test strip.

In the use of said kit, 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.

With the described configuration, during passage of the test sample through the first test strip:

- if the target ligand is completely masked by antibodies present in the biological sample, the target ligand cannot form a complex neither with the labelled anti-ligand nor with the capture anti-ligand,

- if the target ligand is not completely masked by antibodies present in the biological sample, the target ligand forms a complex with the labelled anti-ligand, dragging the labelled anti-ligand therewith, and subsequently forms a complex also with the capture anti -ligand, and

- possible absence or presence of the labelled anti-ligand in the detection zone, at the capture anti-ligand, is optically detectable at the detection zone, to enable to infer thereby information concerning a state of immunization of the subject.

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.

It should be emphasized that the execution of the diagnostic test according to the preferential way (i.e., using saliva as a biological sample, instead of blood), allows to identify the presence of an immune response where this response is most useful - that is, at the oral mucous membranes - for the prevention of an infection of the respiratory tract.

As mentioned, the proposed test is of a functional type and, unlike the binding tests based on RBD and Anti-RBD antibodies, it also allows to detect correct functioning in the ability of antibodies to inhibit the bond between the ligand represented by the viral protein and the anti -ligand represented by the capture antibody, and therefore to evaluate the degree of neutralization of the viral agent.

In order to verify the effectiveness of the methodology in accordance with the invention, Applicant carried out comparative analyses with respect to the current state of the art.

The tests were carried out using four plasma samples containing different concentrations of anti-Spike neutralizing antibody. Said concentrations, identified through previous tests, are equivalent to the concentration of antibody considered inhibiting ("high positive"), to two intermediate concentrations ("medium positive" and "low positive") and one negative ("negative"). More specifically, the concentrations used were as follows:

- high positive: 1 microgram/10 microliters of plasma, - medium positive: 0.063 micrograms/10 microliters of plasma,

- low positive: 0.0156 micrograms/10 microliters of plasma, and

- negative: 0 micrograms/10 microliters of plasma.

The amount of Spike protein used in the tests was 150 nanograms. TEST

Figure 28a schematizes in graphic form the essay in accordance with the invention, with the corresponding elements, represented by:

/ i - the anti-Spike antibody possibly present in the sample,

¨ - the Spike protein added, in a given amount, to the sample to be analysed,

- the anti-Spike monoclonal antibody labelled with an optical detection element, and

Y

- the anti-Spike monoclonal capture antibody immobilized on the substrate (i.e., the test strip). The same graphic symbology as shown in Figure 28a is also used in relation to the further tests proposed below.

As seen above, 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. Conversely, if an optical signal is not observed in the section of the strip to which the capture anti- Spike antibody is bound, the sample is positive, i.e., the neutralizing anti-Spike antibody of the sample has bound to the complex anti-Spike capture antibody + Spike protein, and the labelled anti-Spike monoclonal antibody flowed into the final section of the strip. The proposed use of the Spike protein in free form and its consequent better steric accessibility enable correct (semi -quantitative) determination of the neutralizing antibody present in the sample even at (very) low concentrations. This conclusion is confirmed by the result of the test as represented in Figure 28b, where the numbers 1-4 assigned to the test strips correspond to the respective samples in the aforementioned concentrations of "high positive", "medium positive", "low positive" and "negative", respectively.

As can be clearly seen from the image in figure 28b, starting from the four equal test strips, the increasing presence of anti-Spike neutralizing antibodies in the samples under analysis is directly proportional to the decrease in the optical signal present.

COMPARATIVE TEST 1

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.

In order to implement this comparative assay scheme, the Spike protein and the labelled monoclonal detection antibody were immobilized on the substrate (the strips) by means of the capture antibody, as schematized graphically in Figure 29a. Therefore, in this case, we start from a condition wherein the test line of each strip is already evident.

On the four strips 1-4 thus prepared, equal to each other, the respective four samples were flowed, with the four concentrations indicated above of anti -Spike neutralizing antibodies (high positive, medium positive, low positive and negative), to verify the strength thereof to remove the Spike protein and/or the labelled antibody bound therewith, and then possibly make the test line disappear. This phase is represented graphically in Figure 29b.

As shown in the image of Figure 29c, no concentration of anti-Spike neutralizing antibody was able to compete with the labelled antibody bound to the Spike protein, not even that concentration (high positive) which - in the test according to the invention - is completely inhibiting the bond. Therefore, as can be seen, this combination of techniques known from WO 2020/046857 A1 and US 2012/178105 A1 does not allow to get a working test device.

COMPARATIVE TEST 2

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. While in the previous comparative test 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. Hence, 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.

Also in this case, on the four strips 1-4 thus prepared, equal to each other, the respective four samples were flowed with the four concentrations indicated above of anti-Spike neutralizing antibody (high positive, medium positive, low positive and negative) together with the labelled monoclonal antibody, making these two antibodies compete for the binding to the immobilized Spike protein. This phase is represented graphically in figure 30b.

As illustrated in the image shown in figure 30c, in this case only the highest concentration of anti-Spike antibody, that is, 1 - high positive (i.e., the concentration that, in the test according to the invention, is the only one totally inhibiting the signal), is able to compete, however only partially, with the labelled monoclonal antibody. The test line, despite the high concentration of the anti-Spike antibody contained in the sample, does not disappear completely, but still remains visible. This clearly indicates a low sensitivity of this assay scheme to the detection of anti- Spike neutralizing antibody present in the sample under analysis, compared to the test carried out according to the invention, and therefore the impossibility of a diagnostic use thereof.

As can be seen from the tests referred to in figures 28-30, 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.

It is clear that numerous variants are possible for the person skilled in the art to the devices, kits, process and equipment described as an example, without departing from the scope of the invention as defined by the claims that follow.

In the case of use of saliva as a biological sample, 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.

In this case, in the conjugation part 22, suitable labelled antibodies (with coloured or fluorescent nano-particles, or with chemiluminescence substance) for IgA, indicated with ACTA, will be reversibly associated for possible visualization at the additional test line TA. In such an application, the following cases may occur:

- line T visible, line TA not visible, line C visible: the subject is not immunized;

- line T visible, line TA visible, line C visible: the subject does not have "humoral" mechanisms (IgG, IgA, IgM) available, able to completely inhibit binding of the Spike protein to its antibody; the subject is anyway immunized and could be partially protected; in this case both lines T and TA will be weaker than the control line C; on the test line TA for IgA both the free IgA (in this case very few) and the Spike covered with IgA will bind;

- line T not visible, line TA visible, line C visible: the subject is immunized, although it is not possible to exclude presence of the virus or products thereof in the saliva;

- T line not visible, TA line not visible, line C visible: the subject is immunized but with an incomplete immune response; this eventuality is remote and it would be advisable to repeat the test after a certain time to be established, approximately a few days;

- line T visible or not visible, line TA visible or not visible, line C not visible: the test is invalid and must be repeated with a new kit.

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. However, it should be noted that 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.

In the case of use of the kit with blood, or serum, or plasma, in part 23 of the strip 20 an additional test line with antibodies for the capture of G-type immunoglobulins (IgG), exemplified with TG in Figure 25, may be conveniently added. In this case, in the conjugation part 22, suitable labelled antibodies (with coloured or fluorescent nano-particles, or with chemiluminescence substance) for IgG, indicated with ACTG, will be associated in a reversible way, for possible visualization at the further test line TG. In such an application, the following cases may occur:

- line T visible, line TG not visible, line C visible: the subject is not immunized;

- line T visible, line TG visible, line C visible: the patient does not have "humoral" mechanisms (IgG, IgA, IgM) available able to completely inhibit binding of the Spike protein to its antibody; the subject is anyway immunized and could be partially protected; in this case both lines T and TG will be weaker than the control line C; on the test line TG for IgG both the free IgG (in this case very few) and the Spike covered with IgG will be bound;

- line T not visible, line TG visible, line C visible: the subject is immunized;

- line T not visible, TG line not visible, line C visible: the subject is immunized but with an incomplete immune response; this eventuality is remote and it would be advisable to repeat the test after a certain time to be established, approximately a few days;

- line T visible or not visible, line TG visible or not visible, line C not visible: the test is invalid and must be repeated with a new kit.

To make the test according to the invention more quantitative, it may be preferable to introduce a reference. One possibility in this sense is to prearrange the 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.

In this case, the device 1 includes, in addition to windows la and lb already described, two additional windows la' and lb', respectively for the introduction of a reference liquid and for a reference visualization. The structure of the device 1 in Figure 26 is substantially similar to that shown in Figure 4, but with the difference that, in this case, in addition to the two additional windows la' and lb', the inside of the device 1 will house two strips 20 and 20' substantially identical, in positions substantially parallel to each other, and arranged in such a way that an area of part 21 of each strip 20, 20' is at opening la and la', respectively, and an area of part 23 of each strip 20, 20' is at window lb and lb', respectively.

In this case, 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). Obviously, 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.

In use, 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). On the other hand, 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. From the optical comparison between the portions of parts 23 of the two strips 20 and 20' that are visible at the two windows lb and lb' it is thus possible to infer information relating to the rate of antibodies of the subject, compared to the case of total absence of antibodies.

By increasing the concentration of the inhibitor, the inhibiting concentration in each laboratory can be evaluated directly and analytically, to customize the analytical conditions.

The operating modes of the test device G are similar to those previously exemplified also in relation to the type of labelling used (coloured nano-particles, or chemiluminescence substance or fluorescence substance). In the case of chemiluminescence or fluorescence labelling, the kit including the device G may, if necessary, include two containers 9a for the development liquid, and possibly two containers 9b for the washing liquid.

Finally, it will be appreciated that, although preferable, not all the containers indicated must necessarily be part of the diagnostic kit, and this applies in particular to the "auxiliary" liquids that could be dosed according to the needs of the test starting from commercial flacons or bottles. In this perspective, therefore, the containers 3 and/or 3' for the buffer solution and the containers 9a and/or 9b for the development liquid and the washing liquid, respectively, might not be included in the kit. It is also clear that for some implementations of the test process described, it is not strictly necessary to include in the kit a container for the collection of the patient's biological sample, since other sterile containers normally available in the medical field can be used for this purpose. As mentioned, 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.

Claims

1. A kit for testing a state of immunization in a subject with respect to a pathogenic microorganism, comprising:

- a container (2) containing at least one target ligand (2d), characteristic of the pathogenic microorganism, the target ligand (2d) being in dried or lyophilized form, the target ligand (2d) being intended to be mixed with a buffer solution (3d) and a dose of a biological sample (7f) of the subject, to obtain a test sample (TS);

- a lateral flow test device (1; G) with at least one flow path defined by at least one first test strip (20) which includes at least a receiving zone (21), a conjugation zone (22) and a detection zone (23); wherein at least one labelled anti-ligand (AC_T), specific for the target ligand, is reversibly immobilized in the conjugation zone (22) of the first test strip (20), and wherein at least one capture anti-ligand (T) is irreversibly immobilized in the detection zone (23) of the first test strip (20); wherein the first test strip (20) is configured for causing a flow of the test sample (TS) adducted to the receiving zone (21) through the conjugation zone (22) and the detection zone (23), in such a way that:

- if the target ligand (2d) is completely masked by antibodies (AB) present in the biological sample (7d), during passage of the test sample (TS) through the first test strip (20), the target ligand (2d) cannot form a complex neither with the labelled anti-ligand (AC_T) nor with the capture anti-ligand (T),

- if the target ligand (2d) is not completely masked by antibodies (AB) present in the biological sample (7d), during passage of the test sample (TS) through the first test strip (20), the target ligand (2d) forms a complex with the labelled anti ligand (AC_T), dragging the labelled anti-ligand (AC_T) therewith, and subsequently forms a complex also with the capture anti-ligand (T), and

- possible absence or presence of the labelled anti-ligand (AC_T) in the detection zone (23), at the capture anti-ligand (T), is optically detectable at the detection zone (23) to enable to infer thereby information concerning a state of immunization of the subject.

2. The kit according to Claim 1, wherein the target ligand (2d) is a viral protein, in particular Spike protein, the labelled anti-ligand (AC_T) is an antibody, in particular an Anti-Spike antibody, and the capture anti-ligand (T) is an antibody, in particular an Anti-Spike antibody or hACE2 protein.

3. The kit according to Claim 1 or Claim 2, comprising at least one of the following:

- a container (3) of the buffer solution (3d);

- a collection container (4; 7), for collecting the biological sample (7d) of the subject;

- a tool (5) for dosing the biological sample (7 a) of the subject;

- a tool (6) for taking the biological sample from the oral cavity of the subject;

- a measuring cap (2b, 3b, 7b) associated or associatable with a body (2a, 3a) of at least one container (2, 3, 2’, 3’, 7, 9a, 9b) included in the kit;

- a funnel-shaped element (8) associated or removably associatable with a collection container (7) of the biological sample (7d).

4. The kit according to any one of Claims 1-3, wherein the labelled anti ligand (ACT) is labelled with coloured nano-particles, in particular in gold or in latex or fluorescent, or the labelled anti-ligand (AC_T) is labelled with a substance suitable for developing a chemiluminescence reaction.

5. The kit according to Claim 4, wherein the labelled anti -ligand (AC_T) is labelled by means of a substance suitable for developing a chemiluminescence reaction, and the kit comprises at least one container (9a) of a liquid for developing the reaction of chemiluminescence, and optionally at least one container (9b) of a liquid for washing the first test strip (20).

6. The kit according to any one of Claims 1-5, wherein:

- in the conjugation zone (22) of the first test strip (20) at least one of a labelled control antibody (ACc), a labelled antibody for IgA (ACTA), a labelled antibody for IgG (AC_TG) is also reversibly immobilized, and

- in the detection zone (23) of the first test strip (20) at least one of a control capture antibody (C), a capture antibody for IgA (TA), a capture antibody for IgG (TG), respectively, is also irreversibly immobilized.

7. The kit according to any one of Claims 1-6, wherein the test device (G) comprises at least one second test strip (20’) substantially identical to the first test strip (20), and the kit comprises a further container (2’) containing a respective quantity of the target ligand (2d), in dried or lyophilized form, and preferably a further container (3’) containing a respective quantity of the buffer solution (3d).

8. A process for in vitro testing an immunization state in a subject with respect to a pathogenic microorganism, comprising the steps of: i) providing a dose of at least one target ligand (2d), characteristic of the pathogenic microorganism, the target ligand (2d) being in dried or lyophilized form; ii) providing a dose of a buffer solution (3d); iii) provide a biological sample (7d) from the subject; iv) mixing together the dose of target ligand (2d), the dose of buffer solution (3d) and a dose of the biological sample (7f) to obtain a test sample (TS); v) providing a lateral flow test device (1) with at least one flow path defined by at least one first test strip (20) which includes at least a receiving zone (21), a conjugation zone (22) and a detection zone (23), wherein in the conjugation zone (22) at least one labelled anti-ligand (AC_T), specific for the target ligand (2d), is reversibly immobilized, and wherein in the detection zone (23) at least one capture anti-ligand (T) is irreversibly immobilized; vi) supplying a dose of the test sample (TS) to the receiving zone (21) of the first test strip (20), to cause a relative flow of the dose of the test sample (TS) through the conjugation zone (22) and the detection zone (23), in such a way that:

- if the target ligand (2d) is completely masked by antibodies (AB) present in the biological sample (7d), during passage of the dose of the test sample (TS) through the first test strip (20), the target ligand (2d) cannot form a complex neither with the labelled anti-ligand (AC_T) nor with the capture anti-ligand (T),

- if the target ligand (2d) is not completely masked by antibodies (AB) present in the biological sample (7d), during passage of the dose of the test sample (TS) through the first test strip (20), the target ligand (2d) forms a complex with the labelled anti-ligand (AC_T), dragging the labelled anti-ligand therewith, and subsequently forms a complex also with the capture anti-ligand (T); vii) checking optically in the detection zone (23) possible absence or presence of the labelled anti -ligand (AC_T) at the capture anti-ligand (T), and thereby inferring information concerning a state of immunization of the subject.

9. The process according to Claim 8, wherein the target ligand (2d) is a viral protein, in particular Spike protein, the labelled anti-ligand (AC_T) is an antibody, in particular an Anti-Spike antibody, and the capture anti-ligand (T) is an antibody, in particular an Anti-Spike antibody or hACE2 protein.

10. The process according to Claim 8 or Claim 9, wherein the biological sample (7d) is saliva of the subject.

11. The process according to claim 8 or claim 9, wherein the biological sample (7d) is blood or plasma or serum of the subject.

12. The process according to any one of Claims 8-11, wherein: - in the conjugation zone (22) of the first test strip (20) at least one of a labelled control antibody (ACc), a labelled antibody for IgA (ACTA), a labelled antibody for IgG (AC_TG) is also reversibly immobilized;

- in the detection zone (23) of the first test strip (20), at least one of a control capture antibody (C), a capture antibody for IgA (TA), a capture antibody for IgG (TG), respectively, is also irreversibly immobilized;

- step vii) comprises optically checking in the detection zone (23) possible absence or presence of at least one of the labelled control antibody (ACc), the labelled antibody for IgA (ACTA), the labelled antibody for IgG (ACTG) at the at least one of the control capture antibody (C), the capture antibody for IgA (TA), the capture antibody for IgG (TG), respectively, and thereby inferring information concerning testing invalidity, in case of non-presence of the labelled control antibody (ACc), and further information concerning the state of immunization of the subject, in case of presence or absence of the labelled antibody for IgA (ACTA) or of the labelled antibody for IgG (ACTG).

13. The process according to any one of Claims 8-12, wherein the test device (G) has a further flow path defined by a second test strip (20’) substantially identical to the first test strip (20), and the process further includes the steps of:

- providing a further dose of the at least one target ligand (2d) in dried or lyophilized form;

- providing a further dose of buffer solution (3d);

- mixing together the further dose of target ligand (2d) and the further dose of buffer solution (3d) to obtain a reference sample;

- supplying a dose of the reference sample to the receiving zone (21) of the second test strip (20’), to cause a relative flow of the dose of the reference sample (TS) through the respective conjugation zone (22) and detection zone (23);

- optically comparing a portion of the detection zone (23) of the first test strip (20), at which the relating capture anti-ligand (T) is irreversibly immobilized, with a corresponding portion of the detection zone (23) of the second test strip (20’), at which the relating capture anti-ligand (T) is irreversibly immobilized, to infer information concerning the quantity of antibodies possibly present in the biological sample (7d).

14. The process according to any one of Claims 8-13, wherein the labelled anti-ligand (AC_T) is labelled with coloured nano-particles, particularly in gold or latex, or fluorescent particles, or: - the labelled anti-ligand (AC_T) is labelled with a substance suitable for developing a chemiluminescence reaction, and

- before step vii), at least at the detection zone (23) of the first test strip (20), a development liquid of the chemiluminescence reaction is added, preferably after the addition of a washing liquid in the receiving zone (21) of the first test strip (20).

15. The process according to any one of Claims 8-14, wherein step vii) is carried out by means of a shooting equipment (30), preferably a shooting equipment configured for use in combination with a mobile phone (TC) equipped with a camera and a dedicated software application.