WO2024013713A1

WO2024013713A1 - Point-of-care device for measuring thyroid function regulatory compounds in a biological fluid

Info

Publication number: WO2024013713A1
Application number: PCT/IB2023/057225
Authority: WO
Inventors: Vincenzo PIEMONTE; Tamara BOSCARINO; Andrea PALERMO; Anda Mihaela NACIU; Gaia TABACCO
Original assignee: Università Campus Bio-Medico Di Roma
Priority date: 2022-07-14
Filing date: 2023-07-14
Publication date: 2024-01-18

Abstract

The present invention relates to the field of the point-of-care devices and, in particular, it relates to a new lateral flow device and a reading device of a chromatographic strip, to detect and/or quantify one or more thyroid function regulatory compounds, in particular thyroid stimulating hormone and calcium, in a biological fluid of a subject. The invention also relates to kits comprising the above-mentioned devices, as well as in vitro uses and methods providing the use thereof.

Description

"Point-of-Care device for measuring thyroid function regulatory compounds in a biological fluid"

FIELD OF THE INVENTION

The present invention relates to the field of the point-of-care devices and, in particular, it relates to a new lateral flow device and a reading device of a chromatographic strip, to detect and/or quantify one or more thyroid function regulatory compounds, in particular thyroid stimulating hormone and calcium, in a biological fluid of a subject. The present invention also relates to kits comprising the above-mentioned devices, as well as in vitro uses and methods providing the use thereof.

STATE OF ART

The development of point-of-care testing (POCT) devices has seen rapid growth over the past few years, by facilitating the execution of diagnostic tests in a short time, allowing to monitor continuously and effectively pathologies even in domestic field, and relieving the health care system of services which often require a considerable use of time and resources with qualified personnel. In fact, the simplicity of use, the reduced cost and the absence of complex equipment makes such devices suitable also to the direct use by patients in the home environment.

Over the last years, most efforts have focused on improving the technologies underlying the POCTs with the purpose of increasing sensitivity and specificity thereof.

Although the medical science has evolved to render the POCT devices a fundamental resource in the medical landscape, today there are still different pathologies therefor effective portable diagnostic systems are not available, especially due to a reduced analytical sensitivity. In particular, the patients suffering from thyroid dysfunctions, such as hypothyroidism, hyperthyroidism, hypocalcaemia or hypercalcaemia do not use yet portable devices for measuring online the thyroid stimulating hormone (TSH) and/or calcium. This is caused by the fact that most methods and devices currently present in the field requires a high analytical sensitivity, in antithesis with the portability concept, or once met the portability condition, it has a low analytical sensitivity affecting the detection accuracy. None of the technologies currently existing for diagnosing thyroid dysfunctions has simultaneously the following features:

1. Portability;

2. Execution of a quantitative analysis;

3. Possibility of simultaneous quantification of both pathological conditions, hypothyroidism and hyperthyroidism for thyroid and hypocalcaemia and hypercalcaemia for parathyroids, with a good analytical sensitivity capable of discriminating between the two cuts-off,

4. Timeliness in the use of results;

5. Annual follow-up, that is the use of one single device for the average duration of the post-surgery disease (1 year).

In such context, then, the need remains evident for developing new methods and commercial devices which provide a possibility of rapid diagnosis and reliable and which can be used in home environment from personnel not expert for diagnosing and monitoring thyroid dysfunctions.

SUMMARY OF THE INVENTION

The present invention aims at providing a device of portable point-of-care type to detect and/or quantify regulatory compounds of the thyroid function in a biological sample of a subject and to diagnose or monitor thyroid dysfunctions, allowing to obviate the problems found in the known art, with particular reference to the reduced portability of the devices currently used in the field.

The authors of the present invention have developed two devices which can advantageously be used in combination so as to provide a quali-quantitative analysis of thyroid function regulatory compounds of interest in a biological fluid of a subject, in particular in a blood sample, allowing to assist patients suffering from thyroid dysfunctions, in particular hypothyroidism/hyperthyroidism and hypercalcaemia/hypocalcaemia, both in subclinical and established form.

As it will be better illustrated in details hereinafter in the detailed description, a first device object of the present invention is represented by a lateral flow device (lateral flow assay, LFA) preloaded with a chromatographic strip configured to allow the detection and/or quantification of one or more thyroid function regulatory compounds within a biological fluid of a subject, in particular the thyroid stimulating hormone and calcium. The spread by capillarity of the biological fluid containing the compounds of interest within the chromatographic strip of the lateral flow device object of the invention determines the formation of an immune complex with said one or more thyroid function regulatory compounds at a specific capture area of the strip. The present invention provides that the regulatory compounds of interest migrated through la chromatographic strip towards the specific capture area, as well as immune complexes thereof which in case have formed in this area, could be detected and/or quantified easily by coupling lateral flow device to the second device developed by the authors of the invention.

The latter is represented, specifically, by a reading device configured to detect and/or quantify one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof adsorbed and/or immobilized on a chromatographic strip.

To this purpose, the lateral flow device developed by the authors of the present invention is characterized by a first and a second casing which can be coupled to each other in a removable manner so as to form a cassette which houses the chromatographic strip, and has a lateral slot through which it is possible to expel the strip at the end of the use of the device. A casing of the lateral flow device, in fact, is provided with expulsion means, in particular a spring system, which can be activated at the end of the assay execution to cause the expulsion of the portion of the strip carrying the compound or its immune complex to be analysed, through la lateral slot.

According to the present invention, the strip portion expelled at the end of the use of the lateral flow device can be inserted inside the reading device through a corresponding inlet slot, as illustrated by pure way of example in Figure 4.

The reading device developed by the authors of the invention is characterized by a main body which can be coupled to a support element in the form of a circular crown, which carries a plurality of analysis wells inside of which a solution of a substance can be housed which is capable of reacting with the compounds of interest or immune complexes thereof adsorbed and/or immobilized on the chromatographic strip previously subjected to analysis by means of the lateral flow device, determining the formation of at least a detectable and/or quantifiable product, for example by spectrophotometric or electrochemical route. The presence of suitable cutting means, for example a blade, at the inlet slot of the reading device, allows to cut the chromatographic strip near the end stop, that is to cut the strip portion penetrated inside the slot and carrying the capture area on which the compounds of interest to be detected and/or quantified are adsorbed and/or immobilized, and then to let it to fall into an analysis well. The cutting can be performed by rotating the upper closing element of the reading device by the patient, which is directly connected to a cutting device.

The fall of the strip to be analysed into the well of the reading device determines the desorption and dissolution and/or reaction of the compounds of interest, or of immune complexes thereof, adsorbed and/or immobilized on the strip with the substance present in the solution inside the well. The reaction with the substance present in the well determines the formation of one reaction product which can be suitably revealed and/or quantified by suitable incorporated reading means of the reading device itself. The detection and/or quantification of the compounds of interest into a well of the reading device can be performed, in particular, by spectrophotometric or electrochemical route.

In a first embodiment of the reading device according to the invention, illustrated by way of examples in figures 4-6, the presence of a spectrophotometer, with absorption peak at 690nm, near the well under analysis allows the optical detection of the product which develops in the solution.

In a second embodiment of the reading device according to the invention, illustrated by way of examples in figures 7-8, the presence of a conductivity meter, consisting of control electronics, allows to detect in solution several ions and their differentiation based upon the charge. In order to proceed with the electrochemical analysis of each sample, the reading device can advantageously comprise an electrode for each well present on the circular crown, connected through connectors to the control unit.

The measurements performed with the reading device can be sent to an external interface which allows the display thereof by the patient. In the same way, the motion of the closing element of the reading device, which can be performed manually by the patient as mentioned above, guarantees the availability of a new well for a subsequent analysis.

According to a preferred aspect of the invention, the lateral flow device and the reading device developed by the authors of the invention in particular allow to carry out, simultaneously, a quantitative determination of the thyroid stimulating hormone and of calcium in a blood sample of a subject. To this purpose, the lateral flow device object of the invention can incorporate a chromatographic strip which allows to execute an assay of ELISA Sandwich type on porous membrane, by using specific monoclonal antibodies conjugated to horseradish peroxidase (HRP) as detection agents. The immune complex formed between TSH and monoclonal antibody conjugated to HRP can be analysed, by spectrophotometric or electrochemical route, by means of the reading device object of the invention.

To this purpose, each well of the reading device can comprise a solution of a chromogenic substrate, the 3,3’,5,5’-tetramethylbenzidine (TMB), capable of reacting with HRP which catalyses the oxidation thereof by determining the formation of one product detectable by spectrophotometric or electrochemical route. The calcium ion, instead, since it is not subjected to any reaction on the porous matrix of the chromatographic strip due to its picometric sizes which allow it to reach easily the end stop, and then the capture area of the strip, can be detected simultaneously with respect to TSH. In fact, since the second target analyte (Ca++) is on the same porous matrix which precipitates inside the analysis well, the calcium quantification can be performed simultaneously with respect to that of TSH by electrochemical detection of the cation directly in the solution present in the well of the reading device.

The mechanical coupling of the lateral flow device to the reading device which performs the spectrophotometric or electrochemical measurement of the compounds or their immune complexes of interest, adsorbed or immobilized on the strip, allows then to quantify simultaneously several analytes, such as TSH and calcium, and to monitor at the same time several thyroid dysfunctions. The system of the two devices object of the present invention for example allows to quantify simultaneously both pathological conditions, hypothyroidism and hyperthyroidism (for thyroid) and hypocalcaemia and hypercalcaemia (for parathyroids), with a good analytical sensitivity capable of discriminating between the two cuts-off.

For the first time with respect to the technologies already present on the market, the combination of the two devices developed by the authors of the present invention then allows a simultaneous measurement of the two limit conditions of thyroid and parathyroid dysfunction. The devices, in fact, allow to measure quantitatively the concentration of thyroid stimulating hormone and blood calcium with the purpose of detecting hypo-hyperthyroidism and hypo-hypercalcaemia conditions which can lead to significative symptoms and, in the worst case, to serious pathologies. The triggering factors of subclinical thyroiditis and parathyroiditis are familiarity and, still more often, the post-operative period. In the year or in the two years following thyroidectomy and parathyroidectomy, the patients are subjected to hormone replacement therapy (HRT) and more frequent checks of their blood levels. In such context, the devices object of the invention advantageously allow to assist the patient during the whole duration of the pathology, by providing a clear clinical picture to the physician about the variational trends of hormone and ion levels in blood.

The use of the two devices object of the invention allows to obtain an optimum timeliness in the use of results, equal to about 20-30 minutes. Such timeliness is determined by the reaction time between the immune complex and the substrate present in the solution inside the wells of the reading device, and by the software processing of the piece of data coming from the reading means (estimated at about 10-15 min).

In a preferred embodiment of the reading device object of the present invention, this carries a circular crown containing exactly 25 wells for the fluid analysis. This allows to a patient requiring it to carry out a measurement, of the levels of the compounds of interest, by using the reading device of the invention, every 20 days for 1 year, for a total of about 18 measurements, a target widely fulfilled by the device.

Advantageously, the reading device can be completed by a front-end and back-end application to offer the patient or the clinician who cares for the patient, after authorization to consult the data in compliance with current privacy legislation, to consult the acquired data through graphic interfaces and summary and detailed representations of information.

The devices developed by the authors of the invention can be also used in combination with specific laboratory analyses and, thanks to their portability and operational simplicity, can be used autonomously by a patient requiring them or under the supervision of a physician or a hospital facility. Moreover, the devices can be used in care homes and hospitals to monitor the intake of thyrotropic hormones and synthetic calcium of the inpatients. For this reason, the present invention is of particular interest for the hospital and pharmaceutical sector.

Then, the objects of the present invention are:

1) a reading device of a chromatographic strip, the device being configured to detect and/or quantify one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof adsorbed and/or immobilized on a chromatographic strip, comprising:

- a main body, substantially hemispherical in shape, comprising a base which defines a horizontal resting plane P of the device;

- a support element, in the form of a circular crown, integral with the main body; which support element carries a plurality of wells adjacent to each other, each well being able to contain a solution of a substance capable of reacting with said one or more thyroid function regulatory compounds and/or immune complexes thereof so as to form at least a detectable and/or quantifiable product;

- a closing element, substantially hemispherical in shape, which can be coupled to said support element revolvingly to rotate around a vertical axis V orthogonal to the resting plane P under the manual action of a user; said closing element being couplable removably to said support element in such a way as to allow the closure of the reading device and form an inlet slot of the reading device suitable for receiving a portion of the strip carrying said one or more regulatory compounds and/or immune complexes thereof;

- cutting means associated with an internal surface of said closing element facing at the inlet slot of the reading device when said closing element is coupled to said support element; and - reading means configured to detect and/or quantify said one or more regulatory compounds and/or immune complexes thereof and/or said at least a detectable and/or quantifiable product; characterized in that, when said portion of the strip is inserted into the inlet slot of the reading device 100, the manual rotation of the closing element by the user allows cutting said portion of the strip through said cutting means so as to let said portion of the strip 1 to fall into a well 12’ of said plurality of wells 12. ) a lateral flow device to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject, said device comprising:

- a chromatographic strip carrying a first end and a second end and comprising:

(i) a loading area of said biological fluid;

(ii) a first capture area comprising a detection agent capable of forming a first immune complex with said one or more thyroid function regulatory compounds; and

(iii) a second capture area comprising a capture agent capable of forming a second immune complex with said first immune complex; wherein said first capture area is localized between said loading area and said second capture area; and

- a cassette comprising a first casing and a second casing coupled to each other in a removable manner so as to limit an internal cavity of the cassette which houses said chromatographic strip and so as to define a lateral slot of the cassette suitable for allowing, at the end of the use of the lateral flow device, the expulsion of a portion of said strip bearing said second capture area from the internal cavity of the cassette towards the outside; wherein said first casing comprises expulsion means associated with an internal surface of said first casing facing inside the cassette, configured to cause the expulsion of said portion of the strip from the internal cavity of the cassette towards the outside through said lateral slot; and wherein said second casing comprises a first opening to allow contacting the biological fluid to be analysed with said loading area of the strip, and a second opening, adjacent to the first opening, to allow to display said first and/or second capture area of the strip;

3) a Kit to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject, which kit comprises a reading device and a lateral flow device according to any one of the herein described embodiments;

4) the in vitro use of a reading device and a lateral flow device or a kit according to any one of the herein described embodiments, to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject and/or for the diagnosis of a thyroid dysfunction in said subject; and

5) an in vitro method to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject and/or to diagnose a thyroid dysfunction in said subject, comprising the following steps: a) providing a reading device according to any one of the herein described embodiments in which at least one well contains a solution of a substance capable of reacting with said or more thyroid function regulatory compounds and/or immune complexes thereof so as to form at least a detectable and/or quantifiable product; b) providing a lateral flow device according to any one of the herein described embodiments; c) subjecting said biological fluid to a lateral flow assay by using said lateral flow device; d) connecting, at the end of the assay conducted in step c), said lateral flow device to the reading device in such a way as to detect and/or quantify said one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof, adsorbed and/or immobilized on the chromatographic strip of said lateral flow device, by means of the reading means of the reading device.

Other advantages and features of the present invention will result evident from the following detailed description. BRIEF DESCRIPTION OF FIGURES

The figures of the enclosed drawings will be referred to, wherein:

• Figure 1 shows schematically a front view (A) and a partially exploded lateral view (B) of a lateral flow device 200 according to a first embodiment of the present invention; • Figure 2 shows schematically a detailed view of the spring system (A) and of the lateral outlet slot of the chromatographic strip (B) of the device of Figure 1 ;

• Figure 3 shows schematically three views of the device of Figure 1 assembled with chromatographic strip expelled through the lateral slot;

• Figure 4 shows schematically a reading device 100 according to a first embodiment of the present invention and in particular a detailed view of the main body carrying a plurality of wells (A); a detailed view of the upper closing element (B) and a perspective view showing a support for housing the sensor (C);

• Figure 5 shows schematically a monometric axonometry of the integrated circular crown of the electronic control unit (A), side views with detail of the control electronics (B) and (C), and a plan view with detail of the wells for the analysis of the device of Figure 4;

• Figure 6 shows schematically two views of the assembled device of Figure 4 with detail of the inlet slot for a chromatographic strip;

• Figure 7 shows schematically a plan view with detail of 25 electrodes immersed in 25 wells by electroanalysis (A) and a lateral view with detail of the control electronics (B); and

monometric axonometry of the circular crown, with attached electronic control unit (C) of a reading device 100 according to a second embodiment of the present invention;

• Figure 8 shows a detailed view of the main body and of the support element of the device of Figure 7.

In the above-mentioned figures, the sizes are to be meant as purely exemplifying and not necessarily with components shown in proportion.

GLOSSARY

The terms used in the present description are as generally understood by the person skilled in the art, unless otherwise indicated.

Under the term “point-of-care testing”, in short POCT, in the context of the present invention, a quick and portable diagnostic test is meant, which can be performed in the home environment by a subject requiring it and which is capable of providing results in a short time, without using complex instruments.

The expressions “detection and/or quantification” or “to detect and/or to quantify” used in the context of the present invention with reference to said one or more thyroid function regulatory compounds and/or immune complexes thereof, refer to the determination of the presence of the above-mentioned compounds within the analysed biological fluid and/or the quantification of the levels or the concentration of the above-mentioned compounds within the fluid.

As used in the context of the present invention, the term "thyroid stimulating hormone” also known as “thyrotropin” or “thyrotropic hormone”, in short in the present description and claims as “TSH”, relates to the hormone produced by the pituitary gland which stimulates the release in blood of thyroxine (T4) and triiodothyronine (T3) hormones by the thyroid. T4 and T3 are involved in controlling the use of energy by the organism. The thyroid produces in greater quantity T4, the mostly inactive form of the thyroid hormones, which however is converted into the mostly active form, T3, in the liver and in other tissues. Together with another regulatory hormone, the thyrotropin releasing factor (TRH) produced by hypothalamus, TSH belongs to a feedback system which the organism uses to keep stable the concentration of thyroid hormones in blood.

The term “chromatographic strip” used in the present description and claims relates to any solid support through which a biological fluid according to any one of the herein described embodiments could migrate, spread or distribute by capillary action, such as, in particular, a membrane made of nitrocellulose.

In any point of the present description and of claims, the expression “one or more thyroid function regulatory compounds” can be replaced by thyroid stimulating hormone and/or calcium, preferably thyroid stimulating hormone and calcium.

The term “subject”, used in the context of the present invention, preferably relates to a mammal, still more preferably a human being.

In the context of the present description, the expression "operatively connected" and the like reflects a functional relationship between the several device components according to the present invention, it means that such components are correlated so as to perform a designated function.

The term “substantially hemispherical” as used in the context of the present invention, relates to a structure having shape similar to a hemisphere, and also comprises, for example, structures shaped like hemisphere having a flat resting base such as the base of a main body as exemplified in Figure 6A.

In any point of description and claims, the term “comprising” can be replaced by “consisting of”.

DETAILED DESCRIPTION OF THE INVENTION

Different embodiments and variants of the invention and portions thereof will be described hereinafter, based upon different aspects thereof usable separately or in combination.

Analogous components are designated in the different Figures with the same numeral reference. In the following detailed description, additional embodiments and variants with respect to embodiments and variants already treated in the same description will be illustrated limitedly to the differences with what already illustrated. Moreover, as said, the different embodiments and variants described hereinafter are likely to be used in combination.

By firstly referring to Figure 1 , a lateral flow device (lateral flow assay, LFA) according to a preferred embodiment of the invention is designated as a whole with 200.

As mentioned, the lateral flow device 200 according to the present invention is configured to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject.

To this purpose the device 200 comprises a chromatographic strip 1 carrying a first end 1a and a second end 1b and comprising:

(i) a loading area of the biological fluid;

(iii) a second capture area comprising a capture agent capable of forming a second immune complex with said first immune complex; wherein said first capture area is localized between said loading area and said second capture area.

The lateral flow device 200 according to the present invention further comprises a cassette 4 comprising a first casing 41 and a second casing 42 coupled to each other in a removable manner so as to limit an internal cavity 40 of the cassette 4 which houses a chromatographic strip 1 according to any one of the herein described embodiments.

The coupling of the first casing 41 to the second casing 42 of the cassette 4 defines a lateral slot 4a of the cassette 4 suitable for allowing, at the end of the use of the device 200, the expulsion of a portion of said strip 1 carrying said second capture area from the internal cavity 40 of the cassette 4 towards the outside.

According to the present invention, the first casing 41 of the cassette 4 comprises expulsion means 5 associated with an internal surface of said first casing 41 facing inside the cassette 4, which expulsion means is configured to cause the expulsion of said portion of the strip 1 from the internal cavity 40 of the cassette 4 towards the outside through said lateral slot 4a. Moreover, the second casing 42 of the cassette 4 comprises a first opening 42a to allow contacting the biological fluid to be analysed with said loading area of the strip 1 , and a second opening 42b, adjacent to the first opening 42b, to allow to display said first and/or second capture area of the strip 1 according to any one of the herein described embodiments.

According to an aspect of the invention, said one or more regulatory compounds of a thyroid function which can be detected and/or quantified by using an immunochromatography device 200 according to any one of the variants illustrated in the present description, comprise thyroid stimulating hormone and/or calcium, preferably they comprise thyroid stimulating hormone and calcium.

In other terms, according to a preferred aspect, the device 200 according to any one of the embodiments illustrated in the present description and claims allows to subject to a lateral flow assay the biological fluid of a subject comprises thyroid stimulating hormone and/or calcium, preferably thyroid stimulating hormone and calcium.

A chromatographic strip 1 suitable to be used in a device 200 according to any one of the herein described embodiments is a solid support comprising or consisting of a membrane and/or paper through which the biological fluid to be analysed can migrate by capillary action. Not limiting examples of said solid supports include for example membrane made of nitrocellulose or cellulose acetate. Preferably, said chromatographic strip 1 comprises or consists of a nitrocellulose membrane.

According to an aspect of the invention, the loading area of the biological fluid to be analysed is localized at or near the end 1a of the chromatographic strip 1 according to any one of the herein described embodiments. Preferably, the loading area comprises an element or pad for absorbing or adsorbing the biological fluid, consisting of a material capable of allowing the transfer of the fluid placed on the pad surface within the chromatographic strip by means of capillary action.

Preferably, the element or pad for absorbing or adsorbing the biological fluid comprises or consists of cellulose.

When a biological fluid of a subject to be subjected to analysis is applied, loaded or placed above the loading area of a chromatographic strip 3, in particular above an absorption or adsorption element or pad as exemplified in the present description, the fluid spreads, through the loading area, in the strip 1 and continues to migrate inside the strip 1 in a flow direction towards the first and the second capture area according to any one of the herein described embodiments.

A biological fluid suitable to be analysed by using an immunochromatography device 200, a kit or a method according to any one of the embodiments illustrated in the present application and claims is preferably a sample selected among urine, saliva or blood. Preferably, said biological fluid is a blood sample.

According to an aspect of the present invention, the first capture area of a chromatographic strip 1 according to any one of the herein described embodiments comprises a capture element or pad inside or above which the detection agent capable of forming said first immune complex with said one or more regulatory compounds is incorporated or adsorbed. In this way, said one or more regulatory compounds present in the biological fluid to be analysed, by migrating from the loading area of the fluid through the chromatographic strip 1 towards the first capture area, once reached the first capture area meet and interact with the detection agent so as to form said first immune complex.

Preferably, said capture element or pad comprises or consists of glass fibre.

A detection agent suitable to form a first immune complex with said one or more thyroid function regulatory compounds is an antibody capable of binding said one or more thyroid function regulatory compounds or a subunit, portion or fragment thereof. According to a preferred aspect of the invention, said antibody is conjugated to a marker agent, preferably any enzyme known in the field of the molecular biology which is capable of reacting with a substance to form a detectable and/or quantifiable compound, for example by spectrophotometric and/or electrochemical route.

An example of such enzyme is represented by horseradish peroxidase (HRP), an oxidoreductase capable of catalyzing the conversion of a chromogenic substrate such as 3, 3’, 5,5’- tetramethylbenzidine (TMB), into a coloured compound, or the light production by reaction with chemiluminescent substrates.

In a preferred embodiment according to the invention, the detection agent is a monoclonal antibody capable of binding a first subunit of the thyroid stimulating hormone, conjugated to horseradish peroxidase. Preferably, said detection agent is an anti-p TSH IgG monoclonal antibody conjugated to horseradish peroxidase.

According to an additional aspect of the invention, the second capture area of a strip 1 according to any one of the herein described embodiments is localized at or near the end 1 b of the strip 1 , that is in a portion of the strip 1 localized at or near the end 1 b of the strip 1.

A capture agent suitable to be used for the formation of said second immune complex with the first immune complex which has formed in the first capture area, is any antibody capable of binding said first immune complex, in particular any antibody which is capable of binding a second subunit of said one or more regulatory compounds different from the first subunit of said one or more regulatory compounds bound to the detection agent.

In a preferred embodiment according to the invention, the capture agent then is a monoclonal antibody capable of binding a second subunit of said one or more regulatory compounds, in particular it is an anti-a TSH IgG monoclonal antibody.

A chromatographic strip according to any one of the variants illustrated in the present description could further have a control area carrying at least an immobilized control agent capable of forming a complex with a detection agent.

A detection agent and/or a capture agent according to any one of the variants described in the present description and claims are adsorbed on or incorporated inside, respectively, said first and said second capture area of the strip 1 , preferably in dried form.

The first “capture” area and the second “capture” area as described in the present invention, can be implemented on the chromatographic strip selected to perform the lateral flow assay, in a form selected from circular spot or line.

A suitable chromatographic strip usable in a lateral flow device 200 according to any one of the embodiments described in the present invention, can be manufactured by using one or more available commercial kits and/or by using industrial techniques known in the field.

A chromatographic strip 1 according to any one of the herein described embodiments has standard sizes within lateral flow immunochromatography, in particular it has a lower width than the width of the lateral slot 4a of the cassette 4 of the device 200.

In particular, a strip 1 according to any one of the herein described variants has a length comprised between 3 and 6 cm, preferably equal to 4 cm, and a width comprised between 0.3 and 0.6 cm, preferably equal to 0.4 cm.

The sizes of the first and second casing constituting a cassette according to any one of the herein described embodiments can be adapted to allow housing a chromatographic strip having the wished sizes.

According to an aspect of the invention, the lateral slot 4a of the cassette 4 of a device 200 according to any one of the herein described embodiments has a width suitable to allow the expulsion of the chromatographic strip 1 through the above-mentioned slot at the end of the use of the device 200, that is once the fluid has reached the second capture area of the strip 1 by determining the formation of said second immune complex.

Preferably, said lateral slot 4a has a width between 0.3 and 0.6 cm, still more preferably greater than 0.4 cm.

As already mentioned, the expulsion means 5 of the device 200 according to any one of the embodiments herein described can be activated at the end of the use of the device 200 and it allows to expel a portion of the strip 1 carrying at least said second capture area from the internal cavity 40 towards the outside of the device 200.

According to an aspect of the invention, said expulsion means 5 comprises a spring system. In particular, said spring system comprises at least a spring suitable to come in contact with the end 1a of the strip 1 under the push of an actuation button, so as to allow to expel said portion of the strip 1 carrying the second capture area through the lateral slot 4a.

According to an aspect of the invention, a lateral flow device 200 according to any one of the herein described embodiments is a lateral flow immunochromatography device.

As already mentioned, one of the advantages of the present invention consists in the possibility of combining the execution of a lateral flow assay by using a lateral flow device 200 according to any one of the variants described in the present application, to the analysis of the chromatographic strip obtained at the end of the assay execution, thanks to the combination of the lateral flow device 200 with a suitable reading device of the strip. In fact, the expulsion and the consequent exposure of the portion of the strip 1 carrying said second capture area outside the device 200 according to any one of the herein described embodiments at the end of the use of the same, allows to subject the above-mentioned chromatographic strip to an additional analysis aimed at detecting and/or quantifying said one or more regulatory compounds and/or immune complexes thereof adsorbed on, immobilized and/or incorporated inside said second capture area.

In particular, the portion of the strip 1 exposed towards the outside of a device 200 according to the present invention at the end of the use of the same can be placed in contact with a suitable reading device and/or analysis of the strip 1 as illustrated hereinafter.

A second aspect of the present invention then relates to a reading device 100 configured to detect and/or quantify one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof which are adsorbed and/or immobilized on a portion of a chromatographic strip 1 according to any one of the herein described embodiments.

By firstly referring to Figure 5, a reading device according to a preferred embodiment of the invention is designated as a whole with 100.

A reading device 100 according to the present invention is configured to allow reading a chromatographic strip 1 , that is to detect and/or quantify one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof which are adsorbed, incorporated and/or immobilized on a portion of a chromatographic strip 1 , in particular a chromatographic strip which has been previously subjected to a lateral flow assay.

To this purpose, the reading device 100 according to the present invention comprises the following elements: a main body 10, substantially hemispherical in shape, comprising a base 10a which defines a horizontal resting plane P of the device 100 on a flat surface;

- a support element 11 , in the form of a circular crown, integral with the main body 10;

- a closing element 20, substantially hemispherical in shape, which can be coupled to said support element 11 revolvingly to rotate around a vertical axis V orthogonal to the resting plane P under the manual action of a user; said closing element 20 being removably couplable to said support element 11 so that, when the reading device 100 is in use, it allows to close the device 100 and forms an inlet slot 101 of the device 100 suitable for receiving the portion of the strip 1 to be analysed; cutting means 30 associated with an internal surface of said closing element (20) facing at the inlet slot 101 of the device 100 when said closing element 20 is coupled to said support element 11.

In particular, according to the present invention, the support element 11 of the reading device

100 carries a plurality of wells 12, adjacent to each other, each one thereof is suitable to contain a solution of a substance capable of reacting with said one or more thyroid function regulatory compounds and/or immune complexes thereof so as to form at least a detectable and/or quantifiable product through suitable reading means 35.

As it will be illustrated hereinafter in the present description, the reading means 35 of the reading device 100 according to the invention is configured to allow the detection and/or quantification of said detectable and/or quantifiable product deriving from the reaction of said one or more thyroid function regulatory compounds and/or immune complexes thereof with the substance present in the solution inside a well of the support element 11.

Advantageously, the reading device 100 according to the present invention is characterized in that, when the portion of a strip 1 to be analysed, carrying said one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof adsorbed and/or immobilized thereon, is inserted into the inlet slot 101 of the device 100, the manual motion of said closing element 20, in particular the manual rotation of the closing element 20 by a user, allows to actuate the cutting of said portion of the strip 1 through the cutting means 30 of the device. The cutting of the portion of the strip 1 allows to let said portion of the strip 1 to fall into a well 12’ of said plurality of wells 12 of the support element 11 , ossia it allows to bring in contact the cut portion of the strip 1 with the solution present inside said at least one well 12’ of the support element 11. In other terms, the motion, in particular the rotation, of the closing element 20 of the reading device 100 specially performed by the patient allows to actuate the cutting of the strip portion introduced into the inlet slot

101 of the device, through the cutting means 30 facing at the slot itself. According to an aspect of the invention, the reading device 100 allows the detection and/or quantification of a thyroid function regulatory compound selected from thyroid stimulating hormone, calcium and/or immune complexes thereof, adsorbed and/or immobilized on a chromatographic strip

I according to any one of the herein described embodiments.

A chromatographic strip suitable to be subjected to analysis or reading by means of a reading device 100 according to any one of the herein described embodiments is a chromatographic strip 1 according to any one of the variants illustrated in the present description and claims.

According to an aspect of the invention, all wells of the plurality of wells 12 of the support element 11 of the device 100 include a solution of a substance capable of reacting with said one or more thyroid function regulatory compounds and/or immune complexes thereof so as to form at least a detectable and/or quantifiable product through suitable reading means 35.

A solution suitable to be introduced inside the wells of any reading device 100 according to the present invention is a solution comprising a substrate capable of producing any compound detectable and/or quantifiable by spectrophotometric or electrochemical route by means of reaction with said one or more thyroid function regulatory compounds and/or immune complexes thereof.

According to an aspect of the present invention, said solution comprises a chromogenic substrate or a substrate capable of producing light radiation after reaction with said one or more thyroid function regulatory compounds and/or immune complexes thereof, preferably it comprises a chromogenic substrate.

According to a preferred aspect of the invention, said solution comprises a chromogenic substrate capable of reacting with an enzyme to form a detectable coloured compound, for example optically or electrochemically, and preferably it comprises a chromogenic substrate capable of reacting with horseradish peroxidase.

In a preferred embodiment of the device 100, said solution comprises 3, 3’, 5,5'- tetramethylbenzidine (TMB).

The solution of said substance present in one or more of the wells 12 of the support element

I I of a reading device 100 according to any one of the herein described embodiments can favour the desorption, from the chromatographic strip, of said one or more thyroid function regulatory compounds and/or immune complexes thereof and their dissolution within the solution, so as to allow the direct detection and/or quantification thereof within the solution, by means of any one of the measurement techniques known to an expert in the field, preferably by spectrophotometric route or by electrochemical route.

In an alternative embodiment the wells 12 of a reading device 100 according to any one of the herein described embodiments include a solution suitable to favour desorption, from the chromatographic strip, of said one or more thyroid function regulatory compounds and/or immune complexes thereof and their dissolution, so as to allow the detection and/or quantification thereof according to any one of the techniques known to a person skilled in the art, preferably by spectrophotometric route or by electrochemical route. Said solution then can even not include a substance capable of reacting with said one or more thyroid function regulatory compounds or immune complexes thereof as previously exemplified in the present description.

To this purpose, the wells 12 of the device can include an aqueous solution, preferably a pH- controlled buffer solution.

The support element 11 of a reading device 100 according to any one of the herein described embodiments comprises a number of wells 12 preferably comprised between 20 and 30, still more preferably equal to 25.

According to an aspect of the invention, the reading means 35 of the device 100 according to any one of the herein described variants comprises means for detection and/or quantification by spectrophotometry, in particular comprises at least a sensor configured to perform a spectrophotometric measurement associated with an internal surface of said closing element 20 of the reading device 100, so as to face the inside of said well 12’ when said closing element 20 is coupled to said support element 11.

In a preferred embodiment, said reading means comprises a sensor consisting of a photoemitter and a photodetector. According to an aspect of the invention, the photoemitter is integral to the closure element 20 of the device. To this purpose, the support element in the form of a circular crown of the device 100 can carry a circular path, obtained inside the circular crown and integral therewith, configured to allow the rotation on the path of the photoemitter. In other terms, according to an aspect of the invention, the photoemitter is integral to the closing element 20 and rotates on a circular path obtained inside the crown and integral therewith.

According to an aspect of the present invention, for each well 12 of the support element 20 there is a photodetector positioned on the internal wall of the main body adjacent to the well so that the set of photoemitter and photodetector are in axis with the well itself.

Preferably, each photodetector is connected operatively to an internal wall of the main body adjacent to each well so that the set of photoemitter and photodetector are in axis with the well itself.

According to an additional aspect of the invention, the reading means 35 comprises means for detection and/or quantification by electrochemical route, in particular a conductivity meter and one or more electrodes. Preferably, said conductivity meter can be operatively connected to the support element 11 of the reading device according to any one of the herein described embodiments.

In an aspect of the invention, said reading means 35 is associated with an internal surface of said closing element 20 of the reading device 100 so as to face the inside of said well 12’ when said closing element 20 is coupled to said support element. Alternatively, said reading means 35 is associated with an internal surface of said closing element 20 so as to face the inside of each well of said plurality of wells 12, when said closing element 20 is coupled to said support element 11.

According to a preferred aspect of the invention, the reading means 35 according to any one of the herein described variants comprises a plurality of electrodes or pairs of electrodes associated with an internal surface of said closing element 20 so that, when said closing element 20 is coupled to said support element 11 , each electrode or pair of electrodes of said plurality of electrodes or pairs of electrodes is inserted inside a corresponding well of said plurality of wells 12.

The reading device 100 according to any one of the herein described embodiments can further include one or more housings configured to house the reading means 35. According to an aspect of the invention, the device 100 according to any one of the herein described variants carries a housing associated with a surface of the closing element 20 facing the inside of one of the wells 12 when the closing element 20 is coupled to the support element 11 , which housing is configured to house at least a sensor to detect and/or quantify said one or more regulatory compounds and/or immune complexes thereof, in particular by spectrophotometric route. Cutting means suitable to be used in a device 100 according to any one of the herein described embodiments comprises one or more blades, preferably said blades are placed and/or operatively connected to a surface of the closing element 20 facing at the slot 101 of the device when said closing element 20 is coupled to the main body 10.

The inlet slot 101 of a reading device 100 according to any one of the variants described in the present application preferably has a width suitable to allow the insertion of a portion of a chromatographic strip 1 according to any one of the herein described embodiments through the above-mentioned inlet slot.

Preferably, said inlet slot 101 has a width between 0.3 and 0.6 cm, still more preferably greater than 0.4 cm.

According to an aspect of the invention, the motion of the closing element 20 is performed manually by rotation by a user. The reading device 100 according to any one of the embodiments described in the present description and claims can further comprise an electronic management and control unit 16 operatively associated with said reading means 35, suitable for controlling the activation/deactivation of said reading means 35 and receiving data relating to the detection and/or quantification of said one or more regulatory compounds and/or immune complexes thereof and/or said at least a detectable and/or quantifiable product.

Additionally, the reading device 100 can include at least a storage unit operatively associated with the management and control unit and suitable to store the data measured by the reading means 35, for example in form of a SD-card.

The reading device 100 according to any one of the herein described embodiments can further include one or more additional electrical control means such as, for example, switch devices operatively connected to said reading means 35 and/or to said management and control unit.

According to an aspect of the invention, the management and control unit 16 and the storage unit can be fastened on a surface of the support element 11 facing the inside of the main body 10 of the device 100 when said control element 11 is coupled to the main body 10.

The reading device 100 according to any one of the herein described embodiments can further include means for transmitting the data related to the detection and/or quantification of said one or more regulatory compounds and/or immune complexes thereof and/or said at least a detectable and/or quantifiable product, which transmission means is associated with said reading means 35 and/or to said electronic management and control unit 16. The means for transmitting data can include a serial transfer unit, for example a USB port. Moreover, the means for transmitting data can include or be connected to a unit for displaying data, for example an external display or screen.

Still, the reading device 100 according to the present invention can include a power supply system.

The present invention also relates to a kit to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject, which kit comprises a reading device 100 and a lateral flow device 200 according to any one of the embodiments illustrated in the present description and claims.

According to an aspect of the invention, the kit further comprises one or more replacement chromatographic strips 1 according to any one of the variants illustrated in the present description and claims.

The kit can further include even control reagents and/or instructions for use.

An additional aspect of the invention is represented by the in vitro use of a reading device 100 and of a lateral flow device 200, or a kit according to any one of the herein described embodiments, to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject and/or for the diagnosis of a thyroid dysfunction in said subject.

Preferred examples of thyroid function regulatory compounds which can be detected and/or quantified by using a lateral flow device 200 and a reading device 100 according to any one of the variants described in the present application, include the thyroid stimulating hormone (TSH) and calcium.

Preferably, said reading device 100 and said lateral flow device 200 can be used, in vitro, to detect and/or quantify the levels of thyroid stimulating hormone (TSH) and/or calcium, preferably both TSH and calcium within a biological fluid of a subject.

Advantageously, a reading device 100 and a lateral flow device 200 according to any one of the herein described embodiments can be used to detect and/or quantify the levels of thyroid stimulating hormone within a biological fluid of a subject, preferably in a blood sample, with the purpose of diagnosing a condition of hypothyroidism or hyperthyroidism in a subject.

A reading device 100 and a lateral flow device 200 according to any one of the herein described embodiments can also be used to detect and/or quantify the levels of calcium within a biological fluid of a subject, preferably in a blood sample, with the purpose of diagnosing a condition of hypocalcaemia or hypercalcaemia in a subject.

The diagnosis can be performed by comparing the levels of said one or more thyroid function regulatory compounds, in particular TSH and/or calcium, as determined and/or quantified in the biological fluid subjected to analysis by means of a lateral flow device and a reading device, or a kit or a method according to any one of the herein described embodiments, with a reference value obtained from a healthy subject.

The present invention also relates to an in vitro method to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject and/or to diagnose a thyroid dysfunction in said subject, characterized in that it uses a reading device 100 and a lateral flow device 200, or a kit comprising them, according to any one of the herein described embodiments.

According to an aspect of the invention, said in vitro method comprises the following steps: a) providing a reading device 100 according to any one of the herein described embodiments in which at least one well 12’, preferably each well of the plurality of wells 12, contains a solution of a substance capable of reacting with said or more thyroid function regulatory compounds and/or immune complexes thereof so as to form at least a detectable and/or quantifiable product; b) providing a lateral flow device 200 according to any one of the herein described embodiments; c) subjecting the biological fluid to a lateral flow assay by using said lateral flow device 200; d) connecting, at the end of the assay conducted in step c), said lateral flow device 200 to the reading device 100 in such a way as to detect and/or quantify said one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof, adsorbed and/or immobilized on the chromatographic strip 1 of said lateral flow device 200, by means of the reading means 35 of the reading device 100.

An in vitro method according to any one of the herein described embodiments can be advantageously used to detect and/or quantify the levels of thyroid stimulating hormone and/or calcium in a biological sample of a subject, preferably simultaneously thyroid stimulating hormone and calcium.

The biological fluid of the subject subjected to analysis by means of an in vitro use or method according to any one of the embodiments illustrated in the present description is preferably a blood sample del subject.

According to an aspect of the invention, step c) of the herein illustrated in vitro method comprises contacting the biological fluid of the subject with the loading area of the chromatographic strip 1 of the lateral flow device 200 according to any one of the variants described in the present application, so as to allow the biological fluid to migrate inside the chromatographic strip until reaching the second capture area and forming the second immune complex.

According to an additional aspect of the invention, step d) of an in vitro method according to any one of the herein described embodiments comprises the following steps of: i) expelling, through the lateral slot 4a of the cassette 4 of said lateral flow device 200, the portion of the strip 1 carrying said second capture area; ii) inserting said portion of the strip 1 into the inlet slot 101 of said reading device 100; iii) carrying out the cutting of said portion of the strip 1 through said cutting means 30 of the reading device 100 so as to let said portion of the cut strip 1 to fall inside the well 12’ of the reading device 100 containing the solution of said substance; iv) letting said one or more thyroid function regulatory compounds and/or immune complexes thereof to react with said substance so as to form said at least a detectable and/or quantifiable product; and v) detecting and/or quantifying said at least one product by means of said reading means 35 of the reading device 100.

According to an aspect of the invention, step d) of an in vitro method according to any one of the herein described embodiments comprises the fact of letting said one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof within said solution to desorb and/or dissolve and detecting and/or quantifying said one or more dissolved and/or desorbed thyroid function regulatory compounds of a subject and/or immune complexes thereof by means of said reading means 35 of the reading device 100.

According to the invention, in step iii) of the previously described method, the cutting of said portion 1 of the strip is actuated by the manual rotation of the closing element 20 of the reading device according to any one of the herein described variants, by a user, for example the patient himself/herself.

The detection and/or quantification of said at least one product and/or said one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof dissolved and/or desorbed in the solution can be performed by spectrophotometric route or by electrochemical route, depending upon the specific reading means 35 incorporated in the used reading device 100.

Advantageously, the in vitro method according to any one of the herein described embodiments can further include the step of diagnosing said thyroid dysfunction when the levels of said one or more thyroid function regulatory compounds as quantified in the biological fluid of said subject are higher or lower than a reference value relative to a biological fluid of a healthy subject.

EXAMPLES

Some not limiting embodiment examples of the method according to the present invention are shown herebelow by way of example.

EXAMPLE 1 - Manufacturing of the devices

A system mainly consisting of two distinct and independent devices was developed. The first device is a lateral flow device and it consists of a first and second casing enclosing inside thereof a strip of membrane made of nitrocellulose. The lateral flow device has a spring expulsion mechanism outside the covering casing.

The immunochemical technique used to allow the detection of the TSH target analyte is ELISA Sandwich on porous membrane. A chromatographic strip of membrane made of nitrocellulose (=4x0.4cm) is added on the origin end by two pads, the first one made of cellulose (=1.5x0.4cm) to adsorb the blood corpuscular portion and the second one made of glass fibre (=1x0.4cm) to form the first bond between target analyte and anti-p TSH IgGs conjugated with HRP. At last, on the end stop of the strip made of nitrocellulose the anti-a TSH IgGs are blotted, which represent the formation site of the immune complex, subjected to chemical analysis inside the second reading device.

Once the reaction has occurred, it is possible expelling the strip of membrane outside the cassette and be inserted inside the sensorized slot (Figure 6) of the second reading device. The slot is completed laterally by a blade which cuts the membrane near the end stop, that is only of the portion of the chromatographic strip of membrane made of nitrocellulose which penetrates the reading device. In particular, the cutting is performed by actuating the rotation mechanism by the patient.

The membrane portion having the analyte is let to fall inside one of the 25 wells (Figure 4 or Figure 7) therewith the circular crown of the reading device is provided. The membrane inside the well gravitates towards the bottom. Inside the solution there is a cascade oxidation of the molecules of substrate (3,3’,5,5’tetramethylbenzidine), present in the well, which colour blue the solution, and the molecular spread of the calcium ions from the porous matrix to the solution.

The oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) can be detected spectrophotometrically ore electrochemically.

In a first embodiment of the reading device according to the invention, illustrated by way of examples in figures 4-6, the presence of a spectrophotometer, with absorption peak at 690nm, near the well under analysis allows the optical detection. The measurements are sent to an external interface allowing the display by the user. The motion of the circular crown in steps is performed manually by the patient who, by means of a clockwise/counterclockwise rotation mechanism of the two hemispherical supports constituting the reading device, guarantees the availability of a new well for the subsequent analysis.

In a second embodiment of the reading device according to the invention, illustrated by way of examples in figures 7-8, the presence of a conductivity meter, consisting of control electronics, allows to detect in solution different ions and their differentiation based upon the charge. In order to proceed with the electrochemical analysis of each sample the reading device comprises an electrode for each well present on the circular crown connected by means of connectors to the control unit. The measurements are sent to an external interface which allows the display by the patient. The motion of the circular crown in steps is performed manually by the patient who, by means of a clockwise/counterclockwise rotation mechanism of the two hemispherical supports constituting the second reading device, guarantees the availability of a new well for the subsequent analysis.

The calcium quantification takes place by electrochemical detection of the cation in solution, since the target analyte (Ca++) is on the same porous matrix which precipitates inside the analysis well.

Figures 1 and 2 show the first lateral flow device aimed at containing and expelling the chromatographic strip of membrane made of nitrocellulose. Figure 1A shows the application site of the biological fluid, the blood, to be analysed. The blood runs along the entire length of the membrane made of nitrocellulose until reaching the end stop, where the final reaction takes place in favour of the immune complex formation. Figure 1 B also shows the coupling between the first casing and the second casing to form the cassette of the device, so as to allow to cover the membrane made of nitrocellulose.

In Figure 2 A-B the spring system and the expulsion point of the strip are respectively highlighted. Figure 3 shows details of the first lateral flow device, assembled and ready to be coupled to the second reading device of the chromatographic strip.

Figures 4, 5 and 6 show the device for the analysis and quantification of the sample under examination. Figure 4A and Figures 5 A-D show the details of the circular crown which allows a jerky manual rotation, where even the 25 wells are highlighted. Figure 4B in particular shows the main body and the closing element in the front perspective, by highlighting the inlet slot which allows the coupling of the two devices. Figure 4E mainly shows the main body and the closing element with attached sensor housing.

In Figure 6 the assembly of the reading device with the detail of the hooking slot of the first lateral flow device is represented.

As highlighted in the embodiment illustrated in Figure 7 and in Figure 8, the wells of the device can be complete with electrodes, belonging to the same electronic control unit by means of the connectors unique for each electrode and they allow a detection and quantification of TSH by electrochemical route by means of conductivity meter.

EXAM PLE 2 - Optimization of the development protocol of ELISA assay on chromatographic strip

The used technical protocol provides the development of an ELISA Sandwich on a chromatographic strip consisting of the pads made of cellulose and glass fibre overlapped to a membrane made of nitrocellulose, whereon antibodies, analytes, buffers and substrates adsorb.

Once determined univocally the molar ratios of the different species participating to the process, a method was processed which provides three different variants, aimed at detecting the best experimental conditions. For the target analyte one chose to take into consideration the three pivotal concentrations 5 IU/mL, 2.5μIU/mL and 0.1μIU/mL, respectively for hypothyroidism, euthyroidism and hyperthyroidism.

The first tested protocol provides the deposition of a controlled volume of Ab Anti-a TSH on nitrocellulose, followed by 16h of drying; once the immobilization of Ab on nitrocellulose has occurred, the TSH, in its three different concentrations, reacts with Ab Anti-p TSH-HRP in the well of a 96-we// low attachment plate; the nitrocellulose is immersed inside the wells pre-loaded with the primary immune complex and at last, in equal volumes, the substrate and the stop solution are inserted. This first protocol highlighted the need for coating the nitrocellulose to avoid aspecific bonds of the second Ab on the membrane.

In the second protocol three main modifications were made: activation of the membrane made of nitrocellulose (PBS or ethanol); immobilization through adsorption of aspecific Ab, through suction by capillarity of a controlled volume, and subsequent incubation at 37°C for 2h; locking of the membrane made of nitrocellulose with coating of BSA 1% w/v, followed by a process of washing with PBS buffer to remove the exceeding coating.

The last experimental protocol sees most improvements. Firstly, the parafilm is used to optimize the adsorption of the primary Ab, subsequently incubated overnight at +4°C, differently from the other components incubated for 1 h. Moreover, the washing number was increased from 1 to 3 after insertion of each molecule, excluding BSA. At last, strips of membrane with reduced sizes were adopted for a total immersion of the same, at first within the solution of TMB and then in the stop solution. For sake of completeness, a fourth concentration of TSH (15μIU/mL) was tested, mainly aimed at detecting the maximum reactive capability of the system.

Claims

CLAIMS . A reading device (100) of a chromatographic strip (1), the device (100) being configured to detect and/or quantify one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof adsorbed and/or immobilized on a chromatographic strip (1), comprising: a main body (10), substantially hemispherical in shape, comprising a base (10a) which defines a horizontal resting plane (P) of the device (100); a support element (11), in the form of a circular crown, coupled to the main body (10) in a manner integral with said main body (10); which support element (11) carries a plurality of wells (12) adjacent to each other, each well being able to contain a solution of a substance capable of reacting with said one or more thyroid function regulatory compounds and/or immune complexes thereof so as to form at least a detectable and/or quantifiable product; a closing element (20), substantially hemispherical in shape, which can be rotatably coupled to said support element (11) to rotate around a vertical axis V orthogonal to the resting plane P under the manual action of a user; said closing element (20) being removably couplable to said support element (11) in such a way as to allow the closure of the device (100) and form an inlet slot (101) of the device (100) suitable for receiving a portion of the strip (1) bearing said one or more regulatory compounds and/or immune complexes thereof; cutting means (30) associated with an internal surface of said closing element (20) facing at the inlet slot (101) of the device (100) when said closing element (20) is coupled to said support element (11); and reading means (35) configured to detect and/or quantify said one or more regulatory compounds and/or immune complexes thereof and/or said at least a detectable and/or quantifiable product; characterized in that, when said portion of the strip (1) is inserted into the inlet slot (101) of the device (100), the manual rotation of the closing element (20) by the user allows the cutting of said portion of the strip (1) through said cutting means (30) so as to make said portion of the strip (1) to fall into a well (12’) of said plurality of wells (12).

2. The reading device (100) according to claim 1 , wherein said one or more thyroid function regulatory compounds comprise a thyroid stimulating hormone (TSH) and/or calcium, preferably they comprise TSH and calcium.

3. The reading device (100) according to claim 1 or 2, wherein all wells of said plurality of wells (12) contain said solution.

4. The reading device (100) according to any one of claims 1 to 3, wherein said solution comprises a chromogenic substrate.

5. The reading device (100) according to any one of claims 1 to 4, wherein said solution comprises a chromogenic substrate capable of reacting with an enzyme to form a detectable coloured compound, preferably a chromogenic substrate capable of reacting with horseradish peroxidase.

6. The reading device (100) according to any one of claims 1 to 5, wherein said solution comprises 3,3’,5,5'-tetramethylbenzidine (TMB).

7. The reading device (100) according to any one of claims 1 to 6, wherein said support element (11) comprises a number of wells (12) comprised between 20 and 30.

8. The reading device (100) according to claim 7, wherein said support element (11) comprises a number of wells (12) equal to 25.

9. The reading device (100) according to any one of claims 1 to 8, wherein said reading means (35) comprises means for detection and/or quantification by spectrophotometry.

10. The reading device (100) according to any one of claims 1 to 8, wherein said reading means (35) comprises means for detection and/or quantification by electrochemical route.

11. The reading device (100) according to any one of claims 1 to 10, wherein said reading means (35) is associated with an internal surface of said closing element (20) so as to face the inside of said well (12’) when said closing element (20) is coupled to said support element (11).

12. The reading device (100) according to any one of claims 1 to 10, wherein said reading means (35) is associated with an internal surface of said closing element (20) so as to face the inside of each well of said plurality of wells (12), when said closing element (20) is coupled to said support element (11). The reading device (100) according to any one of claims 1 to 11 , wherein said reading means (35) comprises at least a sensor associated with an internal surface of said closing element (20) so as to face the inside of said well (12’) when said closing element (20) is coupled to said support element (11). The reading device (100) according to any one of claims 1 to 13, wherein said reading means (35) comprises a plurality of electrodes associated with an internal surface of said closing element (20) so that, when said closing element (20) is coupled to said support element (11), each electrode of said plurality of electrodes is inserted into a well of said plurality of wells (12). The reading device (100) according to any one of claims 1 to 14, wherein said cutting means (30) comprises one or more blades. The reading device (100) according to any one of claims 1 to 15, wherein said inlet slot (101) has a width between 3 mm and 6 mm, preferably greater than 0.4 cm. The reading device (100) according to any one of claims 1 to 16, further comprising an electronic management and control unit (16) operatively associated with said reading means (35), suitable for controlling the activation/deactivation of said reading means (35) and receiving data relating to the detection and/or quantification of said one or more regulatory compounds and/or immune complexes thereof and/or of said at least a detectable and/or quantifiable product. The reading device (100) according to claim 17, further comprising means for transmitting said data, associated with said reading means (35) and/or said electronic management and control unit (16). The reading device (100) according to claim 18, wherein said transmission means comprises a serial transfer unit and/or a unit for displaying said data. A lateral flow device (200) to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject, said device (200) comprising: a chromatographic strip (1) bearing a first end (1a) and a second end (1 b) and comprising:

(i) a loading area of said biological fluid;

(iii) a second capture area comprising a capture agent capable of forming a second immune complex with said first immune complex; wherein said first capture area is localized between said loading area and said second capture area; and a cassette (4) comprising a first casing (41) and a second casing (42) coupled to each other in a removable manner so as to limit an internal cavity (40) of the cassette (4) which houses said chromatographic strip (1) and so as to define a lateral slot (4a) of the cassette (4) suitable for allowing, at the end of the use of the device (200), the expulsion of a portion of said strip (1) bearing said second capture area from the internal cavity (40) of the cassette (4) towards the outside; wherein said first casing (41) comprises expulsion means (5) associated with an internal surface of said first casing (41) facing inside the cassette (4), configured to cause the expulsion of said portion of the strip (1) from the internal cavity (40) of the cassette (4) towards the outside through said lateral slot (4a); and wherein said second casing (42) comprises a first opening (42a) to allow contacting the biological fluid to be analysed with said loading area of the strip (1), and a second opening (42b), adjacent to the first opening (42b), to allow to display said first and/or second capture area of the strip (1). The device (200) according to claim 20, wherein said one or more thyroid function regulatory compounds comprise a thyroid stimulating hormone (TSH) and/or calcium, preferably they comprise TSH and calcium. The device (200) according to claim 20 or 21 , wherein said chromatographic strip (1) comprises or consists of a membrane made of nitrocellulose. The device (200) according to any one of claims 20 to 22, wherein said loading area is located at or near said first end (1a) of the strip (1). The device (200) according to any one of claims 20 to 23, wherein said loading area comprises a biological fluid adsorption pad, comprising or consisting of cellulose. The device (200) according to any one of claims 20 to 24, wherein said first capture area comprises a capture pad, comprising or consisting of glass fibre. The device (200) according to any one of claims 20 to 25, wherein said detection agent is an antibody capable of binding said one or more thyroid function regulatory compounds, conjugated to a marker agent. The device (200) according to claim 26 wherein said marker agent is an enzyme capable of reacting with a substance to form a detectable and/or quantifiable compound, preferably said marker agent is horseradish peroxidase. The device (200) according to claims 26 or 27, wherein said antibody is a monoclonal antibody capable of binding a first subunit of the thyroid stimulating hormone conjugated to horseradish peroxidase. The device (200) according to any one of claims 26 to 28, wherein said antibody is a monoclonal antibody IgG anti-p TSH conjugated to horseradish peroxidase. The device (200) according to any one of claims 20 to 29, wherein said second capture area is located at or near said end (1 b) of the strip (1). The device (200) according to any one of claims 20 to 30, wherein said capture agent is a monoclonal antibody capable of binding said one or more thyroid function regulatory compounds. The device (200) according to claim 31 wherein said capture agent is a monoclonal antibody capable of binding a second subunit of the thyroid stimulating hormone. The device (200) according to claim 31 or 32, wherein said antibody is an IgG anti-a TSH monoclonal antibody. The device (200) according to any one of claims 20 to 33, wherein said chromatographic strip (1) has a length comprised between 3 and 6 cm, preferably equal to 4 cm, and a width comprised between 3 and 6 mm, preferably equal to 0.4 cm.

35. The device (200) according to any one of claims 20 to 34, wherein said lateral slot (4a) has a width ranging from 3 and 6 mm, preferably greater than 0.4 cm.

36. The device (200) according to any one of claims 20 to 35, wherein said expulsion means (5) comprises a spring system.

37. A kit to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject, which kit comprises a reading device (100) according to any one of claims 1 to 19 and a lateral flow device (200) according to any one of claims 20 to 36.

38. The kit according to claim 37, further comprising one or more chromatographic strips as defined in any one of claims 20 to 36.

39. The kit according to claims 37 or 38, further comprising control reagents and/or instructions for use.

40. An in vitro use of a reading device (100) according to any one of claims 1 to 19 and a lateral flow device (200) according to any one of claims 20 to 36, or a kit according to any one of claims 37 to 39, to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject and/or for the diagnosis of a thyroid dysfunction in said subject.

41 . The in vitro use according to claim 40, wherein said one or more thyroid function regulatory compounds comprise a thyroid stimulating hormone (TSH) and/or calcium, preferably they comprise TSH and calcium.

42. The in vitro use according to claims 40 or 41 , wherein said thyroid dysfunction is selected from hypothyroidism, hyperthyroidism, hypocalcaemia and/or hypercalcaemia.

43. An in vitro method to detect and/or quantify one or more thyroid function regulatory compounds in a biological fluid of a subject and/or to diagnose a thyroid dysfunction in said subject, comprising the following steps of: a) providing a reading device (100) according to any one of claims 1 to 19 in which at least one well (12’) contains a solution of a substance capable of reacting with said or more thyroid function regulatory compounds and/or immune complexes thereof so as to form at least a detectable and/or quantifiable product; b) providing a lateral flow device (200) according to any one of claims 20 to 36; c) subjecting said biological fluid to a lateral flow assay by using said lateral flow device (200); d) connecting, at the end of the assay conducted in step c), said lateral flow device (200) to the reading device (100) in such a way as to detect and/or quantify said one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof, adsorbed and/or immobilized on the chromatographic strip (1) of said lateral flow device (200), by means of the reading means (35) of the reading device (100).

44. The in vitro method according to claim 43, wherein said one or more thyroid function regulatory compounds comprise a thyroid stimulating hormone (TSH) and/or calcium, preferably they comprise TSH and calcium.

45. The in vitro method according to claims 43 or 44, wherein said thyroid dysfunction is selected from hypothyroidism, hyperthyroidism, hypocalcaemia and/or hypercalcaemia.

46. The in vitro method according to any one of claims 43 to 44, wherein said biological fluid is blood.

47. The in vitro method according to any one of claims 43 to 46, wherein said step c) comprises contacting said biological fluid with the loading area of the chromatographic strip (1) of said lateral flow device (200), so as to allow the biological fluid to migrate inside the strip (1) until reaching said second capture area of the strip to form said second immune complex.

48. The in vitro method according to any one of claims 43 a 47, wherein said step d) comprises the following steps of: i) expelling, through the lateral slot (4a) of the cassette (4) of said lateral flow device (200), the portion of the strip (1) bearing said second capture area; ii) inserting said portion of the strip (1) into the inlet slot (101) of said reading device (100); iii) carrying out the cutting of said portion of the strip (1) through said cutting means (30) of the reading device (100) so as to let said portion of the cut strip (1) to fall inside the well (12’) of the reading device (100) containing the solution of said substance; iv) letting said one or more thyroid function regulatory compounds and/or immune complexes thereof to react with said substance so as to form said at least a detectable and/or quantifiable product; and v) detecting and/or quantifying said at least one product by means of said reading means (35) of the reading device (100). The in vitro method according to any one of claims 43 to 48, wherein said step d) comprises letting said one or more thyroid function regulatory compounds of a subject and/or immune complexes thereof to be dissolved and/or desorbed within said solution and detecting and/or quantifying said one or more dissolved and/or desorbed thyroid function regulatory compounds of a subject and/or immune complexes thereof by means of said reading means (35) of the reading device (100). The in vitro method according to any one of claims 43 to 49, wherein said detection and/or quantification is carried out by spectrophotometric and/or electrochemical way. The in vitro method according to any one of claims 43 to 50, further comprising the step of diagnosing said thyroid dysfunction when the levels of said one or more thyroid function regulatory compounds as quantified in the biological fluid of said subject are higher or lower than a reference value relative to a biological fluid of a healthy subject.