ITCS20120019A1 - DEVICE, METHOD AND KIT FOR DETECTING DIFFERENT MARKERS IN DIFFERENT CELL OR MOLECULAR TYPES AND THEIR QUANTIFICATION - Google Patents

Description

Device, method and kit for the detection of different markers in different cell or molecular types and their quantification

Description of the invention

The present invention relates to:

- devices in the form of microplates or microstrips with elongated wells suitable to accommodate 3 or 4 or 6 immunoadsorbent elements protruding from a rod with a modular distance coinciding with the modular distance of the wells of standard 8 or 12 well microstrips or standard 96 microplates wells;

- the method that uses such devices by placing in competition two solid phases of different conformation, such as the elongated wells, on each of which a different cell or molecular type present in the sample under examination is immobilized, and the immunoadsorbent elements protruding from a rod, which are immersed in each elongated well in groups of 3 or 4 or 6, after one of the same markers to be detected in the sample under examination has been immobilized on each of these elements and after ligands have been inoculated in the elongated wells for the marker to be detected, in liquid phase; these ligands bind to the immunoadsorbent elements in a quantity inversely proportional to the quantity of the relative markers expressed by each cell or molecular type immobilized on the relative elongated well and can be quantified simultaneously through an enzyme immunoassay conducted by immersing the rod bearing the immunoadsorbent elements in a container containing the conjugated and, subsequently, by immersing each single element in single wells of standard microstrips or microplates containing chromogenic substrate, in each of which, finally, the spectrophotometric reading is performed;

- structured kits for carrying out said method through the use of the aforementioned elongated-well devices, for the simultaneous quantification of different cellular or molecular markers and for the concomitant detection of the different cells or molecules that expose them, including enzyme immunoassays for rapid diagnosis of tuberculosis and early stage paratuberculosis, based on the quantification of different markers of infection present in the different cooperating and cytolytic lymphocyte populations, sensitized by different antigens or mycobacterial haptens.

The elongated well conformation of the devices is necessary for the execution of the method aimed at obtaining:

1. the selective immobilization on the surface of each elongated well, mediated or not by antibodies, of a different cellular or molecular type of the test sample of which different markers are to be quantified simultaneously, so that the surface of the elongated wells acquires a function of solid phase for enzyme immunoassays and ELISA, becoming suitable for capturing and immobilizing specific ligands in the liquid phase;

2. the subsequent capture, by cells or molecules immobilized on the surface of the elongated wells, of specific ligands for each of the markers to be quantified simultaneously, which are inoculated into the elongated wells as a mix of known quantity in the liquid phase; these ligands captured by the cells immobilized on the surface of the elongated wells cannot be directly measured by enzyme immunoassays and ELISAs due to the interference of the cell mat with the spectrophotometric reading of the final chromogenic reaction; the technical problem is solved by analytically detecting the proportional fraction of ligands that has been captured by a second solid phase, specifically sensitized, which is introduced in the first by competing for the capture of the ligands in the liquid phase and on which the ligands separate, in so that they can then be individually quantified with enzyme immunoassay and ELISA in wells of standard microstrips and microplates; this second solid phase is constituted by the immunoadsorbent elements protruding from a rod with a modular distance coinciding with the modular distance of the wells of standard 8 or 12 well microstrips or 96 wells standard microplates, in which each element is sensitized with antibodies directed against a specific ligand and competes, for its capture, with one of the markers to be quantified in the sample under examination;

3. the quantification of each ligand captured separately from the immunoadsorbent elements, carried out by enzyme immunoassay or ELISA, which involves the incubation of the elements protruding from the rod in conjugate and, subsequently, in a chromogenic substrate distributed in single wells of standard microstrips or microplates, in such a way that the analytical result is expressed as optical density detected by spectrophotometric reading for each of the markers of interest present in each cell or molecular type of the sample under examination; the quantity of each ligand captured by the specifically sensitized immunoadsorbent element is inversely proportional to the quantity of the respective marker of each cell or molecular type present in the sample under examination, because it is detected with a competitive assay in which two solid phases of different conformation - the elongated well and elements protruding from a rod - compete for the capture of the same ligands in the liquid phase; this competitive assay differs from the assays found in the state of the art, which place different molecules in the liquid phase in competition for binding to the same solid phase and do not allow to set up assays for the double determination of different markers and different cells or molecules that they expose them; the quantification of each ligand captured separately from the immunoadsorbent elements can be obtained with an enzyme immunoassay or other suitable assays, such as chemiluminescence.

The devised devices and method can be applied to the structuring of kits that detect the cellular immune response through an enzyme immunoassay that simultaneously quantifies different lymphocyte markers of tuberculous and paratuberculous infection and simultaneously identifies the different cooperating or cytolytic lymphocyte populations that expose them, allowing the rapid diagnosis of human tuberculosis, bovine tuberculosis and early stage ruminant paratuberculosis.

The present invention was created, in fact, to solve some problems in the diagnosis of human tuberculosis, which is one of the infectious diseases with the highest worldwide incidence:

detect the cellular immune response characterized by the appearance of cooperating T lymphocytes and cytolytic T lymphocytes specific for tuberculous mycobacterium antigens and follow their evolution during disease and therapy, as the detection of antibodies produced in the humoral immune response does not provide diagnostic results reliable;

identify cases of pulmonary, extra-pulmonary infections and latent carriers of infection through an indirect diagnosis test based on the detection of the immune response;

- obtain the analytical result in a few hours, so as to be able to detain infected patients and immediately begin therapy;

- the personnel assigned to carry out laboratory analyzes must be able to be trained in a short time, compatibly with large-scale health campaigns in developing countries, which have a higher incidence of infection;

- complex and expensive laboratory structures, which are not widespread in developing countries, must not be necessary.

These diagnostic problems correspond to the need to solve technical problems for the realization of:

kits capable of detecting and quantifying the presence of infection markers on lymphocyte cells of different types;

kits suitable for detecting the state of the cellular immunity of the subject under examination, which is an indicator of infection regardless of the location of the mycobacterium;

rapid kits, such as those widely used for ELISA assays in other infections diagnosable by antibody detection;

- ready-to-use kits;

kits that can be used by minimally trained personnel;

kits usable in minimally equipped laboratory facilities.

To meet the above needs and adopting as a diagnostic principle that of the simultaneous quantification of different lymphocyte markers that can be highlighted during tuberculous infection and the identification of the different lymphocyte populations that expose them, such as cooperating TCD4 lymphocytes and cytotoxic CD8 T lymphocytes, and the method devised for their use, aimed at the simultaneous quantification of different cellular markers and the concomitant detection of the different cells that expose them, are applied to the structuring of specific diagnostic kits, summarized below.

Useful devices for structuring the kits

In the kit for single sample analysis, the elongated well device consists of 3 elongated well microstrips, each suitable to accommodate 4 immunoadsorbent elements protruding from a rod with a modular distance coinciding with the modular distance of the wells of the standard 96-well microplates ; it is of immunoadsorbent material useful for the adsorption of monoclonal antibodies that selectively capture CD4 T lymphocytes or CD8 T lymphocytes of the sample under examination, immobilizing them separately on two of the three elongated wells, while the third is not sensitized and acts as a negative control.

In the 8-sample test kit, the elongated well device consists of 24 elongated well microplates, containing 3 elongated wells for each row A to H; each elongated well is suitable to accommodate 4 immunoadsorbent elements protruding from a rod and the entire device is made of immunoadsorbent material.

The elements protruding from a rod are of immunoadsorbent material, 12 in number, previously sensitized by immobilization on each of them of one of the same markers to be quantified on each of the different cell types of the sample under examination.

These elements are introduced into the elongated wells that have captured the specific lymphocytes and into which a liquid phase has been added containing a ligand for each marker. The two solid phases of different conformation compete with the immobilized lymphocytes on the elongated wells, possibly they are sensitized by the tuberculous infection, for the capture of ligands for the markers to be quantified.

Method

The method for detecting different markers of tuberculous infection in different cooperating and cytolytic lymphocyte populations and for their quantification involves the use of elongated well devices for the implementation of the competition reaction between two solid phases of different conformation, through following steps:

- on the surface of the elongated wells, thanks to the preventive immobilization of specific capture agents, the different CD4 or CD8 T lymphocytes present in the sample under examination are selectively bound;

- in a subsequent step, different biotinylated ligands for the lymphocyte markers of infection, to be quantified, are added in the liquid phase in the elongated wells, in which the second solid phase is also immediately introduced, consisting of elements protruding from a rod; a capture agent equal to one of the different markers to be detected on the lymphocytes is immobilized on each of the elements introduced into each elongated well;

- the biotinylated ligands therefore bind to the respective tips in an amount inversely proportional to the quantity of each marker expressed on the CD4 or CD8 T lymphocytes captured on the first solid phase, constituted by the elongated well;

- in a subsequent analytical step, the device with elements protruding from a rod is immersed entirely in a container containing the conjugate consisting of enzyme and streptoavidin, which offers an economic advantage over the use of microplates or microstrips and is possible for the the fact that a single type of conjugate consisting of enzyme-streptoavidin is suitable for detecting the presence of the different biotinylated ligands captured by the elements; the quantity of conjugate captured by each element is inversely proportional to the quantity of the respective lymphocyte marker of the sample under examination;

- finally, the elements are individually immersed in the wells of microplates or standard microstrips containing the chromogenic substrate in which the colorimetric reaction that can be measured through spectrophotometric reading takes place.

Kit

The kits for the diagnosis of tuberculosis based on the simultaneous quantification of different markers of infection present on the different lymphocyte populations are structured in such a way as to essentially contain:

- microstrips or microplates with elongated wells, as described above, on each of which a capture agent is immobilized for a specific type of lymphocyte to be detected in the sample;

- rods with protruding elements, each of which sensitized with a capture agent equal to one of the different markers to be detected in each type of lymphocyte of the sample immobilized on the elongated wells;

- liquid phase containing known mix of ligands for the markers to be quantified on the different lymphocyte populations immobilized on the elongated wells, which compete, for the capture of these ligands, with the elements protruding from the rod; these ligands are biotinylated;

- tube containing the horseradish peroxidase / streptoavidin conjugate, in which the rod with immunoadsorbent elements is immersed, after incubation in the mix of ligands in the elongated wells; - microstrips or microplates with chromogenic substrate distributed in the wells where the immunoadsorbent elements are individually immersed, in the final analytical step.

The kits structured in this way allow to propose a solution for the main diagnostic problems of human and bovine tuberculosis, such as the extra-pulmonary localization of the infection, the long times of laboratory procedures, the availability of equipped laboratories, expensive equipment and of experienced staff.

In the veterinary field, the kits structured in this way allow easier execution of bovine tuberculosis prophylaxis operations and respond to the need to identify ruminants infected with paratuberculosis at an early stage, to reduce the spread of infection in the farm.

The devised elongated-well devices and their use in the competition reaction between two solid phases of different conformation, which allows two types of multiple detection, such as the identification of cell types that expose certain markers and the quantitative determination of the different markers of interest , are applicable, mutatis mutandis, to the detection and quantification of cellular and molecular markers, including antibodies, differentiating the cellular or molecular types or the different antibody classes that expose them and which can be selected from a heterogeneous sample, through ready-to-use kits. use that does not require laboratory equipment and specialized personnel for the execution of the tests.

On the basis of the specific analytical and diagnostic needs, the dimensions and the arrangement of the elongated wells that constitute the designed devices can be selected, as described below.

The microstrips or microplates with elongated wells have the dimensions of the standard 8- or 12-well microstrips or the standard 96-well microplates with circular cross section; the modification consists in oversizing the wells in the direction of the length of the microstrip or according to the direction of the rows or, alternatively, of the columns of the microplate, in relation to the desired format, so that each microstrip, or each row or each column of microplate contains wells stretched according to one of the following methods:

- 2 elongated wells, each corresponding to the extension of 4 circular wells, can be present in the microstrip of the size of a standard 8-well microstrip and each elongated well can accommodate 4 elements protruding from a rod, on which said elements are arranged at modular spacing that coincides with the modular well arrangement of standard 96-well microplates or standard 8- or 12-well microstrips; - 2 elongated wells, each corresponding to the extent of 6 aligned wells of a standard 96-well microplate, can be present in the microstrip of the size of a standard 12-well microstrip and each elongated well can accommodate 6 of the above elements;

- 3 elongated wells, each corresponding to the extension of 4 aligned wells of a standard 96-well microplate, can be present in the microstrip of the size of a standard 12-well microstrip and each elongated well can accommodate 4 of the above elements;

- 4 elongated wells, each corresponding to the extent of 3 aligned wells of a standard 96-well microplate, can be present in the microstrip of the size of a standard 12-well microstrip and each elongated well can accommodate 3 of the above elements.

The microplate of the size of a standard 96-well microplate may contain:

- 2 elongated wells, each corresponding to the extension of 4 aligned wells of a standard 96-well microplate, in each of the 12 columns marked from 1 to 12, for a total of 24 elongated wells, each suitable for accommodating 4 of the above elements; - 2 elongated wells, each corresponding to the extension of 6 aligned wells of a standard 96-well microplate, in each of the 8 rows marked from A to H, for a total of 16 elongated wells, each suitable for accommodating 6 of the above elements;

- 3 elongated wells, each corresponding to the extension of 4 aligned wells of a standard 96-well microplate, in each of the 8 rows marked from A to H, for a total of 24 elongated wells, each suitable to accommodate 4 of the above elements;

- 4 elongated wells, each corresponding to the extension of 3 aligned wells of a standard 96-well microplate, in each of the 8 rows marked from A to H, for a total of 32 elongated wells, each suitable to accommodate 3 of the above elements.

The constituent material, the internal surface and the shape of the bottom of the elongated wells can be chosen and combined according to specific analytical and diagnostic needs, such as for the cultivation of cells for which the presence and quantity of certain markers is to be detected. surface, or for the fixation of cells of thin sections obtained with the microtome from frozen material or embedded material, for the adsorption or cultivation or fixation of cells that have undergone infection, to detect the presence of virusspecific or virus-induced antigens following infection with street or vaccine jambs, for the immunocapture of antibodies of different antibody classes of which we want to detect the specificity, for the attraction of paramagnetic beads coated with antibodies or antigens and which have bound the respective targets in immunocomplex molecular or cellular, which are held on the bottom of the well through a magn you are placed outside.

The list is not exhaustive and is provided for illustrative purposes only.

State of the art and innovation presented

The indirect diagnosis of tuberculosis, supported by Mycobacterium tubercubsis, an infection with the highest incidence in the human population worldwide, cannot be performed with normal serological methods and is based on the methods of detecting the cellular response, such as:

- in vivo test with skin test, which must be read after 72 hours, useful for highlighting cases of infection regardless of the location of the mycobacteria, because it is based on a delayed type IV hypersensitivity reaction of the infected host following in vivo inoculation of haptens of tuberculous mycobacterium;

- blood sample test by gamma-interferon test, commercially available, which must be read after 18-24 hours, which highlights the release of interleukins, in particular of gammainterferon, from the cooperating TH1 lymphocytes of the cell-mediated response, if previously sensitized by the tuberculous infection, when they are cultured and stimulated by the presence of tuberculous antigens.

In humans, the distinction between active tuberculosis and latent tuberculosis is important for a targeted therapy of appropriate duration, of 9 or 6 months, respectively, and for monitoring the evolution of the infection during therapy; it is based on the detection of the number of TH1 lymphocytes - sensitized towards specific tuberculous antigens such as ESAT-6, CFP-10, TB7.7 - which secrete gamma-interferon and other interleukins when stimulated by the presence of the antigens with which they are placed in culture.

To circumvent the problem of the long time required for the execution of the analyzes involving the culture of lymphocytes, we propose here the direct detection and quantification of the lymphocyte markers that must necessarily be present on the lymphocyte membrane for the stimulation of the lymphocyte to produce interleukins. when stimulated by a specific antigen.

The structuring of kits for a rapid screening test or diagnosis of tuberculous infection in humans, which can be performed in a few hours and with minimal equipment, would be of great use during large health campaigns, in the event of health emergencies, for health checks. in the case of large movements of people from areas with a high incidence of infection to free areas, for territories where the structural and infrastructural equipment does not allow the capillary monitoring of infected subjects with the diagnostic methods currently marketed.

The indirect diagnosis of bovine tuberculosis, infection sustained by Mycobacterium bovis, constantly subject to eradication plans for the serious diseases it induces in farmed livestock and for being one of the main zoonoses, cannot be performed with normal serological methods and is based on methods cell response detection, such as:

- in vivo or skin test, which must be read after 72 hours, which highlights a type IV delayed hypersensitivity reaction of the infected host, following the inoculation of mycobacterium haptens; - blood sample test by gamma-interferon test, which must be read after 18-24 hours, which highlights the release of interleukins, in particular gamma-interferon, from TH1 lymphocytes cooperating in the cell-mediated response, if previously sensitized by the infection tuberculous, when cultured and stimulated by the presence of tuberculous antigens or haptens.

In cattle, the distinction between active tuberculosis and latent tuberculosis appears superfluous, since the mandatory prophylaxis plans approved at national and international level do not allow the maintenance of infected animals, at any stage. The skin test, which requires the Official Veterinarian to visit the farm twice 3 days apart, is gradually replaced by the commercial gamma-interferon detection test.

The preparation of kits for the rapid screening test for bovine tuberculosis would make the controls established by national and supra-national prophylaxis plans easier and cheaper, allowing a great saving of resources, both in the areas that have already achieved its eradication, and in the areas where tuberculosis is still present and constant control of the animals reared is necessary to stem the spread of the infection among animal populations and to reduce the risk of transmission to humans of this important zoonosis.

Quick and easy-to-perform diagnostic kits are also needed for the indirect early-stage diagnosis of paratuberculosis or Johne's disease, infection sustained by Mycobacterium avium subspecie paratubercutosis, when the immune response of the infected host is cellular and pathogen excretion. with contamination of the environment and transmission in the animal population; the humoral immune response, detectable through normal serological tests, develops in the subsequent stages and presents fewer investigation problems. Paratuberculosis, in addition to affecting the profitability of ruminant farms, is a suspected zoonosis probably correctable to Crohn's disease in humans.

In the state of the art there are no systems for the structuring of rapid diagnostic kits which highlight the host's cellular immune response to pathogenic microorganisms that induce infections that cannot be diagnosed through antibody detection assays; moreover, there are no systems that can be used without laboratory instruments, based on the simultaneous detection of different cells that expose specific markers of infection that can be simultaneously quantified; neither are solid phases found for immunoenzymatic reactions and ELISAs which could be useful for the execution of simultaneous detection assays both of different markers and of the different cells that expose them.

Patent EP 0 154 687 illustrates, in fact, a system characterized by a first level of recess which receives the liquid containing the cells, which submerges the single wells in which, through the immersion-absorbent elements that are inserted, the presence of proteins secreted by the cells themselves and not by markers present on their surface. Furthermore, the cell type could be identified following the recovery of the liquid present in each well, with another test, in ways that are not compatible with the preparation of rapid assays.

WO 03/085401, from which the present invention derives, illustrates a system that offers the possibility of preparing kits that do not require laboratory instruments for carrying out multiple enzyme immunoassays, as they include microplates or pre-filled microstrips of the reagents. necessary for the execution of essays. The advantage is given by the use of the microplate as a mere container of reagents, while the solid phase function is assumed by the spiked device which can be directly immersed in the sample collection tube and then in the wells of the microplates or microstrips containing the reagents. . The device and the method presented in WO 03/085401 and in the scientific publications that illustrate the experimental results obtained with its application in different fields are not, however, sufficient to carry out, in the same analytical procedure, two types of multiple detection: identification of the lymphocyte types that exhibit certain markers and the quantitative determination of the different markers of interest, in order to meet the diagnostic needs for tuberculosis, an infection characterized by the predominant cellular immune response. They are suitable for the diagnostic purposes illustrated when they are used in combination with an innovative solid phase with elongated wells, specially shaped to accommodate the tips in groups.

WO 2207/039400 illustrates a procedure and a kit based on the simultaneous detection of different cytokines produced in excess by lymphocytes sensitized by tuberculous infection when stimulated by certain tuberculous antigens with which they are incubated for 6 -72 hours, compared to other tuberculous antigens used as controls; the execution of the procedure requires expert personnel and equipped laboratories.

DE 103 33 545 illustrates a system with various modules that are composed to obtain multiple levels of withdrawal with variously shaped and arranged elements, which are not suitable for being introduced into a commercially available ELISA reader to proceed with the reading of the analytical results.

WO 96/02836 describes a computerized system, incompatible with the purposes herein expressed, for nucleic acid analysis which are possible only in the case of the presence of the pathogen in the clinical sample and are therefore not applicable to the diagnosis of extra pulmonary tuberculous infection; in the system there is a flat-toothed comb, which can penetrate into specific wells, but which has dimensions and formats such that it cannot be used according to the purposes expressed in the present invention, which also provides for the immersion of the entire device with tips in test tubes, to obtain a reduction in the costs of kits for the diagnosis of tuberculosis, in order to facilitate the diagnosis in developing countries with the highest incidence of infection in the population.

In US 2007/237687 the illustrated devices differ substantially from the microplates and microstrips with elongated wells presented here due to the presence of bottlenecks that would interfere with the homogeneous distribution of the sample inside them and with the homogeneous distribution of the liquid phase in contact with the 2 solid phases, essential for obtaining a reliable diagnostic result.

In US 2010/083778 there are devices suitable for retaining non-porous substrates that are subjected to simultaneous screening, absolutely unsuitable for the preparation of the kits presented here as they do not provide for separation into single wells, which is essential for the correct quantification of the markers of interest. diagnostic illustrated here.

In DE 10 2008 021365 the support suitable for receiving reagents through microcannulas differs from what is conceived and presented here above all due to the fact that it does not have several elongated wells in the same row, useful for the preparation of a kit where it is possible to set up a control test to be performed in parallel to the sample analysis, in a single row of a microplate or in a microstrip entirely dedicated to a sample to be analyzed for the detection of different cell types, each of which exhibits different markers to be quantified.

The present invention was therefore born to satisfy the need to realize a device, a method and kit useful for the detection of different cellular or molecular markers and their simultaneous quantification on the different cells or molecules that expose them; in particular, the kits are useful for the diagnosis of tuberculosis, being easy to use and requiring minimal laboratory equipment.

Construction of kits for rapid diagnosis of tuberculosis based on the simultaneous detection of different markers exposed on CD4 T lymphocytes and CD8 T lymphocytes

Examples of ready-to-use kit structuring using elongated well devices and the competition reaction between 2 solid phases of different conformation are shown below, designed for the rapid diagnosis of human tuberculosis and for the rapid diagnosis of combined bovine tuberculosis. to the diagnosis of paratuberculosis at an early stage, for the issuance of the analytical report within a few hours, based on the detection of cytolytic CD 8 T lymphocytes, TH1 lymphocytes cooperating in the cellular response and TH2 lymphocytes cooperating in the humoral T CD 4 response, sensitized in consequence of tuberculous infection.

An example of structuring a ready-to-use diagnostic kit for human tuberculosis, for single sample, may include:

- 1 elongated 3-well microstrip, each of the extension of 4 circular wells of standard 96-well microplate, coated as specified below:

<■> well 1 is sensitized with anti CD4 monoclonal antibody for binding with cooperating T lymphocytes, which bind in immunocomplex on the surface;

<■> well 2 is sensitized with anti CD8 monoclonal antibody for binding to cytolytic T lymphocytes, which bind in immunocomplex on the surface;

<■> well 3 is not sensitized and acts as a negative control to highlight any non-specific reactions;

- 1 disposable pipette for the distribution of the 3 sample aliquots in the 3 elongated wells; - 1 bottle of washing solution with dispenser for washing the elongated wells after the sample incubation;

- 2 tubes with washing solution of such dimensions that the entire tip device can be immersed in it after the tip incubation phases;

- 3 ampoules containing standardized mix of Mycobacterium tubercutosis antigens, which, by way of non-exhaustive example, can be ESAT-6, CFP-10 and TB 7.7 biotinylated;

- 1 device with 12 immunoadsorbent elements protruding from a rod, sensitized with monoclonal antibodies (MAb) as specified below:

<■> element 1: MAb for ESAT -6;

<■> element 2: MAb for CFP-10;

<■> element 3: MAb for TB 7.7;

<■> element 4: no sensitization - negative control;

<■> element 5: MAb for ESAT -6;

<■> element 6: MAb for CFP-10;

<■> element 7: MAb for TB 7.7;

<■> element 8: no sensitization - negative control;

<■> element 9: MAb for ESAT -6;

<■> element 10: MAb for CFP-10;

<■> element 11: MAb for TB 7.7;

<■> element 12: no sensitization - negative control;

- 1 tube containing conjugate, for example horseradish-streptoavidin peroxidase;

- 1 standard 12-well microstrip;

- 1 bottle with dripper, containing chroniogenic substrate, for example trimethylbenzidine CTMB);

- bibulous paper pads.

Alternatively, a standard 96-well non-immunoadsorbent microplate can be used, pre-filled with washing solution for the elements, conjugate and chromogenic substrate, distributed as follows:

- washing solution in the wells of rows A, B, C, E, F and G;

- streptoavidin / enzyme conjugate in the wells of row D;

- chromogenic substrate in the wells of row H.

1. CAPTURE OF LYMPHOCYTES - The blood sample added with anticoagulant should be dispensed, in three aliquots of 1 mL each, into the elongated wells of the microstrip. It is left to incubate to allow the capture of the cooperating T lymphocytes at the surface of the elongated well 1 and of the cytolytic T lymphocytes at the surface of the elongated well 2.

2. COMPETITION BETWEEN 2 SOLID PHASES FOR THE BINDING WITH BIOTINYLATED ANTIGENS - At the end of the incubation, the elongated wells must be washed 3 times with washing solution; a vial containing the standardized mix of ESAT-6, CFP-10 and TB 7.7 biotinylated antigens is injected into each well.

The 12-element sensitized device is placed on the microstrip so as to immerse 4 elements in each elongated well and it is left to incubate.

Antibodies immobilized on device elements bind biotinylated antigens by competing, in elongated well 1, with tuberculous infection sensitized CD4 T cells captured on the surface and competing, in elongated well 2, with captured tuberculously infected CD8 T cells on surface.

At the end of the incubation, the quantity of biotinylated antigens bound in immunocomplex on each specific element is inversely proportional to the presence of the sensitized and captured lymphocyte markers on the surface of the elongated well, which have bound part of the same antigens through the specific surface receptors, where present as a consequence of tuberculous infection.

3. BINDING THE CONJUGATE TO THE ELEMENTS - At the end of the incubation, the device with immunoadsorbent elements protruding from a rod should be lifted and immersed in one of the two tubes containing washing solution. After drying of the tips, by contact of the distal end with the bibulous paper pad, the entire device is immersed in the tube containing the conjugate and placed to incubate.

Alternatively, if the reagents are pre-distributed in microplates, the elements must be introduced into the wells of row A of the standard 96-well microplate, so that each element penetrates into a well containing washing solution, then the elements must be dried on bibulous paper. . The washing must also be repeated in the wells of rows B and C, always followed by drying. After washing, the elements are immersed in the wells of row D, containing the conjugate and set to incubate.

4. CHROMOGENIC REACTION - At the end of the incubation in the conjugate, the device with immunoadsorbent elements protruding from a rod must be lifted and introduced into the other of the two tubes containing washing solution. After drying of the elements, by contact of the distal end with the bibulous paper pad, they are individually immersed in the wells of the microstrip in which the chromogenic substrate has been previously distributed.

Alternatively, if the reagents are pre-distributed in microplates, the elements are washed in the wells of rows E, F and G, dried each time and finally immersed in the wells of row H, containing the chromogenic substrate.

After incubation, the elements are extracted and the spectrophotometric reading is carried out by means of an ELISA reader of the D.O. of the solution in the wells in which the chromogenic reaction has developed. In the absence of reading tools, visual reading can be performed by comparing the color of each test well with that of the wells containing the negative controls.

5. INTERPRETATION OF RESULTS - The D.O. detected is inversely proportional to the quantity of lymphocytes present in the blood sample, respectively cooperating CD4 or cytotoxic CD8, for the respective tuberculous antigen; the interpretation of the test results must be made in relation to the D. 0. measured in correspondence with elements 9, 10 and 11 which, in the absence of competing lymphocytes in the elongated well 3, bind the maximum quantity of each biotinylated antigen, while the elements 4, 8 and 12 constitute the specificity check.

The positive control must be set up in parallel to obtain the calibration curve useful for the quantitative interpretation of the analytical results and the calculation of the cut-off.

On the basis of scientific advances, different markers and different cell types can be substituted or introduced among those that the kit may be able to detect and quantify and of which only one example has been given.

The structuring of a similar kit for the diagnosis of bovine tuberculosis combined with the diagnosis of early stage paratuberculosis can be achieved with haptens:

- bovine tuberculin as a marker of tuberculous infection to be quantified on the different CD4 and CD8 T lymphocytes;

- avian tuberculin, to detect false positive results due to cross-reaction of the transient infection with Mycobacterium aviurn,

- johnina, as a marker of paratuberculous infection to be quantified on different CD4 and CD8 T lymphocytes.

The ready-to-use kits for numerous samples must be structured to include at least:

- microstrip with 3 elongated wells, each of which is able to accommodate 4 ogival tips of the sensitized devices, or the elongated 3-well microplates arranged in 8 rows corresponding to the rows marked from A to H in the standard microplates, sensitized as above specified for each type of diagnosis;

- vials with the standardized mix of antigens or biotinylated haptens;

- devices with 12 sensitized tips as described above for each type of diagnosis;

- tubes containing washing solution, tubes containing the conjugate, standard 96-well microplates or a sufficient number of standard 12-well microstrips where the chromogenic substrate is to be distributed;

- bibulous paper pads for drying the tips.

In the case of use of pre-filled microplates, standard 96-well microplates or a sufficient number of standard 12-well microstrips pre-filled with conjugate and as many pre-filled must be included in the kits, alternatively to the tubes with conjugate and washing solution. with chromogenic substrate;

- bottles with washing solution equipped with dispenser;

- support for simultaneously containing and moving the 12-element immunoadsorbent devices from the microplate containing the conjugate to the one containing the chromogenic substrate. In this case, the manual execution of the tests requires that, after incubating the blood samples in the elongated wells and washing them, the standardized mix of antigens or haptens of the chosen mycobacteria is inoculated in each of them and positioned the support containing 8 devices with 12 sensitized immunoadsorbent elements, which allows the devices themselves to be moved at the same time, so that the immunoadsorbent elements are: - immersed in the elongated wells and placed to incubate; - washed and dried; - immersed in the standard wells containing the conjugate and placed to incubate; - washed and dried; - immersed in the standard wells containing the chromogenic substrate and placed to incubate; - extracts for reading by ELISA reader of these last wells, in which the chromogenic reaction has developed.

The simplicity of the operations that follow one another in the diagnostic procedure makes it easy to robotize the use of the kits prepared as described.

Brief description of the figures

In Figure 1, the microstrip (11) has the size of a standard 12-well microstrip and contains 3 elongated wells of equal size; each elongated well (10) is suitable to accommodate 4 elements (12) of the 12-element device (13), which protrude from a rod with modular distance coinciding with the modular distance of the wells of standard 96-well microplates and standard microstrips 12-well.

In Figure 2, the microstrip (21) has the size of a standard 12-well microstrip and contains 4 elongated wells of equal size; each elongated well (20) is suitable to accommodate 3 elements (22) of the 12-element device (23), which protrude from a rod with modular distance coinciding with the modular distance of the wells of standard 96-well microplates and standard microstrips 12-well.

In Figure 3, the microstrip (31) has the size of a standard 12-well microstrip and contains 2 elongated wells of equal size; each elongated well (30) is suitable to accommodate 6 elements (32) of the 12-element device (33), which protrude from a rod with modular distance coinciding with the modular distance of the wells of standard 96-well microplates and standard microstrips 12-well.

In Figure 4, the microstrip (41) has the size of a standard 8-well microstrip and contains 2 elongated wells of equal size; each elongated well (40) is suitable to accommodate 4 elements (42) of the 8-element device (43), which protrude from a rod with modular distance coinciding with the modular distance of the wells of standard 96-well microplates and standard microstrips with 8 wells.

In Figure 5, the microplate (51) has the size of a standard 96-well microplate and contains 24 elongated wells of equal size; each elongated well (50) is suitable for accommodating 4 elements (52) of the 12-element device (53), which protrude from a rod having a modular distance coinciding with the modular distance of the wells of standard 96-well microplates.

In Figure 6, the microplate (61) has the size of a standard 96-well microplate and contains 32 elongated wells of equal size; each elongated well (60) is suitable for accommodating 3 elements (62) of the 12-element device (63), which protrude from a rod having a modular distance coinciding with the modular distance of the wells of standard 96-well microplates.

Claims (2)

Claims 1. Device for the detection of different markers in different cellular or molecular types and their quantification consisting of microstrips or microplates with wells and rods with protruding immunoadsorbent elements placed at a modular distance according to the arrangement of wells of standard microplates or microstrips, characterized by the fact that the wells have an elongated shape able to accommodate 3 or 4 or 6 of said elements inside them, that the wells immobilize a different cell or molecular type present in the sample under examination, that the immunoadsorbent elements are sensitized with one of the same markers that are want to detect in the sample under examination, that, when the protruding elements are introduced into the wells, said wells contain a ligand in liquid phase both for the markers of the different cellular or molecular types present in the sample under examination immobilized in said wells both for the markers and sensitize the protruding elements of the rods. 2. Method for the detection of different markers in different cellular or molecular types and their quantification using the devices of the previous claim characterized in that it comprises the following steps: a) on wells of microplates or microstrips, of elongated shape, able to accommodate 3 or 4 or 6 immunoadsorbent elements protruding from rods placed at modular distances, a different cellular or molecular type present in the sample under examination is immobilized; b) said markers to be detected in the sample under examination are immobilized on the protruding immunoadsorbent elements; c) the immunoadsorbent elements protruding from the rods are introduced into the elongated wells filled with a ligand in the liquid phase for the markers to be detected and, therefore, both for the different cellular or molecular types present in the sample under examination immobilized in said wells and for the markers which sensitize the protruding elements of the rods; d) said ligands bind to the immunoadsorbent elements in an amount inversely proportional to the quantity of the relative markers expressed by each cell or molecular type immobilized on the relative elongated well and can be quantified simultaneously by means of an enzyme immunoassay; e) the rod or rods carrying the immunoadsorbent elements are immersed in a container containing the conjugate; f) and, subsequently, each single element is immersed in single wells of standard microstrips or microplates containing chromogenic substrate, g) in each of which, finally, the spectrophotometric reading is performed. Kit for the detection of different markers in different cellular or molecular types and their quantification using the device and the method according to claims 1 and 2 characterized in that it comprises: - microstrip with 3 elongated wells, each of which is able to accommodate 4 sensitized elements protruding from a rod; the first elongated well is sensitized with anti CD4 antibodies for the immobilization of cooperating T lymphocytes present in a blood sample and the second elongated well is sensitized with anti CD8 antibodies for the immobilization of the cytolytic T cells present in the same blood sample; an elongated third well is not sensitized and acts as a negative control, being pre-filled with buffer solution; vials with a standardized mix of antigens or haptens of mycobacteria whose infection is to be diagnosed, able to bind specifically to the markers to be detected on the lymphocytes of the sample under examination; devices with 12 protruding elements, each of which is sensitized with one of the markers to be detected on each type of lymphocytes sensitized by mycobacterial infection; 96-well standard microplates with 8 rows of 12 wells each, identified respectively with the letters A, B, C, D, E, F, G and H, non-immunoadsorbent, or containers in the form of tubes or trays containing conjugate and solution of wash; 96-well standard microplates with 8 rows of 12 wells each, identified respectively with the letters A, B, C, D, E, F, G and H, non-immunoadsorbent, containing chromogenic substrate; bibula paper pads.