ITRM980266A1 - GLOBAL SYSTEM INCLUDING COMPACT AND ROBOTIZED INSTRUMENT AND SPECIAL REACTIVE CHAMBERS FOR COMPLETE DETERMINATION - Google Patents

Description

TITLE: GLOBAL SYSTEM INCLUDING COMPACT AND ROBOTIZED INSTRUMENT AND SPECIAL REACTIVE CHAMBERS, FOR THE DETERMINATION IN FULL AUTOMATIC AND CONTROLLED TEMPERATURE, OF TOTAL OR DIRECT ULINE IMMUNOGLOB AGAINST ALLERGENS OR OTHER MOLECULES (ANTIBODIES OR ALLERGENS).

INDUSTRIAL INVENTION DESCRIPTION FORM

SUMMARY

Instrument to determine the presence of total or direct immunoglobulins against allergens or other molecules (anti-bodies or allergens) in aqueous samples in a completely automatic and temperature-controlled way.

TEXT OF THE DESCRIPTION

The present invention describes a complete system called "ENEASYSTEM II T.C" used to measure analytes present in solution by solid phase separation with immunochemical methodology. The system consists of a single compact and robotic instrument, completely automatic, equipped with particular capillary containers in the shape of a "U" to which a reagent is adhered that reacts specifically with the analyte present in the fluid under examination, such as chambers of reaction. The instrument is able to dispense the samples under examination into the reaction chamber, wait the necessary time for the reaction to take place, remove the non-reagent solution by washing appropriate, technically innovative, circular flow by dispensing from a hole in the "U" chamber "And subsequent aspiration from the other hole, dispense the reagent called tracer, wait the time necessary for the second reaction to occur, repeat the washing procedures of the solution that has not reacted, dispense the appropriate detection solution, wait for the reaction to develop and read the color or energy variation that occurs.

And the field of application of the System essentially refers to the in vitro diagnosis of allergies and autoimmune diseases.

Essential characteristics are represented by:

1. total automation of the procedure using a robotic instrument: "mind that controls a single arm" that performs all the necessary operations on the plate without any movement of the reactive chambers and without any intervention by the operator;

2. control of the reaction temperature throughout the procedure;

3. use of a special reaction chamber with an innovative "U" shape feature that allows for the achievement of particular qualitative performance;

4. innovative circular flow washing technique for reactive chambers;

5. standardization carried out for each group of molecules analyzed (for specific IgE carried out for families of allergens); this standardization is stored by the instrument and can be rechecked by recalibration at the user's discretion according to good laboratory practice;

Basic principles:

the analytes present in biological fluids are generally determined by specific immunological reactions between antibody and antigen that form an immune complex. The reagent that reacts specifically with the analytic is marked so as to allow the detection and measurement of the concentration of the analyte itself. The marking can be done with radioactive, fluorescent, chemioluminescent or enzyme emitters. In the latter case, the signal relating to the enzyme must be expressed by adding a suitable substrate that must change its state so as to become detectable (for example by changing color and energy state).

To facilitate the separation between the analyte that has reacted and that which has not reacted (and which therefore is not specifically related to the test performed), different solid phases are used. The binding of the specific reagent to the chosen solid phase allows to easily separate the immune complex that is formed between the specific reagent and the analyte.

It is possible to use a large number of solid phases, such as paper discs, plastic spheres, microplate wells, test tubes, cellulose capsules and so on. If the solid phase is mobile, there is a risk of inadvertent removal of the same in the course of the reaction with the consequent impossibility of carrying out the analysis successfully. If the solid phase is of the fixed type, the reaction volume is very large (from 50 to 200 ul).

Some of the solid phases mentioned above allow to automate some or even all the procedure steps (sample and reagent dispensing, washing and reaction detection); in the first case with the use of a series of instruments each with a dedicated function such as dispenser, washer and reader and which therefore requires the intermediate manual intervention of the operator, in the second with systems in which total automation is entrusted to the management of different compartments with specific function, that is to transport the work plates from one to another, considered in any case modular; but none of them allows the complete automation of the whole procedure, in the sense of using a single, compact instrument, with a fixed work plate (therefore reaction chambers) on which a robotic arm performs each operation thus acting as a dispenser, washer and reader.

The "ENEASYSTEM II T.C. " comprehends.

1. ENEAS II TC SRA Automatic Robotic Instrument for carrying out the entire procedure at a controlled temperature with acoustic signaling of the out-of-range; 2. ENEAS II TC CR Reactive Chambers: innovative and special reaction chambers; 3. ENEAS II TC TRACER, reagents for the detection of the reaction;

4. ENEAS II TC REFERENCE, reference standard for each different group of molecules,

5. ENEAS II TC CALIBRATORS, control sera for the recalibration of the standard curve, one for each different group of molecules;

ENEAS II TC SA Automatic Instrument

The tool for the automatic execution of the analysis procedure is a compact device with dimensions of 50.00 cm x 60.00 cm x 40.00 cm, including:

• an AT computer, with hard disk, a 3 and 1⁄2-inch disk drive, 9-inch keyboard and video; the computer uses a program dedicated to the management of the mechanical parts of the instrument itself, the execution of the procedure and the management of a patient archive;

• a mechanical working arm equipped with 10 needles for washing the reaction chambers (5 needles for aspiration and 5 needles for injecting the washing solution), a needle for dispensing the sample and the tracer reagent, and a needle for dispensing the detection reagent;

• an optical head for reading the final result directly in the reaction chambers, head which is anchored to the aforementioned mechanical arm;

• two 10 mL syringes, connected to two diluters for dispensing the reaction solutions;

• two peristaltic pumps for dispensing the washing solution and unloading the tubes;

• a vacuum pump (alternatively a peristaltic pump) for suction of the reaction and washing solutions;

• a work surface for chambers and sample holders, made up of 3 central recesses equipped with a thermostated surface with sensors / indicator, for the positioning of the plates - containers of the reaction chambers and 4 lateral recesses for the positioning of the containers for samples and controls;

• plates - containers for the reaction chambers; each container carries 10 rows of 15 reaction chambers;

• sample containers; each container has 10/20 positions indifferently for 1 ml or 2 ml cups;

• containers for controls; each container has 5 positions for 1 ml cups;

• drain holes for the washing solutions of the tubes;

• department for the positioning of reagents and washing solution;

• container for waste solutions;

• printer;

• cover for the front face of the instrument for the isolation of the work area.

The user has the ability to record, through the dedicated program, the information relating to the patient to be analyzed and request, by scrolling through a prepared panel, the analyzes relating to each patient. At the end of the data entry for each patient, the program presents a work plan proposal and indicates how to position the reaction chamber containers in the chamber plates. The program asks if it is necessary to recalibrate the standard curves and if so, it asks to insert the relative cups in the preset positions.

Once the indicated preliminary preparation has been carried out and the analysis send key has been pressed, the instrument first adds the normal temperature and then begins to carry out the procedure. The latter involves dispensing the samples, waiting for the appropriate reaction times, washing and reading the results, after zeroing on each reaction chamber. At the end of the analysis procedure, the program compares each data obtained with the standard curve stored for the group to which the required analyte belongs and calculates the values in Relative Units and Positivity Classes. Through the printer, the response cards relating to each patient are reproduced, showing the patient's data and the results obtained for each of the required analyzes, results that are reported both in Relative Units and in Positivity Classes.

The data relating to each patient and the analysis performed are stored in the journal archive present in the program and can also be sent via an appropriate connection to the user's central computer.

ENEAS II TC CR. Reactive Rooms

The reactive chambers have a capillary-sized “U” shape. Each chamber has a vertical arm with a smaller diameter (1.20 mm) and a larger vertical arm (diameter 2.00 mm), connected to a basic horizontal arm with a diameter of 1.00 mm.

The distance between the center of one arm and the center of the second arm is 2.30 mm. The rooms can be presented in groups of 5 or individually. If in groups of 5 the distance between the center of one arm of one chamber and the center of the arm of the adjacent chamber is 2.45 mm.

Each chamber consists of an upper part in glossy polystyrene and a cap in opal polyethylene of different colors according to the groups of molecules. The material composition of the chambers can change due to production needs.

The shape of the chamber was designed as described to allow the dispensing of samples and reagents without the formation of bubbles and for carrying out an accurate washing of non-reactive fluids. The innovative principle is placed in the circularity of the washing flow which is repeated several times depending on the procedure in question. The effectiveness of this technique allows for high efficiency. The particular geometry of the chambers, in the dispensing phase of samples and reagents; associated with the adequate pressure exerted by the fluid to be introduced, therefore with an instrumental characteristic, it allows the elimination of bubbles and homogeneous filling of the chambers.

It may possibly contain solid parts if these represent a qualitative advantage in the reaction.

The volume of sample or reagent contained in the chambers is between 20 and 50 ul. ENEAS II TC TRACER

The tracer system includes:

• a suitable reagent containing the antibody conjugated to the enzyme, such as Ureasi. The antibody is specific for the analyte in question.

The antibody can be represented indifferently by monoclonal or polyclonal proteins and the enzyme can be modified for production and quality needs; as an alternative to the enzyme, it is possible to use different molecules with their own detectable direct signal, for example Iodine-125 with radioactive emission or fluorescent or chemoluminescent substances;

• the detection reagent consisting of a suitable substrate for the enzyme used. In the case of Ureasis, it is purpura of bromine cresol and urea. The presence of Ureasis determines the transformation of urea and consequently the modification of the pH. The pH variation determines the color change of the pH indicator, bromine-cresol purple, from yellow to purple, a variation that is measured by the photometer placed in the reading head. The substrate may change in relation to the enzyme used, or it may be absent altogether if the tracer is labeled with a direct signal emission molecule (for example RIA, fluorescent or chemoluminescent);

• the washing solution consisting of a buffer and a detergent.

ENEAS II TC REFERENCE

The reference system consists of standard sera with a known concentration of analyte, for different concentrations (from a minimum of 3 to a maximum of 8).

There is a standard curve for each homogeneous group of molecules being analyzed and the sera are calibrated against the international standards of each group, if these have been deposited with the responsible legal organizations (for example the WHO in Geneva, standard for Total IgE n. .75 / 502, standard for IgG n.66 / 233).

ENEAS II TC CALIBRATORS

The calibrators consist of a standard serum with a known concentration of analyte for each homogeneous group of molecules being analyzed. The number of different types of calibrators available is equal to the number of reference curves available.

Claims (16)

CLAIMS 1. Instrument for the fully automatic and temperature-controlled determination of molecules and immunoglobulins directed against other molecules (antibodies or allergens) in aqueous samples, comprising: to. reaction chambers to which specific antibodies or other specific molecules are attached or in which segments with these molecules are inserted, b. a first reagent comprising antibodies directed against the analytes to be determined, antibodies made capable of emitting a direct signal, as they are marked by a detectable radiation or other energy emitter, or indirect, as they are marked by an enzyme capable of developing detectable reactions with a suitable with a suitable substrate; said first reagent is intended to come into contact, in vitro, with the aqueous test sample already reacted in the appropriate reaction chambers. Overall formed between the solid phase with the specific molecules attached, substances possibly attached to them and liquid phase irradiating or marked with enzyme, the amount of signal emitted is a function of the concentration of analyte under examination in the aqueous solution tested; c. a second optional reagent capable of expressing the signal given by the presence of the enzyme. 2. The instrument of claim 1 wherein the reaction capillary chambers have a special "U" shape as described above. 3. The instrument of claim 1 and 2 wherein the "U" reaction capillary chambers are single. 4. Instrument of claim 1. and 2. wherein the reaction capillary chambers are in groups of 5. 5. The instrument of claim 1 and 2 wherein the reaction capillary chambers contain reactive segments. 6. The instrument of claim 1., 2., 3. and 4. wherein the reaction capillary chambers have specific molecules attached to the surface. 7. The instrument of claim 1., 2. and 5. wherein the segments contained in the ration chambers have specific molecules attached to the surface. 8. The instrument of claims 1., 2., 6. and 7. wherein the molecules are attached to the surfaces both passively and covalently. 9. Instrument of claims 1., 2. and 4. wherein all 5 reaction chambers contain the same molecules attached to the surface. 10. Instrument of claims 1., 2. and 4. wherein each of the 5 chambers contains a different molecule attached to the surface. 11.Method for the determination and quantification of analytes in aqueous solutions and biological fluids by immunoassay using the means of claims 1., 2., 3., 4., 5., 6., 7., 8., 9. and 10. in an absolutely automatic way through a tool capable of proceeding with all the necessary steps to obtain the result. The method includes: (a) reaching the temperature appropriate to the procedure on the work surface; (b) filling the U-shaped capillary chamber with the sample; (c) waiting for the analyte in the sample to react with the specific molecules attached to the reactive surfaces; (d) removing the sample from said reaction capillary chamber; (e) internal washing of said reaction chamber to remove components not specifically bound to reactive surfaces; (f) filling said reaction chamber with a second reagent capable of reacting with one of the reagents of point (b), namely the reagent present in the sample or the first reagent bound to said reactive surfaces; (g) waiting for the reaction involving the second reagent to take place; (h) removing the second reagent from said reaction capillary chamber; (i) internal washing of said reaction capillary chamber to remove the second non-specifically bound reagent; (j) measuring the amount of said second reactive specifically bound to the reactive surfaces. 12. The method of claim 11 wherein the said second reagent is labeled with the radioactive, fluorescent or luminescent molecule and the amount of said second reactive specifically bound to the reactive surfaces can be measured by counting the amount of radioactivity, fluorescence or luminescence emitted through said reaction capillary chamber. 13. The method of claim 11 wherein said second reagent is labeled with an enzyme and the amount of said second reagent specifically bound to the reactive surfaces can be measured, after addition to said reaction capillary chamber of a substrate, through the quantity of product formed by enzymatic catalysis. 14. Method according to claims 11., 12. and 13. in which the final measurement is quantitative as it is compared with a specific standard curve for each group of molecules analyzed, in which each standard serum is calibrated against the international standard of the his group, if this exists deposited with the competent legal organizations. 15. Tool referred to in claims 11., 12., 13. and 14. capable of automatically carrying out all the necessary reaction steps without any intervention by the operator. 16. Means according to claims 11., 12., 13., 14. and 15. capable of automatically carrying out all the reaction steps at a controlled temperature