PROCEDURE WITHOUT CENTRIFUGATION FOR IMMUNOLOGICAL TESTING OF MATERIALS.

Description

TITLE INV. DES. PRIORITY CAIS MICHAEL SHIMONI MOSHE TECHNION RESEARCH 8 DEVELOPMENT FOUNDATION LTD HAIFA ISRAEL CENTRIFUGATION-FREE PROCEDURE FOR IMMUNOLOGICAL TESTING OF MATERIALS. MICHAEL CAIS MOSHE SHIMONI ISRAEL SUNDAY 9 REV. N. 63363 OF 20 JULY 1981 Ck>i N Rome, li_______________ Register A Protocol No. 3 LI 40 Ministry of Industry, Trade and Crafts 332# Provincial Office of Industry, Commerce and Crafts of Milan COPY OF THE MINUTES OF FILING FOR INDUSTRIAL INVENTION PATENT The snno 1SS2 11 3 ì0rn0 1ICHAEL CAIS Pt Gentlemen of nationality' TWENTY-NINE month of 21VOSHE S^IMONI and xtsjixsffoeJUNE ( the TECHNIQN RESEARCH & DEVELOP- I MENT FOUNDATION LTD. i . lux tut'.i in: HAIEA iiorasleì resident r Via ----------- through agent EL'GNION SPA and elected domiciled * for legal purposes in Milan - Via Carlo Farini, Yes at the agent they presented to me, the undersigned: - Stamp application for the granting of a patent for an industrial invention entitled: Inventors designates: f'.'ENTO WITHOUT CENTRIFUGATION FOR TESTING THE .'NUNOII MATERIALS” VII^AEL CAIS s VOSHE SIVONI Priority of patent application in: ISRAEL No. 63363 of 20 July 1951 CO'.'UN ! the two applicants are 25% co-owners while the applicant is 50% co-owner; corrected by: - Duolo description of η. a6 pages of writing. - Drawings, plates re. ~ in duplicate. Letter of appointment - ^θ(τ»3(Κ»Κΐ)©ίθ(^Κϊβ|!βΒ)ββ>)ΜΧΚίίίχκβ8Χ«8. - Priority document and Italian translation Κθ< fi xjx e «sswxtex - Act of designation of the inventor. - Proof of payment on postal account no. 30663004 in the name of the Tax and Concession Registry Office Rome by L. - Revenue stamp of L. 3.800 issued at the Milan Post Office 39 on 29 / 6 / 82. The finding of which the present application does not constitute any deposits of equal content, issued on the same date, n. 191 by the same owner. .upon request, the descriptions and drawings listed above have been signed by the applicant and countersigned by me and stamped with the office seal THE DEPOSITANT v This is a certified copy of the original THE L'FFtCtALE ROGANTE Idillia Russo p. the Director (Salvatore Ravalli) THE BOSS 22140A / 82 Hon. Ministry of Industry, Commerce and Crafts CENTRAL PATENT OFFICE - ROME -° oo o 0221 ίδ ί _ ______ Messrs. MICHAEL CAIS and MOSHE SHIMONI entered ramPil'cri—naI »*>·* Israeli nationality both residing in HAIFA (Israel) and the company TECHNION RESEARCH fi, DEVELQPMENT FCUNDATIOi. LTD. with registered office in HAIFA (Israel) the first two applicants being co-owners each of 25% and the third applicant being co-owner of 50% and electively domiciled in Milan, Via C, Farini, 81 at the agent and domiciliary BUGNION SpA are applying for a patent certificate for INDUSTRIAL INVENTION entitled: PROCEDURE WITHOUT CENTRIFUGATION FOR TESTING IMMUNOLOGY OF MATERIALS” Designated Inventors: MICHAEL CAIS and MOSHE SHIMONI Priority of patent application in: ISRAEL 63363 of July 20, 1981 For this purpose, the following are attached: ι Description of the finding in duplicate no. 46 pages 2' Lo. 7_duplo drawing boards 3) Att. of vers. in c / c ρ. n. 00668004 of L. 336,000 4) Letter of appointment __ 5) Act of designation of inventors 6J Priority document and Italian translation 2Milan, June 29, 1982 more than Messrs. MICHAEL CAIS and MOSHE SHIMONI and the Company r TECHNION RESEARCH & DEVELOPMENT FOUNDATION LTD. In Faith THE AGENCY BUGNION SpA 24.C057.12IT.3 2Z / es BUGNION SPA DESCRIPTION attached to a patent application for an INDUSTRIAL INVENTION entitled: CENTRIFUGATION-FREE PROCEDURE FOR IMMUNOLOGICAL TESTING OF MATERIALS Applicants: Mr. MICHAEL CAIS and Mr. MOSHE SHIMONI, both of Israeli nationality and both residing in HAIFA (Israel) and the TECHNION RESEARCH & DEVELOPMENT company FOUNDATION LTD. of Israeli nationality with headquarters in HAIFA (Israel) Marciata'· I : EUGNION SpA - Via Carlo Farini, 81 M i 1 aro Deposited J9 61U.19& at No. 22140A / 82 _ 0 _ SUMMARY The present invention relates to a process that eliminates the centrifugation and decantation steps that must be performed automatically to perform specific binding test experiments, in which liquid and solid phases are present. In accordance with the invention, use is made of a specially designed device, which consists of a mixing tank into which a mixing separator having a channel in the axis is inserted with play. BUGNION SpA, vertical mixer-separator. A rack holding a number of such mixing tanks containing the incubated reagents and analytes, covered with separator mixers, is placed in a press device designed to achieve a controlled downward movement. The separator mixers are pushed downward into the mixing tanks at a selected speed for a predetermined distance to complete the separation and mass transport operations. The separation devices are removed and one of the separated phases can be measured in the desired analytical instrument for quantitative or qualitative determination. The results obtained in accordance with the present invention compare very favorably with those determined by other procedures requiring centrifugation and / or decantation in the test protocol. DESCRIPTION The present invention relates to an improved process for performing specific binding test experiments. More specifically, the invention relates to an improved process that must be carried out automatically for performing specific binding test experiments involving both liquid and solid phases. As is known, specific binding tests are based on the principle of controlling specific binding reactions, where the degree of binding is a function of the amount of the known ligand present, by means of a classified component. Among the known procedures, the following specific binding assay techniques can be mentioned: radioimmunoassay (RIA), metalloimmunoassay (MIA), free radical assay technique (FRAT), hemagglutination inhibition (HI), multiple enzyme immunoassay technique (EMIT), fluorescence immunoassay (FIA) and luminescence immunoassay (LIA). In some of these techniques (RIA, MIA, FIA, LIA) the mixture comprising the unclassified binder, the classified binder and the antibody is allowed to reach equilibrium and the antibody-bound ligand is separated from the free binder. In the radioimmunoassay, the ligand or antibody is labeled with a radioactive isotope, while in the metalloimmunoassay the ligand is labeled with a metal-containing reagent which also contains a suitable functional group by which the metal reagent can be attached to the hapten that is to be tested. A complete description of the latter is given in the previous Italian Patent Application of the same Applicant No. 49584A / 77. In FIA the classifier is a fluorescent compound and in LIA the classifier is a cheminoluminescent or bioluminescent agent. The operation of separating the free fraction from the bound fraction is of great importance and its precision determines the sensitivity and accuracy of the entire specific binding test technique. When choosing and testing a separation operation, it is useful to consider the criteria that must be satisfied in order to achieve the desired result. The following main requirements for an ideal separation can be mentioned: It should completely separate bound and free fractions with a large margin for error under the conditions used for separation; (ii) It should not interfere with the primary antigen-antibody binding reaction; (iii) It should be simple, easy and quick to ;0N SpA TO. use ; (iv) It should be inexpensive and use reagents and equipment that are readily available; (v) It should not be influenced by plasma or serum; (vi) All manipulations should be performed in a single tube; (vii) It should be suitable for automation; (viii) It should be applicable to a broad range of antigens; (ix) Handling steps in radioimmunoassays should be designed to ensure maximum safety from radiation hazards resulting from handling of the radioactive reaction system. A critical examination of the variety of available procedures and the degree to which each procedure achieves all or some of the ideal requirements mentioned above is beyond the scope of this description. The most widely used techniques mentioned in the prior art are adsorption processes (soft coal, silicates), fractional precipitation processes (ammonium sulphate, ethanol, dioxane, polyethylene glycol), double anti-staining processes s. PA and solid-phase processes (immunosorbents) all of which end with a system of particles suspended in a liquid medium. The selection of a particular technique is determined by the consideration of many interrelated factors such as the solubility of the compound, the characteristics of the antiserum, the fraction to be counted, the degree of non-specific binding, the type of radioisotope. However, a feature that is common to all the aforementioned processes is the need for a centrifugation phase to carry out the aggregation of the suspended solid particles followed by a decantation (or aspiration) phase to physically separate the liquid and solid phases. In a previous Italian Patent Application No. 239OOA / 8O on behalf of the Technion Research & Development Foundation Ltd., a procedure for use in the specific binding test has been illustrated, in which the separation of the bound fraction from the free fraction is performed by a solvent extraction technique, using organic solvents as extractants. In another previous Italian Patent application No. 26541A / 80 to M. Cais, M. Shimoni and TechΟ Λ / 3-ρΛ nion Research & Development Foundation Ltd., a newly designed device referred to as LIDEX for carrying out the said solvent extraction technique has been described. In accordance with this invention, the Lidex device consists of a mixer-tank (A) into which a mixer-separator (B) is inserted with play, having a channel in the vertical axis of the mixer-separator. The two substantially immiscible liquid solutions are introduced into the mixing tank, the phases are thoroughly mixed by moving the mixer-separator (B) in and out of the mixing tank (A). After spontaneous separation into an upper and lower phase, the upper phase is removed by pushing the mixer-separator, said upper phase being accumulated in a collection container (E). Ir another previous Italian Patent Application No. 22446A / 81 of the same Applicants as the one mentioned above, mass transport separations for various purposes including specific binding tests to be performed through selective barriers have been illustrated. The invention discloses a novel Lidex device similar to that described in the Patent Application. Italian No. 26541A / 80, a barrier being positioned in the mixer-separator. The resistance presented to the flow of the liquid phase through the membrane in the mixer-separator will generally cause the fluid to penetrate around the sealing element located on the mixer-separator, as the mixer-separator slides downward, which will of course completely interfere with the test. In order to remove such inaccuracy, the device is provided with means for accumulating a gas pocket such as one or more horizontal, vertical or spiral grooves on the mixer-separator in which the air placed therein will decrease the pressure exerted on the barrier such that such penetration of the fluid around the sealing element is avoided. The invention has been found to give excellent results in various systems and for different membranes and types of solvents and / or precipitates. One of the main requirements met in immunological tests is the reproducibility of results with a minimum difference between two duplicates, which implies a complete standardization of the procedure with minimum handling and minimal labor but SUGNION SpA, a company without dependence on external factors. An example of such an extraneous factor is the degree of phase mixing in the test. Another extraneous factor is the separation rate of the desired phase to be analyzed later. In the barrier-free Lidex device used in immunoassay, the experiment requires vigorous and thorough shaking to allow complete mass transfer and accurate separation between the two liquid phases. As can be understood, the agitation obtained by manually moving the mixer-separator (B) in and out of the mixer-tank (A) cannot be interpreted quantitatively, being actually of a subjective nature in agreement with the technician who carries out the immunological test. The problem is even more complicated in the case of a Lidex device with a membrane, when each different system may require a specific membrane and / or a specific solvent and therefore will require different degrees of mixing and / or different separation speeds. This will of course be very difficult or even impossible with manual handling, especially for immunological test, when high precision is required with results that are as reproducible as possible. Even a technician highly experienced in immunoassay technique can hardly guarantee that complete mass transfer has been achieved after a certain period of shaking. On the other hand, prolonged agitation may interfere with easy phase separation, when two liquids are involved, or cause damage to the membrane, When precipitates are present. The object of the present invention is to provide an improved method for carrying out the immunoassay technique. Another object of the present invention is to provide an improved method for performing the immunoassay technique which eliminates both centrifugation and decantation manipulations. A further object of the present invention is to provide an improved method for performing the immunoassay technique which eliminates the subjective determination of the degree of agitation and improves phase separation. It is yet another object of the present invention to provide an improved method for making » 11 S.pX the immunoassay technique that avoids the laborious manual process for the thorough mixing required for effective mass transfer. Thus, the invention consists of an improved method for carrying out the immunological testing technique in a specially designed device consisting of a mixing tank into which a mixer-separator having a channel in the vertical axis of the mixer-separator is inserted with play, which consists of the combination of the following steps: ia) arrange the mixing tanks in a rack suitably designed to hold a number of said mixing tanks with the separating mixers; (b) introduce the reagents and analytes into said mixing tanks; (c) cover said mixing tanks with said mixer-separators; ,d) leave the reagents and analytes to incubate for a required period of time in the aforementioned mixing tanks covered with the mixing-separators; te) place the rack containing the aforementioned separation devices with the analytes and reagents ÌC ί ; incubated in a press device specially designed to provide a controlled rate of downward motion whereby the mixer-separators are pushed downward into the mixer tanks at a selected speed and for a predetermined distance to complete the desired separation and mass transport operation; (f) operate the downward movement of said pressing device at the predetermined distance and speed; (g) remove the rack upon disengagement of the press device; and (h) place the separator devices in the desired analytical instrument for quantitative or qualitative measurement of one or both of the separated phases as required. The process is very simple to carry out as it is characterised by the absence of any centrifugation or decantation phase, the entire operation taking place in a single tube device. Furthermore, the results obtained with the process according to the present invention compare very favorably with the known processes described in the prior art. One of the elements that enable the performance of the process according to the present invention is the press device used in step (e) referred to in the present description as press belt. Indeed, the manual operation in steps (e), (f) and (i) is very simple and can be easily performed by a technician in a laboratory where there are a relatively small number of test tubes. However, since each pipe must be individually slurried, this operation can be time-consuming in a routine laboratory performing a large number of analyses. Arche a standardization of the procedure would be more desirable in order to ensure complete independence of extraneous factors. Various prototypes of the press carpet based on either a pneumatic mechanism or an electric motor have been found equally satisfactory. Figure 1a illustrates the pressure mat at the start of separation with two racks of experiment tube, in situ, each holding 20 Lidex devices. Figure 1b illustrates the same instrument at the end ÉJUtàNlON S.pAj of the operation, when the separators B in all 40 tubes were pushed down from the moving platform to the required terminal position and the stops S in all separators have hermetically closed all collection containers E. The last phase occurs only in the final stage of the downward movement of the pressing platform, in order to allow the displaced air in the Lidex separator to escape. Upon completion of this operation (measured in our experiments to take less than 3 minutes in total) the pressing platform automatically reverses direction of movement and returns to the starting position of the instrument. The racks of experiment tubes can be removed from the press mat to be brought to the counter as soon as the upward movement indicated on the LED indicator screen begins. The press belt (figure 1) is constructed in the form of a closed press in which the movement of the platform (pressing plate a) is produced by a motor connected to a running plate which presses on the Lidex separators in the rack (b) by direct contact. The body of the press carpet can withstand the stresses15 BUG Ν ΙΟΝ SpA deformation actions produced as a result of the pressure that is exerted during operation. The motor can be pneumatic, hydraulic, a pneumatic-hydraulic combination, or electric and can be connected either directly or through a transmission system to the moving plate. The motor is equipped with a motion control mechanism (not shown in the figure) which allows the adjustment of the rising or falling speed of the running plate according to the requirement of the operation. The operation of the carpet press is very simple. By pressing the start button, the moving plate begins its downward movement and starts pressing on the Lidex separators with a preset descent speed and a preset pressure. When the platform (a) reaches its pre-set lower point (figure 1b) of descent, a delay mechanism begins to operate which holds the platform in that position for a required and pre-set period of time in order to complete the equal closure of all the Lidex separators in the racks of experiment tubes (b) previously introduced into the press belt. THE BUGNION SpA Upon completion of this delay time, the follower plate (a) disengages from the Lidex separators and begins its movement in the upper direction at a desired speed and returns to the start position (figure 1a). At this stage the pressure mat is ready for the next operation. Figure 2 is a schematic representation of the Lidex PS separator particularly useful for immunological testing at the beginning (2a) and at the end (2b) of the separation operation. The solid particles (P) initially suspended in the liquid phase (L) are completely separated and retained at the bottom of the mixer tank A. The additional feature of the system illustrated in figure 2C is the plastic rod R placed in the axial channel C of the separator B. The purpose of this rod is to move its equivalent volume of the liquid phase to the collection container E. The dimensions of the rod are such that there should be no interference with the free flow of the liquid phase as it passes through channel C, while at the same time only an insignificant amount of liquid will remain in channel C at the end of the separation. BUGNION S pA As a result, the radioactivity partitioned between the solid and liquid phases can be physically separated virtually entirely. This, combined with the hermetic seal of the S-stopper, provides significant added flexibility to the test protocol. With gamma emitting tracers it is possible to count either the liquid phase and / or the solid phase by simply placing the separator in the wall of the counter in the normal or inverted position respectively. Figure 3 shows an illustration of the rack with the devices, designed to allow maximum viewing of the mixing tanks. A during the manual pipetting phases of the test, and to fit appropriately into the press carpet instrument. The test protocol using the Lidex PS separator methodology described in the description consists of the following: a series of reservoir: A mixers, in duplicate, were placed in the racks of experiment tubes and test reagents, standard and clinical samples were added according to the box instructions; (ii) the mixing tanks were left alone for SUGNiON SpAJ the expected primary incubation time; iii) the precipitation (or adsorption) reagent for the test was added, ensuring that the total reaction volume was 1.5-2.0 ml; (iv) separators B, equipped with O-ring, membrane M, disk D, rod R and slightly covered with stopper Yes, they were introduced into tubes A, as illustrated in figure 2(a) and left alone for the second incubation (if the latter was not required by the test, we proceeded directly to the next phase); there two racks of experimental tubes (figure 3) holding the Lidex separators were placed in the pressure mat and the instrumental operation began; (vi) upon completion of the operation (3 minutes) the racks of the mixing tanks were removed from the press belt and the separators Lidex have been transferred to the counter. The problem encountered by diagnostic laboratories performing competitive protein binding assays is multifaceted. Therefore, it is necessary to combine a large sample pool submitted from several sources; interpret the significance of the results for the less experienced clinician; BUGNION S.p.A. provides a wide range of tests; returns results quickly; and, above all, ensures that each test is accurate. This must be done despite the economic difficulties encountered with a technique that is labor-intensive, complex, and expensive compared to certain other forms of testing tested in clinical biochemistry. The increasing availability of RIA reagents in commercial kits may alleviate some of these problems, provided the analyst can rely on the quality of the reagents and the accuracy of the test protocol. Assuming high quality reagents are available, bond separation of the free moiety becomes, in the applicants' opinion, the most important step in the testing procedure. The effectiveness of the new methodology according to the present invention, which is based on the Lidex PS separation device and the automation features introduced by the press belt instrument {Figure 1) has been compared with some of the most widely used reagent separation systems in commercially available boxes. 125 Commercial I-RIA boxes chosen to provide a variety of commonly used separation reagents, all requiring centrifugation and decantation in the box protocol, were grouped into four categories according to the separation reagent: (a) double-antibody (DAB) (Prolactin and FSH boxes); (b) double-antibody / polyethylene glycol (DAB / PEG) (ferritin, estriol, cortisol, testosterone, progesterone, / 9-hCG, insulin, and hPL boxes); (c) solid phase (insoluble T-antibody); (d) activated charcoal (digoxin box). All of these provide evidence for the practicability and potential of the centrifugation-free Lidex separation methodology. It is important to emphasize that all results presented here were obtained without any previous work to optimize the adoption of commercial box reagents for use with the Lidex PS separator device. In cases where experiments were performed with high quality reagents and optimization of the Lidex test protocol was performed (incubation time, reaction volumes, precipitation reagent), the test results with the Lidex PS methodology were very favorable compared to those obtained with the commercial box test protocol. BUGNION SpA Preliminary results indicate that with suitable interceptor membranes it may be advisable to use the Lidex separator device immediately after the primary incubation step, without requiring the addition of a precipitation or adsorption reagent. In the preamble to the description, the main requirements of an ideal separation technique, as formulated by the prior art, have been numbered. Based on the results obtained, the methodology according to the present invention will have the following advantages: ii: separates completely or nearly completely bound and free fractions with a large margin for error under the conditions used for separation; (ii) does not interfere with the primary antigen-antibody binding reaction; (iii) it is simple, easy and quick to use; (iv} is inexpensive and uses reagents and equipment that are (or can become) readily available; (v) is not influenced by plasma or serum; (vi) all manipulations are performed in a single tube separator device; (vii) it is highly suitable for automation; (viii) is applicable to a broad range of antigens; ( ix '> The methodology and shape of the separator device practically eliminate potential contact with the radioactive reaction mixture, thus ensuring maximum safety from radiation risks. While the invention has been described with specific embodiments thereof, it will be understood that it is susceptible to further modification, and the patent is intended to cover all variations, uses or adaptations of the invention and includes all that arises from the present description or from known or usual practice in the art to which the invention pertains and as may be applied to the essential features described herein above and as falling within the scope of the invention. In order to further illustrate the nature of the present invention and the manner of practicing it, the following examples are presented for clarity of illustration only and without any limiting intent. EXAMPLES The methodology according to the present invention was used in conjunction with the following separation reagents: a) double antibody; BUGNION SpA b) double antibody / PEG; he tries solid phase, and activated soft carbon. In each case, at least two parallel experiments were performed: one experiment was performed exactly according to the instructions in the box, including the centrifugation and decantation steps; in the other experiment, the instructions in the box were used with regard to the addition of reagents, clinical samples, standards, and incubation times, except that Lidex PS separators, experiment tubes, and the press belt instrument were used for cell separation between free and bound parts. Additionally, total reaction volumes were adjusted as needed; 125 to The standard curves obtained for the I-FSH tests 125 and 1 Prolactior (HPRL) are shown in Figures 4b and 4a, respectively. The clinical serum values ​​detected in the two tests are compared in Tables 1 (FSH) and 2 (HPRL). The precipitating agent was double antibody. b) Double antibody / PEG (20%) was used as precipitation reagent for the ferritma I test which produced the standard curves shown in figure 4c the values ​​of clinical sera collected in table 3. The use of the precipitation system required two pipettings and an additional 15-minute incubation (after the addition of the double antibody). It was found that a single pipetting of a premixed double antibody / PEG reagent (at 8°C) produced immediate precipitation at room temperature and no secondary incubation was necessary. The effectiveness of this reagent in conjunction with the Lidex separator methodology is similarly demonstrated by the standard curves obtained for estriol. 125 I (figure 6a), cortisol (figure 6b), testosterone I (figure 6c), progesterone I 5 (figure 6d), β HCG I (figure 5c), insulin 125 I (figure 5d) the hPL 125 I (Figure 5a), hFSH (Figure 5b), gastrin (Figure 7a), hLH (Figure 7b), PAP (Figure 7c) and alpha FETO protein (Figure 7d). o, The Lidex separator methodology is particularly suitable for use with solid-phase immunoassays. An example is shown for the solid-phase 125 assay of 1 thyroxine (immobilized antibodies). The standard curves are shown in Figure 4d. eUGN / QN SPA Although the two curves (Lidex box and cross-sectional procedure) are not superimposable as in the previous examples, the values ​​of the clinical sera calculated from their respective curves are almost identical (table 4). d) The use of activated carbon as an adsorption reagent in conjunction with the Lidex separation methodology was demonstrated with a box of 125 Digoxin. In this example, three parallel experiments were performed in which the results obtained by following the instructions on the box exactly (with decantation and centrifugation for 20 minutes) were compared with those resulting from a test using a solvent extraction procedure with Lidex LS separators and with one using the new separators. Lidex PS with selective barrier. In the last experiment, the bound fraction transported with the liquid phase in the collecting vessel Ξ of the separators was counted by placing the covered Lidex PS separators upside down in the wall of the counter. The standard curves of three experiments are shown in Figure 16. Digoxin concentrations in clinical serum samples were determined in all three tests in a blinded experiment by comparison with results obtained for the same sera in another laboratory (Sheba Government Hospital) with another commercial kit (Diagnostic Products). The data are summarized in Table 5. Similar experiments for comparison data with known boxes were performed with various clinical sera and are presented in Tables 1 to 14. Table 1 FSH Table 2 HPRL (prolactin) Table 3 Ferritin Table 4 Thyroxin Table 5 Digoxin Table 6 Estradiol Table 7 Progestin Table 8 hPL Table 9 hUH Table 10 Total urinary estrogen Table 11 T3 Table 12 hTg - Ab Table 13 TSH Table 14 FSH (Biodata box) Similarly, comparison experiments were performed with: guóNfON S.pA; - HPL system with enzyme immunoassay, and - solid-phase GENTAMISIN fluorescence immunoassay. fìUGA / / Oii •PA - 28 Cs'frc-tc ce: heats of serum and lyric FSTll / rl, obtained with box protocol (centrifugation) and Lidex PS methodology ·* Serum code Box protocol HYPOLAB Protocol Lidex K 9 15.67 16.03 L ♦ Γ75 Π7 M 59Ϊ48 £7753 ί : ; 59.68 53.45 - r — - 4, 19 3' ' ' • · 1:2 53.10 57.04 II 1:4 50.04 44.04 0 2 4 6.10 7.01 2 6 0.61 0.62 *· 01 3.07 3.32 σ' 7 4.19 5.19 LHRH Ste 0' 8. 11 9.10 30' 11.69 10.78 r » 60' 14.98 14.35 Ortho III 5.69 4.31 Ortho IV 4.21 3.84 Ortho 10T10 2A 9.26 11.87 Ortho 10T10 2B 4.45 4.62 Ortho 10T10 2C 3.64 2.77 ·* BUGNiON SpA. TABLE 2 Comparison c: hPRL clinical sera values ​​•ng / mli obtained with box protocol (centrifugation) and Lidex PS methodology Serum code Hypnlah protocol box protocol lo-Lidex M 9 11.66 13.96 M 7 10.00 9.67 L 4.07 5.08 76.42 ICO.77 75.64 75.80 0' 2.40 2.6S 0 ' 5.28 6.18 2 4.02 4.45 0 * 9.94 10.73 oenop. 2 7.31 7.71 “ £T· rn * 4.70 6.78 Orthc- Binder 10T10 2A 1.89 1.90 10T10 2B 1(58 1.53 10T10 2C 1.84 2.76 0U6N1ON * TABLE 3 - Comparison of clinical serum ferritin values ​​(ng / nl) obtained with the box protocol <centrifugazic re) e cor metodologia Lidex PS Serum No. Hypolab Box Protocol Licex Protocol 1 20 s 20 2 . 20 < 20 3 71.4 70.3 4 32.1 36.3 Li 135.7 132 F 5G.7 65.4 - 20 20 8 - 20 _ 20 BUGNION S pA - 31 .* TABLE Λ Comparison of clinical serum thyroxine values ​​(ug%) obtained with box protocol (centrifugation) and with Lidex PS methodology Box Protocol Lide HyjP.OlAb Protocol K 9 6.76 6.62 1.70 1.30 K 22.44 20 Plasma-5 9.66 9.80 Plasna-18 16.84 17.80 Plasma-6 9.76 10.58 FIasma-21 5.05 4.53 Flasna-20 7.00 7.75 Ortho-alloy JOTIO 2A 0.61 0.67 1CT1O 23 7.24 e.74 10710 2C 13.61 15.25 ouGNIQN SpA - 32 TABLE 5 - Comparison of clinical serum digoxin values ​​(ng / ml) obtained with box protocol (centrifugation); Lidex LS (solvent extraction); Lidex PS methodology and (independently) Sheba Hospital Laboratory (different box with centrifugation) Digoxin concentration (ng / ml) Serum No. Test Directions BECTON-DICKINSON -LIDEX PS LIDEX LS (membrane) (solv. extraction) Test Directions Sheba Hospital (Diagnostic Products 1 0.5 0.3 0.5 0.4 2 l.ts 2.3 1.9 2.2 3 C.7 0.9 0.9 1.0 4 . 0.5 0.7 0.6 0.6 S 1.4 lS 1.6 1.6 6 1.0 1.4 1.4 1.3 7 *-0.5 0.7 0.7 0.5 S 0.7 1.1 1.2 1.4 9 4.1 4.1 3.4 5.7 t TABLE 6 - Comparison of clinical serum estradiol values ​​(pg / ml) with box protocol Biodata and methodology Lidex bvgnion S PA eIODATA 20¾ PEG -LIDEX TEST SYSTEM — PEG 8Ϊ7δΑΒ REFERENCE VALUES Max. binding (%) \ S 9 32.2 29.0........ 45 ' 3. 7 6.9 i ? . 5 STANDARD CURVE CONCENTRATIONS 5 I. C ri 62.5 7C.0 68.0 6S.4 57.S 125.0 ' 250 54.7 50.1 42.2 » * 40.7 h 5 - . 3 28.9 33.2 . ?4.7 ........... 2 OC 19.7 1 19.3 CLINICAL SAMPLES ΐ:.'' '',' :, ' / ·* ^'^JÌ 'V 1· ÀJ^''' 1 ' ΓΟΝΙΟΝ»:. - 34 TABLE 7 — Comparison of PEG / STAT clinical serum values ​​obtained with the Serono test protocol and the Lidex methodology V BUGNION S4MV >«“ -ΐ*»· ; _ Ί h .. X^w < τ. A. j:.„Ai .. There- 35 TABLE fi Comparison of hPL clinical sera values ​​(ng / ml) with Hypolab box protocol and Lidex methodology HYPOLAB LIDEX SYSTEM OI VALUES TEST PEG IU_FEG 81 / DAB rjffr. MAX. BOND (V) 69.8 73.8 65.7 NSB 5.7 6.4 4.1 CONCENTRATIONS__STANDARD CURVE 12.5 ng / zl 102 94.2 25 » » 90.4 83.2 SG 1 » 79.1 73.8 100 11 62 62 200 tl 42 40 400 lf 26.5 26 eoo II 17 14.5 * SAMPLES £UiATiC. Serum Test 94.7 77.9 1) >800 >800 2) 8.1 15.6 3) 16.2 19.0 ♦) ' 800 >800 5) 66.5 98 6) 68.0 61.3 «9.1 »00 ► - * * F*· / , . _;rr X BUGNìQN SPA' TABLE 9 - Comparison of HlH clinical serum values ​​(rrlu / ml) obtained with Hypolab box protocol and Lidex methodology TEST SYSTEM L:ypolas L1PEX PEG S' / DAB --- — REFERENCE OI VALUES Max. bond .23.6........ . 28.S . 5. ? .35-2 . ... V c P ! 1 . ' STANDARD CURVE CONCENTRATIONS ---,--------- «- I 1 S S. “ Si. Divide x 1.5 .r :j' gUGNlQll S^Aì - 37 TABLE 10 - Comparison of TUE clinical sera values (total urine estrogens)£ng / ml) with Hypolab box protocol and Lidex methodology HYPOLAB TEST SYSTEM -JJPEX _ PEG «VDAB Reference Values ​​Serum 4» <kptant * PEG 20¾ ( LEGAME MAX. (%) 39.4 22.8 35.9 n s e l 5.5 STANDARD CURVE CONCENTRATIONS . 5 r.£ / xì . . 52.6 I » SA A...... 7 » « f ' 71.6 « ! • J 75.5- .... Ϊ 2 - r 62.6 64.2 II 4 55.4 5'.S ..... ( ? I 14.6 Ito. 1 . . -. - ìó 7.2...... •ss . . . - ... CLINICAL SAMPLES ; Serum tests ! 44.9 ί 1 49.14 . . 29.4 4(!J>7 mean.. . 35.0 4 7.5 54.82 l > • ' L ''•V ι ' ' ,.· / :'* ir3Jl ' ri*i ... ' . ·*. - ..... ' ,.r A4 _ ,. '.ST*?'ψ ' Τΐί,-Ι eo Q HlQt, s. A4, - 38 TABLE 11 Comparison of T-3 clinical serum values ​​(ng / ml) with Biodata box protocol and Lidex methodology TEST SYSTEM AND IODATA P£C 20% LIDEX PEG 8% / DAB REPEAT VALUES MAX. BINDING (%) 62.9V 60.2% ....... NSB 2.0% 4.4 CONCENTRATIONS STANDARD CURVE '0.125 ng / ml; 96.2 Ό.25 j 92.6 j°.5 j 82.1 Ho ·· I S9.9 - ) . T 25.9 15.4 96.0 94.-5 83.2 61.9 58.9 26.1 14.3 CLINICAL SAMPLES ι- • Test serum r [ fì S2 0,SR . . ί DI L 1.2 : .02 0-6 ng g / ml 0.96 b ' Ί. 52 0.7] and; 0.77 0.92 of 1.54 1.33..... . . — e) - 0.69- 0.68 9 0.86 0.95 Norm j values: 0.6-1 .7 nn / inlj -...... τ~ ι 'hi· ùw liN.Ori 3.pAj - 39 TABLE 12 - Comparison of hTg-Ab clinical serum values ​​with Hypolab box protocol and Lidex methodology SYSTEM OF . --- TEST _HYPCLAB .Counts / average * CPU 4BOND LIDEX Counts- average ΓPM % ---7-, Bond TcTC ! : ct-55 1S' . 9 STANDARD CURVE CONCENTRATIONS ί HiC 2 Γ 3.9¾ 2625.7 *3.9% l : 2 a 2014.6 10.6% 5902.5....... 20.6%.. .. ; : ρ Ί 2917.05 . 15.4 V 5952.25....... .31.6% i : mor 3326.95 . 17.6% ' . 7120.85 ..... . .37.7% . * ' ; 5 1 — — 5247.35 ...... 44.1¾ . The:zccac » I 6269.2 . . . . 33.1%..... - -9102.55........ . 48.-1% -7 ! : : 57730 7524.25 33.7% 9259.1 48.9% CLINICAL SAMPLES — 47.0.9 25.02 7611.5 - 1 41.77 . NC SS6.9 4.6..... 1350.15 ?.9% . a) 1650.15 8.7 1540.45 . 6.1%. t; 1570.96. 8.3. . . 1955.9 10.3% J ci . 1777.25 ....... - 9.4% .- ... * . . , • AV · θυ® ΝίβΗ 3^, - 40 TABLE 13 - Comparison of clinical serum TSH values ​​(yulu / nl) cc r Hypolab box protocol and Lidex methodology HYPOLAB SYSTEM . LIDEX TEST VALUES DAB / H 0 PEG YES / PAB REFERENCE MAX. BINDING (%) NSB 40.3¾ 41.Yes 5816¾ 7.0¾ 4.8¾ 2.3¾ STANDARD CURVE CONCENTRATIONS - -- - n - r » l ί 0.62 μ!..- PS.6 - .....................T ti P5.8 1 1.25. .. .. 2.5 SS.7 .87 2 . 76.3 78.6 S .0 49.7 59.8 'i'.r ' 28.1 ' · 29.6 - .n. . . '·. 14.-0 · 18.-2 . . . ... CLINICAL SAMPLES ,y . ;>* -,Γ . ....v I ί*β· i -Iti-Stsfc· ! ..... At£u: . etJnw, nM ©UGNIQN s.pA -41 TABLE 14 - Comparison of FSH clinical serum values ​​(miu / ml) with Biodata box protocol and Lidex methodology I- » I biodata LIDEX VAL. OITRIF.. RAMBAN / HYPOLAB PEG II / DAB PEG II / DAB Sereno * . - ....... ..a} 5? • . .151. . . . 168 2.79 . . 3.53. . . .... 2.7........ 8.78 9.48 6.6 -) 227 5 6 : d) 228 ...... 7.51 5.6 ( ! ej 25 o 51.9 34.74 ............ . . 34....... C ->λ C L. 81.18 72.75 - -6T.......ii ; ε l 2~~ 33.17 31 .C • 25....... h' 6 5 2 ' .4 2.66 * — i 1 ..... 1.35 ........... 2.56 -* T t 1 I 1 661 1 -.98 . 2.64 X ' · · · - - ' 2.4 7.5 ........ ! k' r 6Ξ1 8.95 10.56 - .... - « · · · - · ' .* - - - / -* ' ,β· η jb · , - ·*-·;. β'·2**· 5.:.. \ h ,* Λ17 Ι r-Ji*» --. / - < ' 7 > Ì -:-·\ιχ3Λ· - - buoniON S.p.A

Claims

1. CLAIMS 1) Improved method for performing immunological testing without centrifugation in a specially designed device consisting of a mixing tank into which is inserted with a gap a mixer-separator having a channel in the vertical axis of the mixer-separator, characterized in that it comprises in combination the following steps: (a) arranging the mixing tanks in a rack appropriately designed to hold a number of said mixing tanks with the mixer-separators; (b) introducing the reagents and analytes into said six mixing tanks; (c) covering said mixing tanks with said mixer-separators; (d) allowing the reagents and analytes to incubate for a required period of time in said mixing tanks covered with the mixer-separators;(e) placing the rack carrying the said separator devices with the incubated analytes and reagents in a press device specially designed to effect a downward movement at a controlled rate whereby the mixer-separators are pushed downward into the mixer tanks at a selected rate and for a predetermined distance to complete the desired separation and mass transport operation; (f) effecting the downward movement of said press device at the predetermined distance and rate; (g) removing the rack upon disengagement of the press device; and (h) placing the separator devices in the desired analytical instrument for quantitative or qualitative measurement of one or both of the separated phases as required.; 2) A process according to claim 1p characterized in that the immunological test process is selected from radioimmunoassay, cheminoluminescence, enzyme test, bioimmunoassay, fluorescence test, metalloimmunoassay and combinations thereof. 3) Method according to claim 1, characterised in that the pressing device is constructed in the form of a closed press in which the movement of the pressing plate is produced by a motor. 4) Method according to claim 3, characterized in that the motor is operated pneumatically, electrically, hydraulically or by a co-inaction thereof. 5) Method according to claim 1, characterized in that the rack is designed for maximum visibility of the mixing tank during the test. 6) Process according to claim 1, characterised in that the mixer-separator is equipped with a barrier. 7) Process according to claims 1 to 6, characterised in that it is performed automatically. 8) Process according to claims 1 to 7, characterised in that said immunological test is applied for the determination of antigens, hormones, bariums, steroids, vitamins, tranquilizers, drugs and alkaloids. 9) Process according to claim 8, characterised in that it is applied to FSH. 10) Process according to claim 8, characterised in that it is applied to HPRL. 11) Process according to claim 8, characterised in that it is applied to ferritin. 12) Process according to claim 8, characterised by the fact that it is applied to thyroxine. 13) Process according to claim 8, characterised in that it is applied to digoxin. 14) Process according to claim 8, characterized in that it is applied to estradiol. 15) Process according to claim 8, characterised in that it is applied to the Pregstat. 16) Process according to claim 8, characterised in that it is applied to hPL. 17) Process according to claim 8, characterized in that it is applied to the total estrogen titrated. 18) Process according to claim 8, characterized in that it is applied to the T. w 19) Process according to claim 8, characterised in that it is applied to Tg-Ab. 20) Process according to claim 8, characterised in that it is applied to TSH. 21) Process according to claim 8, characterized in that it is applied to the eliza nosticon HPL enzyme immunoassay system. 22) Process according to claim 8, characterised in that it is applied to the solid-phase GENTAMISI fluorescence immunoassay. 23) Improved process for carrying out immunological tests without centrifugation substantially as described and claimed above. by Mr. MICHAEL CAIS, Mr. MOSHE SHIMONI, and the company TECHNION RESEARCH & DEVELOPMENT FOUNDATION LTD. THE BUGNION SpA AGENT 1000 (ng / rnl) Fig.4 $ 0,ￎﾛ BUCM.C,ￂﾷ,' 0] ^TABLE