FR2576418A1 - NEW SUPPORT FOR IN VITRO ORGANIC TESTS AND APPLICATIONS OF THIS SUPPORT - Google Patents

Abstract

The medium according to the invention is characterized in that it comprises a sheet of transparent plastic material such as polyester or polyvinyl (101) and a hydrophobic coating showing a plurality of hydrophilous uncoated regions (102) intended to be sensitized. The medium is particularly applicable to immunofluorescent tests, immunoenzymatic tests and radio-immunological tests.

Description

 New support for in vitro biological tests and applications of this support.

 The present invention relates to a new support for in vitro biological tests, in particular immunofluorescence tests, ELISA tests, radioimmunoassays, etc. as well as to the applications of this support.

 The development of immunofluorescence techniques has greatly improved the serum for the detection of antigens or antibodies with increased sensitivity. Currently used for its implementation, glass slides covered on one side by a thin hydrophobic coating, such as in particular silicone revealing circular hydrophilic uncoated areas intended to be sensitized, that is to say to fix a certain quantity of antigens, in the case of tests for the detection of antibodies or, conversely, fix antibodies in the case of detection of antigens.

Due to the increasing number of samples to be tested, it would be desirable to develop the automation of the different stages of the tests. However, the glass supports currently used do not lend themselves to automation owing in particular to the difficulties of handling, the time spent wicking and the difficulties of locating.
The same applies to the immunoenzymatic tests, called ELISA tests, which use sets of plastic cups which are difficult to handle, long to wash and of very high cost price.

The present invention proposes to remedy these drawbacks and to provide a new support for in vitro biological tests which has, compared to known supports, the following advantages
- possibility of automatic continuous or discontinuous use,
- elimination of cleaning and recycling operations for media,
- significant time savings, particularly in terms of assembly, drying, reading and immediate availability,
- gain in reagents,
- safety,
- transmission to UV light greater than that of glass,
- very significant weight gain,
- reduced risk of tracking error,
- easy storage and design,
- possibility of dimensional adaptation,
- possibility of simple manufacturing,
- easier handling for the installation of reagents and products to be tested, such as washing, drying, reading, etc.
- ease of transport for use in the field.

 The subject of the present invention is a new support for in vitro biological tests and in particular immunological tests, characterized in that it comprises a substantially transparent plastic sheet carrying, on one of its faces, a hydrophobic coating leaving one and preferably several hydrophilic uncoated areas, preferably circular in shape.

 By plastic material, in the sense of the present invention, is meant a synthetic material capable of being produced easily in the form of sheets of ribbons or sheets and having either spontaneously or after treatment, a hydrophilic character allowing sensitization operations. , that is to say the establishment of molecules, complexes or particles, such as antigens or antibodies, as well as the collection and distribution of liquid media such as, for example, serums or blood.

Preferably, the plastics according to the invention are produced in sheets or films of polyvinyl acetate or polyester such as for example the films sold under the name 63610 DT2 by the company
STAEDTLER NURNBERG RFA

 The sheets according to the invention can be more or less flexible depending on their nature and the thickness chosen. Preferably, they are relatively flexible while tending to be substantially maintained without collapsing when held by an edge. Preferably, the thickness is between 0.05 mm and 0.5 mm. According to the invention, the films used may have undergone an anti-thermal and / or anti-static treatment such as, for example, the films sold. under the name 63610 DT2 by the company STAEDTLER.

 The hydrophobic coating may for example be a silicone coating. This can be done for example by placing drops of glycerol on the sheet placed horizontally at the locations of future hydrophilic zones and by spraying a silicone aerosol on the entire sheet, the location of the drop remaining hydrophilic while its periphery becomes hydrophobic. This process can be automated by using, for the depositing of the drops, an automatic reagent dispenser.

 However, in accordance with a particularly advantageous improvement of the invention, the coating can be obtained by a simple photocopying process using for example xerography or similar techniques. It then suffices to make a model printed on plain paper and to make photocopies of it on the aforementioned plastic sheets, allowing the formation of a hydrophobic carbon coating.

 This improvement of the invention also makes it possible to obtain quickly and at particularly low costs, a large number of supports with all the desired spatial arrangements, these supports being able, moreover, to present directly on the one hand all the inscriptions necessary for locating the various hydrophilic zones, on the other hand, all useful information, such as dates, nature of the deposited antigen (s), etc.

 The diameter of the hydrophilic pads is, according to the invention, between 2 and 20 mm.

 The sheet support according to the present invention can be presented in extremely diverse dimensions. Thus, it may be in the form of blades of small dimensions, comparable to glass slides, or of sheets of large dimensions capable, if necessary, of being cut into subassemblies used separately or even in the form of continuous rollable tape particularly suitable for automatic continuous processing.

Other plastics can also be used, including polyvinyl chloride, polyvinyl, polystyrene, polyethylene polyearbonate,
Cellophane (registered trademark) and derivatives of these polymers.

 The subject of the invention is also the application of these supports to immunofluorescence tests, characterized in that one sensitizes one nu several plaques or hydrophilic antibodies of a support using an antigen / tales in a liquid drop, that the support is washed with a reduced quantity of a usual buffer (for example phosphate buffer), that the support is dried, preferably by letting it float in a flow of hot air while maintaining it at one end , that a drop of serum or substance to be tested and a drop of fluorescent conjugate is applied to the said area or areas with repetition of the above-mentioned washing and drying operations and the reading (s) is carried out under a UV microscope after having deposited on the track (s) a drop of oil covered with a transparent plastic sheet, which may in particular be made of the same material as the support.

 Preferably, a support will be used comprising a plurality of ranges in rows and columns in step with the already known drop dispensing devices providing means for automatic advancement of the supports according to the invention. Automation can also be practically total by providing, between different drop dispensing stations, automatic washing and drying means.

 A subject of the invention is also the application of these supports to ELISA tests, characterized in that one or more hydrophilic plaques of an or antibody are sensitized - support for ltalae falun antlgene tcontenu in a liquid drop, that the the support is washed with a reduced quantity of a usual buffer (for example phosphate buffer), which is dried the support, preferably by letting it float in a flow of hot air while maintaining it at one end, that the 'is applied to said area a drop of serum or test substance and a drop of a chromogen, preferably of a type which polymerizes while it is colored, with repetition of the above-mentioned washing and drying operations, after which a colorimetric measurement is made of the said area or areas.

 Preferably, the chromogen used is diaminobenzidine when the enzyme used for the conjugate is peroxidase.

 In this application, the entire test can be automated, including reading and color measurement.

 The invention also relates to the application of these supports to radioimmunological tests, characterized in that one sensitizes one or more antigen or nyaropniia planes to I1aicie oun / antibody contained in a liquid drop, that the support is washed with a reduced quantity of a usual buffer (for example pH 7.2 buffer), let it dry the support, apply a drop of the antigen to be titrated to said area to which a known quantity of the same antigen is added labeled with a radioactive isotope. After repeating the above-mentioned washing and drying operations, a radioactivity measurement of the said area (s) is carried out. This latter operation can be carried out in two different ways: 1) either by perfect contact - between the support and a sensitive film (autoradiography) , 2) either by cutting out each of the tracks and by carrying out a radioactivity coupling in appropriate flasks.

 This antigen titration method is said to be by competition.

 The application of the support to radioimmunological tests can also be extended to titrations according to a process analogous to that of the above-mentioned ELISA.

Other advantages and characteristics of the invention will appear on reading the following description, given by way of nonlimiting example and referring to the appended drawing in which
Figure 1 shows a view of a support according to a first embodiment of the invention.

 FIG. 2 represents a view of a support according to a second embodiment of the invention.

 Figure 3 shows a view of a support according to a third embodiment of the invention.

 Figure 4 shows a view of a support according to a fourth embodiment of the invention.

 Fig-ure 5 shows a view of a continuous support in the form of a ribbon.

 FIG. 6 represents a view of a set of supports when the test is ready for reading the results under a UV microscope (supports covered with mounting liquid and coverslip).

 FIG. 7 represents a view of a support adapted to the use of the micrometric displacement of the stage of a microscope.

 Referring to Figure 1, there is shown a support according to the invention comprising a plastic sheet 1, more specifically polyester, having a thickness of 0.1 mm and dimensions of 100 x 150 mm.

On this transparent sheet 1, six coated zones, of rectangular shape, numbered 1 to 6, have been materialized and each provided with ten circular uncoated pads forming the hydrophilic pads, referenced 102 and having a diameter of 6 mm. These ranges are distributed in the form of two columns of five ranges 102.

 Outside of said coating zones, of references 1 to 6, the support also has an inscription ("P. Falciparum") which designates the antigen with which, in the example described, the areas 102 are sensitized once the support has been produced. . The production of the support according to the invention is carried out by preparing a matrix or original made of paper having an appearance identical to that shown and by scrolling the plastic sheets in a conventional photocopying machine of the type of a xerography machine so applying the coating appearing in black, a coating consisting of the dried ink of the xerographic process.

 We understand that we can manufacture at very low cost a large number of supports and this in all the desired forms and on demand.

 Referring to Figure 2, we see a support having, on a rectangular sheet 103 of the same kind as the sheet 101, two areas of each time forty tracks 104 arranged in eight rows of five tracks referenced 1 to 8 and 9 to 16 The pitch of the pads 104 is adapted to the pitch of an automatic droplet dispenser.

 In FIG. 3, another support is shown comprising a sheet 105 with a single coating area of ninety-six areas 106 arranged on a rectangular matrix of eight rows and twelve columns referenced a to h and 1 to 12.

 Such a support is particularly advantageous for carrying out an immunofluorescence scan after a cloning of a cell producing monoclonal antibodies.

 We now refer to Figure 4.

 We see a support having the same conformation as the support of Figure 3 but in which we applied on the sheet 107 a silicone area 108 leaving areas 109. Markers or other indications can be set up for example by xerography . To make zone 108, first of all, for example using a drop dispenser of the "autodrop" type sold by the company FLOW LABORATORIES, a plurality of drops of glycerol diluted 1/4 in distilled water having a volume of 13 μl at the step of the desired ranges. Then sprayed on the sheet an aerosol of silicone sulfur. Then the sheet is washed to remove the glycerol and, if necessary, inscriptions are applied, for example by xerography or by any other process, for example by printing.

 Of course, various other techniques for applying coatings are possible. Thus, the coating by xerography can be replaced by other types of coating either using similar principles but with special inks, or even by printing or screen printing.

 Once the supports such as those described have been produced, hydrophilic areas such as 102, 104, 106 and 109 are sensitized.

 Referring to Figure 5, there is shown a support in the form of a polyester or polyvinyl tape having a thickness of 0.1 mm to which has been applied, by screen printing or in any other way, a number of zones 111 leaving hydrophilic areas 112. Graphical lines of separation 113 may possibly be provided. Different inscriptions can of course be made. To make such a support, a polyvinyl acetate film having a thickness of 0.1 mm is preferably used, making it possible to wind the ribbon.

 The various supports according to the invention can be packaged in sterile form in sachets or other packaging made of paper or plastic. They are suitable for transport by post and can therefore be used for field sampling and sending to the center or laboratory carrying out the tests.

An example of the use of these supports will now be described.
A liquid suspension of antigen of
Plasmodium falciparum as follows
A support such as that shown in FIG. 3 is used. This support is placed under an automatic drop dispenser of the "autodrop" type which distributes, in the middle of each of the circular hydrophilic areas, a drop of antigen. The sensiDilisasiot lasts 15 hours3 at room temperature. Washing is then carried out in phosphate buffer at pH 7.2 for a period of 10 min.

 After e washing, drying is carried out, for example by maintaining the support by one of these edges of the sheet inside a stream of hot air, for example coming from a fan, the sheet floating freely in the air stream. In less than two minutes, the leaf is dried.

A blood sample is taken from a person suspected of being parasitized and this sample undergoes the following dilutions: 1/20, 1/40, 1/80, 1/160, 1/320
One drop of each of the dilutions is applied to the different ranges. It is left to incubate for 30 minutes in an oven at 370C. The above-mentioned washing and drying operations are repeated. Apply a drop of fluorescent anti-immunoglobulin conjugate hu maines, previously brought to its optimal use dilution.

The above-mentioned incubation, washing and drying operations are repeated.

Reading can be visual or, if necessary, made automatic by optical analysis means sensitive to the variation in light intensity depending on the agglutinations performed.
For a reading under a microscope, a standard mounting liquid (glycerin) is spread over the different areas, for example using a drop dispenser, or directly over all of the area (s) comprising the areas of the support. buffered at pH 7.2 for example) and the support is immediately immediately covered with its mounting liquid with a second transparent polyester sheet without any coating. A strip of mounting liquid is thus formed between the two sheets of polyester acetate allowing easy reading under the microscope. FIG. 6 shows a support 114 provided with eight zones, 1, 2, 3, 4, 11 , 12, 13, 14, of two rows of five hydrophilic pads 115, each zone having been covered with mounting liquid and then with a 116 lamella / of slightly larger size. These strips are bonded by the mounting liquid which gradually dries. Reading under the microscope can be carried out in the usual way.

 If necessary, the supports can be fixed on the stage of the microscope to be adapted to the micrometric displacement of the stage, for example as shown in FIG. 7 on which we see a rectangular sheet of narrow width of plastic material 117 provided with two holes for the passage of the screws of the plate 118. Thanks to a sheet of conventional self-adhesive material 119, it is possible to secure a support 120 according to the invention (of which only a part without coating is seen), this sheet then being able to be easily separated by removing the adhesive tape 119.

 The invention lends itself to tests by immunofluorescence of practically all antigens, antibodies or other elements capable of being checked by this kind of tests and in particular indirect immunofluorescence.

 Among these applications, the following conditions may be mentioned in particular: toxoplasmosis, rubella, syphilis, trypanosomiasis, amebiasis, malaria, viral diseases, hydatidosis, bilharziasis and other parasitic diseases, aspergillosis and other fungal diseases.

 The invention is however not limited to immunofluorescence techniques but can also extend to other testing techniques. Thus, by way of example, the invention can be used for radioimmunoassay tests by replacing the immunofluorescence marker with a usual radioactive marker. Once the substance to be tested is placed, at suitable ailutions, on the hydrophilic pads, it suffices to apply a sensitive film closely against the sheet constituting the support. The sensitive film being thus brought into perfect contact with the support, an impressed film is then obtained immediately supplying the test result without being obliged to use the heavy current radio-activity counting apparatus, owing to the fact that, thanks to the immediate contact with the film, the area of lateral blurring due to radiation emission. In a perfectly quantitative assay, each of the hydrophilic ranges is cut and a coupling is carried out by sticking them in an appropriate counting liquid.

In the case of an application to type tests
ELISA, an enzyme-labeled conjugate (such as peroxidase, phosphatase, p-galactosidase) is used and a single dilution of the serum is sufficient, the reading being carried out by determination with the concentration of reacting antibodies.

 It has been found that it is possible to use the supports according to the invention by using a chromogen which polymerizes over the range of the support as it becomes colored by oxidation (for example diaminobenzidine in Tris HC1 + water buffer). oxygenated). For example, in the case of a serum reacting with a hydatid cyst antigen, a brown coloration is obtained on the hydrophilic range of the support.

 Of course, the invention also applies to indirect or reverse tests with initial sensitization of antibodies on the hydrophilic areas of the support.

 Although the invention has been described with regard to a particular embodiment, it is understood that it is in no way limited thereto and that it can be made various modifications of shape or material without thereby move away neither from its frame, nor from its spirit.

Claims (18)

 1. New support for in vitro biological tests and in particular immunological tests, characterized in that it comprises a plastic sheet (101, 103, 105, 107, 110, 114), substantially transparent, bearing, on one of its faces, a hydrophobic coating leaving at least one uncoated hydrophilic surface, in particular of circular shape (102, 104, 106, 109, 112).  2. Support according to claim 1, characterized in that it is made of polyester or polyvinyl acetate.  3. Support according to one of claims 1 and 2, characterized in that said hydrophobic coating is a silicone coating.  4. Support according to claim 3, characterized in that the coating is obtained by placing glycerol drops on the sheet and by spraying a silicone aerosol on the whole sheet, the location of the drop remaining hydrophilic then that its periphery becomes hydrophobic.  5. Support according to one of claims 1 and 2, characterized in that the coating is obtained by a copying or printing technique.  6. Support according to claim 5, characterized in that the coating is a coating obtained by a - - process / xerography.  7. Support according to any one of claims 1 to 6, characterized in that it further comprises inscriptions and / or markings.  8. Support according to any one of claims 1 to 7, characterized in that the diameter of the hydrophilic areas is between 2 and 20 mm.  9. Support according to any one of claims 1 to 8, characterized in that it comprises several sets separated by several coatings delimiting several signs; nbles of areas, possibly capable of being cut to form individual supports.  10. Support according to claim 9, characterized in that it is in the form of a rollable tape.  11. Application of a support according to any one of claims 1 to 10 to immunofluorescence tests, characterized in that one or more hydrophilic plaques of a support are sensitized using an antigen or antibody / contained in a liquid drop, which the support is washed with a reduced amount of a usual buffer, that the support is dried, which is applied according to said. or said areas a drop of serum or substance to be tested then after washing and drying a drop of fluorescent conjugate with repetition of the above-mentioned washing and drying operations and the reading (s) are carried out under a UV microscope after being deposited on the or the pads a drop of mounting liquid covered with a transparent plastic sheet (116).  12. Applieation according to claim 11, characterized in that, for displacement on the mieroscope, one connects the support (120) to a plastic sheet (117) provided with holes for the passage of the screws of the microscope stage by means of a sheet of self-adhesive material (119).  13. Application of the support to any one of claims 1 to 10, to ELISA tests, characterized in that one or more hvdro plaques - or - phile antibodies - of a support are sensitized using an antigen / contained in a liquid drop, which the support is washed with a reduced quantity of a usual buffer, that the support is dried, that a drop of serum or substance to be tested is applied to said area or said areas , then a drop of a chromogen with repetition of the above-mentioned washing and drying operations, after which a colorimetric measurement is made of the said area or areas.  14. Application according to claim 13, ca ractériseeen that a chromogen of a type which polymerizes while it is colored is used.  15. Application according to claim 14, characterized in that diaminobenzidine is used as chromogen.  16. Application of the support according to any one of claims 1 to 10 to radioimmunological tests, intended for the titration of antibodies or antigensZ  characterized in that one or more hydrophilic plaques are sensitized using an antigen or an antibody in a liquid drop, that the support is washed with a reduced quantity of a usual buffer, only after washing and drying, the serum to be studied or a previously diluted antigen is made to act, then, after incubation, drying and washing; a radio-labeled conjugate and that the reaction is revealed by bringing the support into perfect contact with a sensitive film  17. Application according to any one of claims 11 to 16, characterized in that one proceeds to the operations of drying the support while holding it at the ends to float it in a flow of hot air.  18. Application according to any one of claims 11 to 17, characterized in that supports are used having areas with the pitch of automatic drop dispensing devices.