WO1983003677A1

WO1983003677A1 - Analysis device for biological symbols

Info

Publication number: WO1983003677A1
Application number: PCT/CH1983/000046
Authority: WO
Inventors: S.A. Genefusion
Original assignee: Boquet, Patrice
Priority date: 1982-04-16
Filing date: 1983-04-15
Publication date: 1983-10-27
Also published as: EP0106855A1

Abstract

A system allows to carry out simultaneous analysis and detections of a factor in a plurality of biological samples. It is characterized by the combination on one hand of a plate comprising a set of cavities distributed in a plurality of distinct series and rows, said plate being made of a material having no adsorption capacity for the factor to be tested for and, on the other hand, of a comb comprising teeth (4) of which the spacings allow to insert them simultaneously in as many cavities of the plate, the teeth of said comb being themselves made of a material retaining or capable of retaining, particularly by absorption or adsorption, either the factor or the reactant for detecting said vector.

Description

Analysis device for biological samples

The invention relates to a system allowing simultaneous analyzes and detections of a factor, for example a tigene year or an antibody, in a plurality of biological samples. More particularly, it uses plates comprising a set of cavities suitable for receiving the samples to be assayed as well as the reagents useful for detection.

Systems using such plates have already been proposed, more particularly plates containing in advance the reagents necessary for the reaction, for example the antibody corresponding to the antigen sought in the samples to be assayed. It has in particular been suggested to use plates formed from a plastic material, characterized by a capacity for adsorption or absorption of proteins, for example an antibody, the plates then being delivered to users already provided in advance. reagents necessary for the assays, for example antibodies, in some of their cavities. The user can then introduce the sample to be studied into the corresponding cavities, if necessary diluted in an appropriate buffer. It is also known that carrying out an assay can involve a series of successive operations, which may involve successive transfers of the reaction medium from one cavity to another, either on the same plate, or on different plates. , in particular by pipetting operations.

Although such operations have already been automated, they can remain of a rather complex implementation. Furthermore, it may happen that the presence of certain reagents, initially introduced into the medium to be transferred with a view to carrying out a first useful reaction, becomes a cause of disturbance of the posterior reactions to operate on the biological sample during a posterior stage of the assay.

The object of the invention is to remedy these difficulties, more particularly to provide a system which, while allowing simultaneous detections of a factor, or even of several factors in a plurality of biological samples, is nevertheless of manipulation extremely simple. In addition, it aims, in the case of assays comprising a series of operations to be carried out in separate cavities to obviate the difficulties that may result from the forced transfers, from one cavity to another, of some of the reagents used in the first cavities, with all of the sample then transferred to the following cavities, with a view to carrying out the subsequent steps of the assay.

The system according to the invention is characterized by the combination, on the one hand, of a plate comprising a set of cavities distributed in several distinct series, respectively comprising equal numbers of cavities, and forming as many rows of tubes in mutual correspondence , said plate being formed of a material having no adsorption or absorption capacity, both with respect to the factor to be detected and the reagents necessary for its detection and, on the other hand, a comb comprising teeth, the spacings of which allow them to be introduced simultaneously into the cavities of each of the above rows, or to be removed therefrom simultaneously, the teeth of said comb being themselves formed from a retaining material or able to retain, in particular by absorption or adsorption, either the factor, or the detection reagent of said factor, or both at the same time.

It goes without saying that the aforementioned plate may be constituted in any way known per se with regard to its structure, that the respective numbers of series and rows of cavities may vary from one plate to another, in particular according to the nature of proposed dosages; that these cavities can be of any shape, semi-spherical, conical, tubular, etc.

The non-adsorbent or absorbent nature of the material from which the walls of the cavities are made allows the possible extraction from the medium contained in these cavities of one of the principles which it contains, more particularly either of the factor to be assayed, reagent characteristic of this factor, ie of the reagent on which the factor has come to be fixed, for example when the factor to be assayed consists of an antigen and the specific reagent consists of an antibody.

In particular, this extraction from the culture medium can be carried out by the teeth of the comb, if these are made of a material capable of retaining either the factor, or the reagent, or both at the same time, as required. dosage.

Advantageously, the materials in question are made of plastics well known to the specialist.

To constitute the material of the walls of the cavities of the plate, it is possible to have recourse to plastics such as polystyrene or non-ab = absorbent and smooth PVC.

As plastics likely to be used to form the teeth of the comb, we can use key similar materials, however pretreated with oleaginous compounds or surfactants, available commercially.

It is understood that the expression "comb" covers any system comprising a plurality of teeth or rods, kept apart from one another by any appropriate means, the simplest being constituted by a common support, of which they are permanently or temporarily attached, so to be able to be introduced simultaneously ment, during a movement of these means towards the plate, in a plurality of cavities, preferably in the same row. It goes without saying that one could also design an equivalent system allowing the simultaneous introductions of the teeth of the comb into cavities belonging to distinct rows of the plate. This would naturally result in an equivalent operating system, allowing each tooth to pass from one cavity to another, on the occasion of the movements of withdrawal, translation with respect to the plate, and reintroduction of the teeth. of the comb in separate cavities of the plate, and having similar relative arrangements.

If we now consider that the comb can be moved from one row to another of the cavities of the plate, we can see that it allows the simultaneous production of as many dosages as the plate has cavities in the same row and / or the tooth comb. Insofar as the comb has qualities of absorption or adsorption with regard, for example, to the antibody used as reagent, the comb will allow the transfer of the antibody possibly present in the cavities of the first row, after modification or not by the antigen, of these cavities to those of the second row, then from the latter to the third, etc., and this. the number of times necessary for the completion of all the assays required by the detection, as will be illustrated by way of example below, as regards the detection of the presence of cholera toxin taken by way of example , in a biological liquid. It goes without saying that if we have considered in the above the case of simultaneous detections of the same factor in a plurality of biological samples, using a reagent, the invention would apply from the same way to the simultaneous detections of several distinct factors possibly contained in fractions of a biological liquid coming from the same sample, the reagents used being then adapted to each particular factor sought. Of course this variant is only an equivalent of the previous one and therefore enters the scope of the claims attached to this request.

A system according to a preferred embodiment of the invention is characterized by consecutive rows, cavities or groups of rows of cavities having sections of reduced dimensions alternating with consecutive rows or groups of rows whose cavities have more elongated sections , in particular in the directions of the series to which they belong, these longer sections allowing limited relative displacements in the same directions of the teeth of the comb, when these have been introduced therein. These rows of cavities of distinct sections will be respectively designated below under the designations of rows "of the first type" or of rows "of the second type", or else "first rows" and "second rows".

The above preferred arrangement makes it possible, when necessary, either to keep the teeth of the comb in a well-defined position with respect to the internal volume of the cavities already in question, in particular when one is not looking for a stirring effect and that the teeth of the comb with the walls must be avoided. Conversely, the elongated dimensions of the other cavities allow a certain level of agitation of the liquid contained in these cavities, via the teeth of the comb or possibly facilitates the rinsing of the products badly or insufficiently fixed, entrained by the teeth of the comb. upon removal ^of a previous row of cavities. Again, it will be appreciated that there is a large choice of sizing possibilities for the different cavities, so that each type of dosage can meet a specific type of plate. II. It goes without saying that the directions corresponding to the largest dimensions of the sections of the elongated cavities can be any, for example perpendicular to the directions of the series in question. All these variants are naturally equivalent to the more particularly claimed embodiments and to this title falls within the framework of the protected invention. Even the plates of the systems according to the invention could also include rows of cavities having sections distinct from that of the rows "first type" or "second type", etc.

To ensure the correct positioning of the teeth of the comb in the cavities of each of the rows, while avoiding possible contact of the teeth of the comb with the walls of these cavities, the plate is advantageously provided with guide means coagulating with members guided by the comb, to ensure the establishment, the maintenance in position (and if necessary limited movements of the teeth of the comb) and the withdrawal of these teeth out of the cavities in question. The movements of introducing the teeth of the comb into the successive cavities can be carried out manually. Of course, the system can also be automated, the comb being able in particular to be associated with a device known per se making it possible to control the successive movements of the comb, in particular the successive introductions of the teeth in distinct rows of the plate, keeping them in position in the cavities in each row (or the

limited movements of the teeth of the comb ---------------------- inside said cavities), then the removal operations, according to a predetermined program. Means available in the prior art may be used for this purpose.

Additional characteristics of the invention will become apparent during the description, given for information only, of a preferred construction of the system according to the invention, as well as of an example of implementation of this construction, to achieve , here again only by way of example, simultaneous detections of a determined antigen, here cholera toxin, in a plurality of biological samples presumed to be able to contain it. This des cription. will also be made with reference to the drawings in which: - fig. 1 is a diagram of a plate seen in perspective,

- fig. 2 is a section through a particular embodiment of the comb,

- fig. 3a, 3b, 3c illustrate shapes advantageously imparted to the ends of each of the teeth of the comb.

The plate of fig. 1 comprises a set of four series S1, S2, S3, S4 of cavities 2a, 2b: thirteen cavities per series in the example considered. It follows that the plate also includes, in the example considered, thirteen rows of cavities. As indicated above, this plate, in any case the walls and the cavities, are made of a material, in particular a plastic material having no absorption or adsorption capacity, both with respect to the factor to detect only reagents necessary for its detection.

- Fig. 2 schematically shows the comb associated with the plate in the system according to the invention, this comb comprising four teeth 4 having spacings similar to those of the cavities in each of the rows, so that they can be introduced if simultaneously in the cavities 2a or 2b, be held there and be withdrawn simultaneously, the teeth of the comb being, as indicated above, made of a material, in particular a plastic material, retaining or capable of retaining , preferably by absorption or adsorption, either the factor or the detection reagent of said factor.

In the case shown, the cavities 2a are circular and the cavities 2b have an elongated section in the longitudinal direction of series, these more elongated sections allowing limited relative movements in the same directions of the teeth 4 of the comb, when these have there been introduced.

Advantageously, the plate and the comb are provided with mutual guiding means allowing the easy positioning of the comb with respect to the cavities of each row, each time it is brought opposite the cavities of this same row, in particular vertically. In particular, the guide means carried by the plate consist of two types of notches 6a, 6b, formed by the lateral sides 8 of the plate, on either side of said series, these notches of the first type (6a ) and of the second type (6b) being in correspondence with the rows formed of circular section cavities (2a) and the second rows of elongated section cavities (6b). The guided members carried by the comb consist of complementary rods or teeth 10 ₁ , 10 ₂ , preferably on either side of the teeth (4) and capable of interacting with the notches, during the insertion movement. teeth (4) in successive rows of cavities. Advantageously, the rods have a section substantially complementary to the notches 6a of the first type, so that the engagement of said rods in these. notches, on the occasion of the introduction of the comb teeth into cavities of type 2a, ensures the guidance of said teeth in a well-defined direction, in particular along the axis of the cavities 2a. The notches of type 6b, in corres laying with the second rows, have a greater width, the edges 6b ₁ , 6b _{2 of which} define stops, interacting with the rods 10 ₁ , 10 ₂ , in order to limit the limited relative displacements then authorized of the teeth of the comb, when these these are or have been introduced into the cavities of the corresponding row of the plate.

It is advantageous that the teeth of the comb have at their ends 12 bulging parts or having larger sections than the intermediate zone 14 of the teeth, between said ends 12 and the support 16 of said teeth, support which optionally includes a part 18 allowing its connection to a device desmisesen position and movements of the comb vis-à-vis the plate. We can give the ends 12 any shape, as shown for example in Figs. 3a, 3b and 3c, it being understood that these shapes (pallets or fins) have in common the ability to prevent capillary action of the liquid possibly contained in the cavities into which they penetrate, along the narrower intermediate portions 14 of said teeth, towards the support. This system can then be implemented to carry out assays of the type which will be described below by way of example.

EXAMPLE OF EVIDENCE OF AN ANTIGEN WITH THE SYSTEM ACCORDING TO THE INVENTION.

We want to look for the presence of cholera detoxin in a biological fluid (element A). In this example, a plate and a comb having the following characteristics are used:

1 °) plastic plate with cavities 2 °) comb with a special plastic plastic (in ....... ....................... ..). The non-adsorbent plastic plate comprises at least three rows of thirteen holes of circular and elongated sections respectively according to the modulation shown in fig. 1.

The sequence is always as follows:

A cavity where a reaction takes place, followed by three larger cavities where the washes are carried out.

The comb is made of specially treated plastic, in particular ........................................ to adsorb proteins.

We have: - rabbit-type antibodies directed against cholera toxin (element B) .;

- rabbit-type antibodies directed against cholera toxin, on which an enzyme has been covalently attached, for example lactoperoxidase (lacto-peroxidase catalyzing the peroxidation of a substrate (for example: orthophenylenediamine) passing it from colorless to dark brown (element C);

- of this substrate (for example: orthophenylenediamine) undergoing peroxidation (element D).

Cholera toxin is also available in order to make the positive controls (element E).

The research methodology is as follows: a) the anti-cholera antibodies (element B) are dissolved in a "buffer", in the first row of cavities; b) the teeth of the comb are introduced into these cavities and brought into contact with this solution for a defined time and at a defined temperature, to effect the absorption or adsorption, preferably until saturation, of the antibodies on the material of the comb teeth; c) the comb is then washed so as to remove the excess of antibody which would not be sufficiently solidly absorbed; this is done in the second, third and fourth rows of cavities each containing a "pad" and a detergent. The limited movements which can be printed on the comb under the conditions described above, without the teeth hitting the end walls of the cavities, facilitate washing. d) the comb is then introduced or deposited in the fifth row of cavities ("reaction cavities") into which the biological liquid to be assayed is introduced (element A).

One or more cavities in this row were filled with solution comprising either cholera toxin (positive control: element E), or simply "buffer" (negative control).

The teeth of the comb remain in contact with these solutions for a defined time and at a defined temperature. e) the teeth of the comb are then washed in the sixth, seventh and eighth rows of cavities, as indicated in paragraph c. f) the teeth pass into the ninth row of cavities (or third row of "reaction cavities") which contains anti-choleric antibodies coupled to the enzyme lactoperoxidase (element C) in a "buffer" solution.

The teeth of the comb are also left in contact with this solution for a defined time and at a defined temperature. g) washing in the tenth, eleventh and twelfth series of cavities, as indicated in paragraph c). h) we finally arrive at the last row of cavities (or fourth row of "reaction cavities") in which the substrates of the enzyme lacto peroxidase are located (element D).

Where there is the presence of cholera toxin (fixed on the antibodies absorbed on the corresponding teeth of the comb), the cholera antibodies coupled with lactoperoxidase can be fixed on the substrate and the colored reaction (dark brown) can take place.

If the biological fluid examined does not contain cholera toxin, the reaction producing colo dark brown ration can not take place, and the liquid in the cavity remains colorless.

This example shows the ease with which it is possible to carry out detections of one or more determined factors, by operations involving extremely simple equipment and entirely reduced manipulations, which can be either manual or partially or completely automated. The technique according to the invention also makes it possible to promote successive chemical or immunological reactions, in different media, proteins, such as antigens or antibodies which can, after absorption or adsorption on the teeth of the comb, pass successively into cavities containing different environments, each appropriate to the nature of the operation to be carried out. By transferring the factor to be detected or the corresponding reagent, or both at the same time, from one cavity to another, via the teeth of the comb, it is possible to prevent successive reaction liquids from being soiled by the others, especially if recourse is had to steps for washing the teeth of the comb, under the conditions which have been indicated in the example. Of course, the detection described above is only an example and the specialist will be well aware. multiple variations as to the nature of the dosages to which the system according to the invention can lend itself.

For example, antibodies against an X antigen can be assayed, for example by using, in a first step, the fixing of this X antigen on the teeth of the comb, then in a subsequent row of cavities when the retaining comb is brought into contact. antigen X with antibodies carrying a marker, such as lactoperoxidase, against the type of antibody to be detected or assayed. The system is obviously not limited to assays involving couplings between antigens and antibodies. It can be used for any assay involving the detection of a particular protein, such as an enzyme, provided that a molecule affine with respect to this protein or enzyme is available, capable of selective retention on the teeth. of the comb, for example on the occasion of bringing it into contact with said affine molecule in a first row of cavities in the plate.

The technique according to the invention allows not only qualitative detections, but also quantitative assays. In the latter case, for example, a colored reaction may be used which can be made either visually or by spectrophotometry with digital or printed optical reading. One can naturally use any other conventional technique leading to the detection in particular of a concentration, of active principle. In the foregoing examples, the case of the detection or the determination of the same active principle in a plurality of biological samples has been considered. It goes without saying, of course, as has already been indicated above, that it is also possible to envisage carrying out simultaneous assays of distinct factors originating from one or more biological samples. In the latter case, the reagents intended to be removed by the teeth of the comb must be chosen. accordingly and arranged in the first reaction cavities (or other cavities) also accordingly.

Of course, it can be chosen to reverse the nature of the products to be absorbed first on the teeth of the comb, when this seems desirable, taking into account the particular dosage envisaged. - In the foregoing, we have essentially described the combination of a plate with a comb. It goes without saying that we can also consider systems associating several combs to a plate or vice versa.

For example, several combs will be used when it is a question of successively introducing at the level of a determined row of "reaction cavities" several distinct reagents, respectively extracted from distinct anterior rows of cavities, under the conditions described in relation to the single comb referred to above.

In a particularly advantageous variant of the invention, the support plates of the cavities can also be supplied pre-filled with different reagents capable of forming buffers or reaction media as the case may be, either in liquid form or in lyophilized form, the plates being then generally made available to the public protected by a cover or by a film adhering to the intermediate zones of the plate, separating the cavities from one another. These plates can be delivered to users with one or more combs, so that they will only have to add the biological sample (blood, serum, urine, etc.), possibly diluted, in those of the rows suitable for receive them and complete the other reaction cavities, either with water or with a solution meeting the conditions required by the detection method put forward.

In yet another variant of the invention, the comb or combs are delivered with the pre-absorbed or pre-adsorbed detection reagent on their teeth. Such an arrangement could prove to be particularly advantageous in the case of the detection of specific enzymes to which affine molecules correspond, for example inhibitors of activation of these enzymes, the affine molecule then being able to be retained on the teeth of the comb under conditions similar to those prevailing in the case of affinity chromatography substrates.

As is obvious and as it already follows from the above, the invention is not limited to zero ment to those of its modes of application and embodiments which have been more especially envisaged; on the contrary, it embraces all its variants.

Claims

1. System allowing analyzes and simultaneous detections of a factor, in a plurality of biological samples, characterized by the combination, on the one hand, of a plate comprising a set of cavities (2a, 2b) distributed in several distinct series (S ₁ , S ₂ , S ₃ , S ₄ ) respectively comprising equal numbers of cavities, and forming as many rows of tubes in mutual correspondence, said plate being formed of a material having no adsorption capacity or absorption, both with respect to the factor to be detected and the reagents necessary for its detection and, on the other hand, of a comb comprising teeth (4) whose spacings allow them to be introduced simultaneously into the cavities (2a, 2b) of each of the above rows, or to be removed therefrom simultaneously, the teeth of said comb being themselves formed of a retaining material or able to retain, in particular by absorption or adsorption or the fact r, or the detection reagent of said factor.

2. System according to claim 1, characterized in that the plate is formed from a plastic material or the like absorbing or not absorbing the proteins and that the teeth of the comb are formed from a plastic material or the like absorbing n not absorbing proteins.

3. System according to claim 1 or claim 2, characterized in that the cavities or consecutive groups of first rows (2a) having reduced dimensions sections alternate with second rows or consecutive groups of second rows (2b) including the cavities have more elongated sections, in particular in the directions of the series to which they belong, these more elongated sections allowing limited relative displacements in the same directions of the teeth of the comb, when these have been introduced into the corresponding cavities of the same second row.

4. System according to any one of claims l to 3, characterized in that the plate is provided with mistletoe means dage (6a, 6b) coacting with guided members (10 ₁ , 10 _b ) carried by the comb, to ensure the correct positioning of the comb with respect to each row of cavities, during the movements of introduction of the comb teeth into the cavities of the same row and their withdrawal movements out of the cavities thereof.

5. System according to claims 3 and 4 taken together, characterized in that the guide means carried by the plate consist of two types of notches (6a, 6b) formed in the lateral sides of the plate, on the side and d 'Another of said series, these guide means being in correspondence with the above first and second rows, and in that the guided members (6b ₁ , 6b ₂ ) carried by the comb consist of rods or the like capable of engaging in said notches, the notches of the first type in correspondence with said first rows advantageously having a section substantially complementary to that of said rods, so that the engagement of said rods in notches of the first type, on the occasion of the introduction of the teeth of the comb in the cavities of one of the said first rows guiding the said teeth in a well-defined direction, the notches in correspondence with the second ra nées having a greater width and edges (6b ₁ , 6b ₂ ) defining stops interacting with said rods to limit the limited relative movements then authorized of the teeth of the comb, when these are or have been introduced into the cavities of one of the second corresponding rows of the plate.

6. System according to any one of claims 1 to

5, characterized in that the teeth of the comb having at their end a portion (12) swollen or of larger section than the intermediate zone (4) of said teeth between said end and the support part (16) of the comb carrying said teeth.

7. System according to any one of claims 1 to

6, characterized in that some of the cavities of the plate contain in advance the reagents necessary for carrying out the user extemporaneous dosage.

8. System according to any one of claims 1 to 7, characterized in that one or more reagents necessary for the detection of a pu or several factors are prefixed on the teeth of the comb.