WO2002016942A1 - Immunoassay method by adherence reaction - Google Patents

Abstract

This patent is an extension of patent n<o> FR 9405123. It concerns a method using adherence or adherence inhibition reactions for carrying out an immunoassay used for researching natural (Simonin test) or immune antibodies. The method is derived from the observation that test-globules in weak suspension (0.2%) in the presence of diluted plasma (1/4) do not agglutinate visibly to the naked eye before at least one hour of incubation, despite the presence of natural anti-A and anti-B antibodies. However, said antibodies can be properly fixed on their corresponding antigens. It is thus possible to apply the adherence technology without differential filtering effect despite the presence of a hydrating pad comprising a Sephacryl gel and hence, to demonstrate the fixing of anti-A and anti-B antibodies by adherence or adherence inhibition on a sensitized solid phase formed by the base of wells. Said technology involves the press effect of the gel which increases the forced contact of red blood cells with the solid phase and hence, enhances the sensitivity of the test. The hydrating pad prolongs the stability of the fixed antibodies and eliminates the washing phase as a result of the gradient effect of the liquid contained in the sealed wells.

Description

 IMMUNOLOGICAL ANALYSIS PROCESS by ADHESION Reaction

The present invention is an extension of patent n ° FR 9405123: "Device and Method for immunological analysis" and an extension of patent n ° FR 99 02523 "Method of Immunological Analysis by Adhesion Reaction". It allows the search for IgM antibody in binders biloba ^® device and in particular the implementation of the reaction Simonin, also called against-test, serum test, indirect test or "ABO Reverse", used in the groupage blood.

Traditionally, blood groupings are carried out by two complementary tests.

The first is the direct test also called Beth-Vincent test. It consists in looking for the presence of antigens A and B on the surface of the red cells of a given propositus. This test uses anti-A and anti-B test serum.

The second is the indirect test or counter-test also called Simonin test.

It consists in looking for the presence of anti-A and anti-B antibodies in the serum or plasma of the propositus. This test uses Ai, A ₂ , and B test globules.

The results observed during the indirect test confirm those observed during the direct test because it is known that in human blood, anti-A and anti-B antibodies are systematically and naturally present each time the corresponding antigen is absent on red cells.

The techniques used to demonstrate either antigens with known antibodies or test serum, or antibodies with known red blood cells or test cell are multiple. The most traditional are those made on slides, tubes or microplates (also called microtiter plates). The techniques of the new generation, more recent, are micro-column filtration techniques also called micro-filtration techniques. However, whatever the medium used by each of them, all these techniques exploit the agglutination phenomena according to which, each time that an antigen carried by a red blood cell is brought into contact with an antibody of the same specificity, it there is formation of agglutinates. In slide techniques, agglutination is spontaneous, that is to say visible to the eye after a few seconds or minutes of gentle agitation: a simple movement of the slide with a "boat" is sufficient due to the high concentrations of globules used: 10%. In tube and microplate techniques, agglutination is amplified and accelerated by centrifugation.

Micro-filtration techniques, originally developed by Yves Lapierre and used by Diamed, use a Sephadex gel in a micro-column. Agglutinates, which are formed either spontaneously in the reaction chamber located at the top of the column, or in contact with reagents present in the gel column (by simple imbibition, or even by coating of gel particles, which makes n 'is that a variant of the basic process of Lapierre), are pushed by centrifugal force through the so-called gel contained in the micro-column. The gel then acts as a filter letting free red cells which come to concentrate at the bottom of the micro-column. On the other hand, the gel retains the agglutinated red cells with all the more resistance as the agglutinates are important. The position of the agglutinates thus retained in the micro-column is related to the intensity of the agglutination. To date, no solid phase technique, using adhesion reactions, allows the simple realization of a counter-test. Only the BilOba ^® process allows such an approach.

The BilOba ^® process (as described in the initial patent FR 9405123) is characterized by the association of two sub-assemblies: "Bil" or incubation chamber and "Oba" or reaction chamber. These two elements associated at the time of the analysis allow in particular to reveal an antigen-antibody reaction by simple centrifugation and adhesion reaction on the bottom of the "Oba" chamber.

Solid phase techniques make it possible to demonstrate the presence or absence of adhesion reactions between fixed molecules (antibodies or antigens) on a solid support such as (but this is not limiting) on the bottom of cuvettes made of plastic and molecules present on red cells

(or particles) directed and / or sensitized against fixed molecules

(antigens or antibodies). In general, the presence of adhesion reactions indicates a positive biological reaction and the absence of adhesion reaction indicates a negative biological reaction.

In the "BilOba" process, the presence of a moisturizing cushion in the "Oba" part ensures three functions:

. hydration of the sensitive layer fixed on the bottom of the cuvettes providing the antibodies or antigens fixed with stability of several months (whereas it is only a few weeks for dehydrated antibodies). The hydrating gel never contains antibodies and never reacts with red cells. . a gradient effect, as described in the European patent for STOCKER

(81 110 764.8) but today abandoned. This gradient effect contributes to provide washing steps to remove proteins not attached to the red cells. The (low) height of the gel does not affect the gradient effect.

. a "press" effect: during centrifugation, the gel ensures a red blood cell plating effect against the bottom wall of the cuvettes, and this significantly increases the conditions of contact between the erythrocyte antigens and the antibodies coated on the bottom of the bowl. The moisturizing cushion thus promotes antigen-antibody bonds and consequently, increases the sensitivity of the reactions and therefore the performance of the process.

We recall below, by two nonlimiting examples, the operating methods of the BilOba process, as described in the initial patent, in order to fully understand the operation of the process extended to the ABO counter-test.

Example 1: In the context of the direct test, if the molecule attached is an antibody molecule (serum-tests), an adhesion reaction (noted by one to four crosses depending on the intensity of the reaction) between the red blood cells and the antibodies attached, will translate a positive biological reaction indicating that the red blood cells carry antigens against which the serum tests are directed. On the contrary, the absence of an adhesion reaction (noted -) will translate a negative biological reaction meaning that the red blood cells do not carry the antigens against which the test serum is directed.

Example 2 Likewise, in the context of the Search for Irregular Antibodies, if the fixed molecule is an antiglobulin molecule, an adhesion reaction between Pantiglobulin and red cells sensitized by an antibody directed against an antigen present on these red cells , will reflect a positive biological reaction. In contrast, the absence of an adhesion reaction will mean that the red cells have not been sensitized by an antibody directed against the antigens present on these red cells and therefore will reflect a negative biological reaction.

The technique described here is original and innovative because it uses a solid phase technique adapted to the counter-test, which has never been described or used in practice and in such a simple way. This technique is therefore an extension of the applications described in the initial patent FR 9405123.

This technique follows from the following original observation: when, in a "Bil" cuvette of the "BilOba" device, a serum or a plasma from a blood donor or a patient is brought into contact ((which is will name propositus here) and Red blood cells (A1, A2, B, and O), in suspension at 0.2% (concentration recommended in the BilOba technique), it is remarkable that no agglutination appears visibly before at least one hour incubation at laboratory temperature. On the other hand, if natural anti-A and anti-B antibodies are present in the serum of the propositus, these are capable of binding to the corresponding antigenic sites of the erythrocytes. The demonstration of this fixation, also called "sensitization" is carried out in two ways:

. by direct adhesion in the presence of anti-IgM antibodies or by coating the cuvettes with protein A or G (which have the ability to associate with immunoglobulins), or any other means of binding to proteins and specifically globulins of antibody

. by inhibition of adhesion in the presence of anti-A and anti-B antibodies The natural anti-A and anti-B antibodies mask the antigens of the same specificity and, therefore, inhibit their adhesion capacity with antibodies of same type fixed on the bottom of the bowl "Oba".

Two methods of demonstrating the antigen - antibody reaction in the ABO system, and in the original BilOba technology then become usable:

. an adhesion inhibition method using anti-A and anti-B antibodies

The erythrocyte antigens thus "capped" with an antibody molecule become inaccessible to antibodies of the same type fixed on the plastic support. Adhesion is therefore no longer possible. On the other hand, the non-sensitized antigens can adhere if the antibodies fixed on "Oba" correspond to them.

In this way, it is possible to demonstrate, in a perfectly adjusted system, a reduction or even a complete inhibition of adhesion in the presence of antibodies of the same specificity.

. a direct adhesion method using reagents capable of associating with the antibodies fixed on the red blood cells, such as antibodies against human globulin or of protein A or G, and this, in a nonlimiting manner.

The adhesion inhibition reactions are explicitly described in the table below:

AGAINST TEST BilOba ^® by inhibition of specific adhesion

A positive reaction between the antibody of a given subject and αlobule-tests results not in an adhesion reaction with the test serum attached to the plastic but in an inhibition of this adhesion reaction (denoted -): reactions 3, 5, 6, 13, 14 and 15. This results, after centrifugation, by the presence of a central pellet of red blood cells, in the form of a more or less concentrated point. - A negative reaction between the antibody of a given subject and test globules results in an adhesion reaction (noted ++++ to +) if the antigens present on the red cells correspond to the test serum attached to the plastic : reactions 1, 2, 7, 9, 10 and 11. It is possible to validly add red cells for control systematically giving a negative reaction, making it possible to ensure that the correct quantity of red cells has indeed been added during the procedure and its progress. See reactions 4, 8, 12 and 16

The reading of the BilOba ^® counter-test in adhesion inhibition technique is therefore reversed compared to that of the traditional counter-test:

AGAINST TEST BilOba by direct adhesion using anti-globulin anti-IgM or anti-heavy chains, or Proteins A or G or finally equivalents.

These red cells can therefore be centrifuged before one hour, before sedimentation, through a moisturizing cushion in which no retention takes place, which is a fundamental difference with gel techniques (such as those implemented in the process Diamed) whose foundation is based on the differential migration of red blood cells through a gel according to their degree of agglutination.

These are the foundations of this adhesion technology applied to the Simonin test.

Competition reactions of antigens or antibodies are known in immunological practices such as ELISA but none has ever been successfully applied to blood groupings by inhibition of adhesion. First, because only BilOba ^® technology makes it possible to perform blood groupings in wet adhesion with sufficient and demonstrated sensitivity. Secondly, because the absence of spontaneous agglutination in the middle of a weak red blood cell suspension has never been reported or exploited. In the case of the counter test taken here as a non-limiting example, the bottom of the "Oba" cuvettes is sensitized by antibodies of the same type as those reacting with antigens A and B, ie anti-test serum -A, anti-B and anti-AB. It will be advantageous to use monoclonal antibodies of the IgM or even IgG type, as widely available on the international market.

To allow the analysis of a single sample, two, three or four tests are possible. The anti-A test serum can be fixed on a cuvette if only one type A red cell is used or on two separate cuvettes (carrying out two tests, one with erythrocytes Ai, the other with erythrocytes A), anti-B and anti-AB serum tests each on a cuvette (carrying out a test with B red cells and another with O red cells).

The sensitive layer is then covered with a moisturizing cushion which can be a single liquid, with a gradient effect or this same liquid to which a dextran, Sephadex or Sephacryl gel or any other gel providing an increased retention capacity is added. water to increase the conservation of the product and therefore improve its stability.

The "Oba" bowl is finally sealed with an aluminum seal as described in the basic patent and can be stored for several months between 2 and 8 ° C.

To fully understand the chronology of the phases of this technique, we give below, by way of nonlimiting example, the methodology for using a counter test carried out with red blood cells Ai, A ₂ , B and O. 1 - A "Bil" plate is set on an "Oba" plate.

2 - 30 μl of a dilution medium, physiological water for example, are placed in four "Bil" cuvettes. These four "Bil" cuvettes correspond to four "Oba" cuvettes sensitized by anti-A antibodies for Oba 1 and Oba 2, an anti-B antibody for Oba 3:: and an anti-A + B antibody for Oba 4.

3 - 10 μl of plasma or serum from a patient or a blood donor are deposited in these same four cuvettes: either the plasma of subject A for this example.

4 - 30 μl of red cells Ai, A ₂ , B and O in suspension at 0.2% in the same dilution medium as that previously deposited (physiological water in this example) are added respectively to the first, the second, third and fourth bowl.

5 - A 5-minute incubation at room temperature is observed.

6 - The BilOba ^® device is centrifuged for 4 minutes at 1000 g. 7 - Reading takes place as follows:

During centrifugation, the red cells will diffuse through the moisturizing solution, disperse homogeneously there and come into contact with the bottom walls of "Oba". If the "styling" or the sensitization of the red cells took place in "Bil" during the incubation, the red cells will no longer be able to fully adhere to the antibodies fixed on "Oba" and will slide along the "V" walls to form a button globular clear in the center of the bowl: reaction noted -. If no "styling" or sensitization took place in "Bil" during the incubation, the red cells will be fixed on the antibody fixed on "Oba" if it is directed against their antigens, to form a carpet more or less homogeneous according to the intensity of the reaction (noted ++++ to +) but will not be fixed on the antibodies not directed against their antigens in which case they will form here again a very clear globular button in the center of the cuvette: reaction noted - .

The reactions observed in the four basins of our example are therefore the following:

Basins 1 (Bil 1 / Oba 1) and 2 (Bil 2 / Oba 2) give an image of a more or less homogeneous pink carpet, a reaction noted from ++++ to + depending on its intensity, because the A cells having no not sensitized by the anti-B antibodies present in the plasma of subject A adhered to the anti-A test serum fixed on the bottom of the Oba 1 and Oba 2 cuvettes.

Cuvette 3 (Bil 3 / Oba 3) gives an image of a central button reflecting the inhibition of adhesion of red cells B sensitized by the anti-B antibodies present in the plasma of subject A and which can no longer adhere to the test serum anti-B fixed on the bottom of the Oba 3 bowl.

Cuvette 4 (Bil 4 / Oba 4) gives an image of a central button reflecting the absence of an adhesion reaction between erythrocytes O (sensitized by any antibody) and the anti-AB test serum fixed on the bottom of the cuvette Oba 4.

The advantages of the BilOba ^® cross-proof technique over other cross-proof techniques are manifold. They reside in a simplification of the technical procedure and an absence of contradictory phenomena leading to interpretations of the results which are sometimes erroneous.

1 - Elimination of the risky steps involved in carrying out the counter-proof, such as the stirring steps which are difficult to standardize.

If we compare the procedure for using a microplate technique and a solid phase technique (see table below), we see that three risky steps necessary in the microplate technique are unnecessary in the BilOba ^® technique. COMPARISON OF THE IMPLEMENTATION PROCEDURES

MICROPLATE STRAWBERRY AND SOLID PHASE STRAWBERRY BilOba ^®

* Etapes à risques.

* Risky steps.

In the microplate technique, the homogenization step of the serum + red blood cells mixture is carried out either by manual tapping of the microplate, or on a vibrating agitator. After centrifugation, globular pellets are formed in the center of the cuvettes. To differentiate the positive reactions from the negative reactions, it is then advisable to agitate the microplate more or less strongly for one minute to peel off the globular disc then to agitate the microplate in a gentle way for another minute to collect in the center of the cuvettes, the agglutinates which will have survived the agitation.

We understand the limits of this technology, the results of which may vary depending on the parameters applied to its implementation. Too strong agitation, destroying weak agglutinates can lead to a false negative interpretation. Insufficient agitation (in time and / or speed) which only partially separates red blood cells concentrated by centrifugation can lead to a false positive interpretation.

There is no standardized device today, allowing identical cycles of agitation to be reproduced. This poses a big problem of reproducibility.

In addition, certain microtitration plates can sometimes present phenomena of spontaneous bonding of red blood cells which imply more vigorous agitation for their resuspension. This is therefore contrary to the search for great sensitivity. 2 - Simplification of reading

In the microplate technique, the arrangement of agglutinates in the center of the cuvettes is important for automated reading, which, apart from reading by more recent and still confidential image analysis, performs a measurement of differential optical density between the center of the bowl and its periphery. A shift of the agglutinates from the center of the cuvettes can lead to a false negative interpretation.

3 - Better sensitivity with red cells A?

In the traditional techniques in tubes or in microplates, weak reactions are often observed in particular with red cells A ₂ , because the natural antibodies are often of weak title and not very eager with respect to the A2 antigenic sites leading to fragile associations. In the BilOba ^® technique, this weakness is not taken into account because the red cells are not agglutinated and sensitization, even partial, immediately results in an inhibition of perfectly visible adhesion.

4- Significant reduction in consumption of test globules

Being diluted to 0.2%, the consumption of test globules is reduced by 50 times compared to traditional tube techniques, and by 4 times compared to micro-column techniques. With a blood bag we can consider more than three million tests in BilOba technique.

Claims

1 - Method of immunological diagnosis by adhesion in micro-cups characterized in that the sensitive layer located on the bottom of each cup is kept moist by a moisturizing pad and in that the cup is hermetically closed by a waterproof and tearable membrane. 2- A method according to claim-1 characterized in that the moisturizing cushion is a complex based on dextran gel of the "Sepharose" or "Sephacryl" type or equivalent and of a solution of intermediate density between that of plasma and that of red blood cells providing a gradient effect which contributes to the dynamic separation of these elements during centrifugation. 3- A method according to claims 1 and 2 which allows immunological reactions without washing phase. 4- A method according to claim- 1 characterized in that the red cells which are taken into account in the immunological reaction are never agglutinated and cross the gel without differential filtration effect to come into contact with the bottom of the wells. 5- A method according to claim-1 characterized in that the reduced height of gel in the cup does not give it a sufficient chromatographic effect to separate alone the constituents of the blood. 6- A method according to claim-1 characterized in that the gel is neutral and never contains antibodies capable of reacting with red cells diffusing through the gel under the effect of centrifugation. 7- A method according to claim- 1 characterized in that the gel constituting the moisturizing cushion causes a press effect on the red cells during the centrifugation phase and during their flow on the bottom of the wells. This press action amplifies the contact conditions of said red cells with the sensitized solid phase from the bottom of the wells and therefore significantly increases the specific adhesion and therefore the sensitivity of the test. 8- A method according to claim-1 characterized in that even natural antibodies of the IgM type do not cause spontaneous agglutination and therefore do not give rise to differential filtration phenomena during their passage through the moisturizing cushion under the effect of centrifugation. 9- A method according to claims 1 and 7 characterized in that it allows the detection of anti-A and anti-B type antibodies by direct adhesion reaction or by adhesion inhibition reaction (test of Simonin). 10 - Process according to claims 1, characterized in that the solid phase can be sensitized by antibodies of different types, without limitation, and by antigens. 11- A method according to claim-1 characterized in that the red cells-tests can be replaced by inert particles covered with antigens or antibodies. 12- A method according to claim- 1 characterized in that the reaction is read by observing the underside of each well. AMENDED CLAIMS [received by the International Bureau on July 5, 2001 (05.07.01); claims 1-12 replaced by claims 1-8 amended (1 page)] - Method of immunological analysis by adhesion in a humid environment in which the low concentrations (0.2%) of red cells test lead to the absence of spontaneous agglutination of these red cells during the incubation phase, even in the presence of antibodies IgM type. (zone effect). - Immunological analysis method in which the adhesion layer is kept moist by a neutral moisturizing cushion, never containing an immunologically active substance. - Method according to claim-2 characterized in that the moisturizing cushion may preferably comprise a Sephadex or Sephacryl gel or any other micro-particulate suspension formulated in a liquid with a gradient effect (such as albumin, sugar, etc.). - Method according to claims 2 and 3 which gives the moisturizing cushion, during the centrifugation phase, a press function which forces the red cells to close contact with the sensitive layer fixed on the bottom of the wells, which promotes antigen- antibody and provides a noticeable gain in sensitivity. - Method according to claims 1 and 2 characterized in that the moisturizing cushion has no red blood cell retention property due to the absence of spontaneous agglutinates after the incubation phase, and the absence of immunologically active component in its formulation. - Method according to claims 1 to 5 which allows to highlight the natural anti-A and anti-B antibodies by direct adhesion in the presence of a coating of anti-IgM antibodies or immunologically active proteins such as protein A or G. - Method according to claims 1 to 5 which makes it possible to demonstrate the presence of natural anti-A and anti-B antibodies by inhibition of adhesion in the presence of a coating produced with antibodies of the same nature (anti-A and anti-B). - Method according to claims 1 to 6 characterized in that the reading of the adhesion reaction is carried out by observation of the underside of each cup.