FR2790093A1 - Blood typing by erythrocyte adherence to coated microwell comprises a sensitizing antibody kept moist by hydrating cushion, and improves storage stability and sensitivity - Google Patents

Abstract

In an immunological diagnosis involving adherence to microtiter wells, the sensitive layer at the base of each well is kept moist by a hydration cushion and the cell is hermetically sealed by a protective cover.

Description

IMMUNOLOGICAL ANALYSIS PROCESS

  by ADHERENCE reaction The present invention is an extension of patent n FR 9405123: "Device and method of immunological analysis" It allows the search for agglutinating antibodies of the IgM type in the BilOba device and in particular the implementation of the Simonin reaction, also called a counter-test, serum test or indirect test, used in

io blood groupings.

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

by Simonin.

  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 globule tests A ", A2, and B. 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 are systematically and naturally present

  whenever the corresponding antigen is absent on red cells.

  This rule leads to the following interpretation table: Red blood cells of type Ai A2 B A1B A2B O Antibodies present in Anti-B Anti-B Anti-A none none Anti-A + B serum The techniques used to demonstrate either antigens with known antibodies or test sera, either antibodies with known red blood cells or test cells are multiple. The most traditional are those35 made on slides, tubes or microplates (also called microtiter plates). New generation techniques, more recent, are

  micro-column filtration techniques also known as micro-

  filtration. However, whatever the medium used by each of them, all of 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 a

  antibodies with the same specificity, 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 "rowboat" movement of the slide is sufficient due to the strong

  concentrations of globules used: 10%.

  In tube and microplate techniques, agglutination is amplified and

  accelerated by centrifugation.

  Microfiltration techniques, originally developed by Yves Lapierre and used by Diamed, use a Sephadex gel in a micro-column. Agglutinates, which form either spontaneously in the reaction chamber o 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 them is only a variant of the basic process of Lapierre), are pushed by centrifugal force through the said - gel contained in the micro-column. The gel then acts as a filter letting the free red cells pass 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 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 carrying out an analysis make it possible in particular to reveal an antigen-antibody reaction by simple centrifugation and adhesion reaction.

on the bottom of the "Oba" room.

  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 reaction.

negative biological.

  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 for several months (whereas it is only a few weeks for dehydrated antibodies a gradient effect, as described in the European patent of STOCKER (81110764. 8) but now abandoned. This gradient effect helps to dispense washing steps to remove proteins not attached to red blood cells. A "press" effect: during centrifugation, the gel ensures a plating effect of red blood cells against the bottom wall of the cuvettes, and this significantly increases the contact conditions between the erythrocyte antigens and antibodies coated on the bottom of the cuvette. The hydrating 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 cross-proof.

  Example 1: In the context of the direct test, if the fixed molecule is an i5 antibody molecule (serum-tests), an adhesion reaction (marked with one to four crosses depending on the intensity of the reaction) between red blood cells and attached antibodies, will translate into a positive biological reaction indicating that

  red blood cells carry antigens against which the serum is directed

  tests. On the contrary, the absence of an adhesion reaction (noted -) will translate a negative biological reaction meaning that the red blood cells are not carriers of

  antigens against which the test serum is directed.

  EXAMPLE 2 Similarly, in the context of the search for irregular antibodies, if the fixed molecule is an antiglobulin molecule, an adhesion reaction between the antiglobulin and red blood cells sensitized by an antibody directed against

  an antigen present on these red cells, will translate 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 translate 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 patent

initial 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

  fix on the corresponding antigenic sites of red blood cells.

  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 s '' associate with immunoglobulins), or any other means of binding to proteins and specifically globulins of antibody A by inhibition of adhesion in the presence of anti-A and anti-B antibodies The natural anti-A and anti-B antibodies come to mask antigens with the same specificity and, therefore, inhibit their adhesion capacity with antibodies of the same type fixed on the bottom of the "Oba" cuvette Two methods of demonstrating the antigen-antibody reaction in the ABO system , and in the original BilOba technology then become usable: 1 5. an adhesion inhibition method using anti-A and anti-B antibodies The erythrocyte antigens thus "capped" with an anticor molecule ps become inaccessible to antibodies of the same type attached to 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. It is thus possible to demonstrate, in a perfectly adjusted system, a reduction or even a complete inhibition of the adhesion in the presence

of antibodies with the same specificity.

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

not limiting.

  The adhesion inhibition reactions are described explicitly in the table below: AGAINST TEST BilOba by specific adhesion inhibition Group blood Solid phase Globule- AB AB O Serum- test tests Ai 1 ++++ 5 9 ++++ 13 - Anti-A A2 2 +. + 6 10 +++ 14 Anti-A B 3 - 7 .. ++ 1i ++++ 15 - Anti- BO 4 - 8 - 12 - 16 - Anti-AB - A positive reaction between the antibody of a given suiet and test globules results not in an adhesion reaction with the test serum attached to the plastic but in an inhibition of this adhesion reaction ( noted -) reactions 3, 5, 6, 13, 14 and 15. This is reflected, 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 suiet 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.

  Red cells can be validly added 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 Reading the BilOba cross-proof in adhesion inhibition technique is therefore reversed compared to that of the traditional counter-test: COUNTER TEST BilOba by direct adhesion using anti-IgM or anti-heavy chain antiglobulins, or Proteins A or G or

finally equivalent.

  Group blood Test cell A B ABO

A1 - ++++ - ++++

A2--

B - ++++

  _ At - +++ ± ++++ _ Io i - - - - _ 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 Diamed process) whose foundation rests on the differential migration of red blood cells through a gel in

  depending on their degree of agglutination.

  These are the foundations of this adhesion technology applied to the Simonin test. Antigen or antibody competition reactions are known in immunological practices such as ELISA but none has ever been

  successfully applied to blood groupings by inhibition of adhesion.

  Firstly, because only BilOba technology makes it possible to carry out blood groupings in wet adhesion with sufficient and demonstrated sensitivity. Then because the absence of spontaneous agglutination in the middle of

  weak red blood cell suspension has never been reported or used.

  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 red cells A1, the other with red cells A2), o anti-B and anti-AB test serum each on a cuvette (carrying out a test with

  red cells B and another with red cells O).

  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 product conservation and

so 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, A2, 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, a

  anti-B antibody for Oba 3 :: and an anti-A + B antibody for Oba 4.

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

example.

  4 - 30 μl erythrocytes A ,, A2, B and O suspended 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, the

  third and fourth bowl.

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

  6 - The BilOba device is centrifuged for 4 minutes at 1000 g.

  4S 7 - The reading takes place as follows: During centrifugation, the red cells will diffuse through the hydrating solution, disperse therein in a homogeneous manner and come into contact with the walls of the

"Oba" background.

  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 clear, globular button in the center of the bowl: reaction noted -.

  s The reactions observed in the four cuvettes of our example are therefore the following: Cuvettes 1 (Bil 1 / Oba 1) and 2 (Bil 2 / Oba 2) give an image of a more or less homogeneous pink carpet, reaction noted by + +++ to + depending on its intensity, because the globules A which have not been sensitized by the anti-B antibodies present in the plasma of subject A have adhered to the anti-A test serum fixed on the

bottom of Oba 1 and Oba 2 bowls.

  Bowl 3 (Bil 3 / Oba 3) gives an image of a central button translating the inhibition of adhesion of red cells B sensitized by anti-B antibodies

  present in the plasma of subject A and which can no longer adhere to the serum-

  anti-B test fixed on the bottom of the Oba 3 cuvette.

  The bowl 4 (Bil 4 / Oba 4) gives an image of a central button reflecting the absence of adhesion reaction between the red cells O (sensitized by

  no antibody) and the anti-AB test serum fixed on the bottom of the Oba 4 cuvette.

  The advantages of the BilObae cross-proof technique over other cross-proof techniques are manifold. They reside in a simplification of the technical procedure and an absence of phenomena

  contradictory leading to sometimes erroneous interpretations of the results.

  1 - Elimination of the risky steps involved in carrying out the counter

  test, such as the agitation 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 BACKPLATE AND

  SOLID PHASE COUNTER-TEST BilOba.

  COUNTER-TEST STEPS IN STREET-TEST

SOLID PHASE PLATE

  Deposit of 30 pil of medium of 1 dilution 1 Deposit of 30 μl of serum or Deposit of 10 μl of serum or 2 plasma to be tested plasma to be tested 2 Deposit of 30 μl of suspension Deposit of 30 μl of suspension 3 of globule-tests to 3% 0.2% test globule 3 Homogenization * Incubation 5 minutes 4 4 Centrifugation 2 to 3 minutes Centrifugation 4 minutes 5 - 200 g 1000 g Strong shaking 1 minute * 6 Slow shaking 1 minute * 7 Reading Reading 6 * Steps at risk. In the microplate technique, the homogenization step of the serum + red blood cells mixture is carried out either by manual tapping of the

  microplate, either on vibrating agitator.

  to After centrifugation, globular pellets are formed in the center of the cuvettes. To differentiate the positive reactions from the negative reactions, it is then necessary to agitate the microplate more or less strongly for one minute to peel off the globular disc and then to agitate the microplate gently for another minute to collect in the center of the cuvettes,

  1s 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 the identical agitation cycles to be reproduced at 2s5. 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

  looking 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, measures the differential optical density between the center of the cuvette and its periphery. A shift of the agglutinates relative to the center of the cuvettes can

  lead to a false negative interpretation.

  ] 0 3 - Better sensitivity with red blood cells A, In traditional tube or microplate techniques, weak reactions are often observed, in particular with red blood cells A2, because natural antibodies are often of low titre and not very avid towards Site (s

  A2 antigens 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, occurs.

  immediately reflected in perfectly visible adhesion inhibition.

  4- Significant reduction in the consumption of test cells Being diluted to 0.2%, the consumption of test cells is reduced by 50 times compared to traditional tube techniques, and by 4 times compared to micro-column techniques . With a blood bag you can

  consider more than three million tests in BilOba technique.

Claims (9)

  1 - Method of immunological diagnosis by adhesion in cuvettes characterized in that the sensitive layer located on the bottom of each cuvette is kept moist by a moisturizing cushion and in that the cuvette   is hermetically closed by a cover.   2- method according to claim-1 characterized in that the moisturizing cushion is a complex based on dextran gel of the "Sepharose" or o "Sephacryl" type or equivalent and of a solution of intermediate density between that of plasma and that 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 reactions   1 immunological 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 background of the cups.   5- A method according to claim-1 characterized in that the gel constituting the hydrating cushion causes a press effect on the red cells during the   centrifugation phase and when they flow over 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 increases   notable the specific adhesion and therefore the sensitivity of the test.   6- A method according to claim-1 characterized in that even the 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.   7- method according to claims 1 and 6 characterized in that it allows the   detection of anti-A and anti-B type antibodies by adhesion reaction   direct or by adhesion inhibition reaction (Simonin test).   8- Method according to claims 1, characterized in that the solid phase   can be sensitized by antibodies of different types, without limitation, and by antigens.   9- A method according to claim-1 characterized in that the reading of the   reaction is carried out by observing the underside of each cup.