RU2818259C2 - Bead-containing device for in vitro diagnostics and use thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to an in vitro diagnostic device for detecting and/or identifying an antigen and/or antibody from a biological fluid sample, in particular, from a blood sample or a sample of blood components and use thereof. An in vitro diagnostic device for detecting and/or identifying an antigen and/or antibody from a biological fluid sample, preferably from a blood sample or a blood component sample comprises a substrate and a hydrophobic porous membrane, disposed on said substrate, comprising at least one hydrophilic reaction region for receiving said sample, wherein the surface of said hydrophilic reaction region, which is smaller than the surface of the hydrophobic porous membrane, comprises beads, on the surface of which at least one antibody or antigen is fixed or adsorbed, wherein said beads feature size exceeding that of porous membrane to avoid absorption of at least one fixed or adsorbed antibody or antigen by hydrophobic porous membrane.

EFFECT: high sensitivity and specificity of the device.

22 cl, 19 dwg, 4 tbl

Description

FIELD OF THE INVENTION

The present invention is in the field of immunodiagnostics. In particular, the present invention relates to an in vitro diagnostic device for detecting and/or identifying an antigen and/or antibody from a sample of a biological fluid, in particular from a sample of blood or a sample of blood components. The present invention also relates to various applications of this device, such as the detection and/or identification of red blood cell antigens or anti-red blood cell antibodies, platelet antigens or anti-platelet antibodies, viral antigens or anti-viral antibodies, bacterial antigens or anti-bacterial antibodies, parasitic antigens or anti-parasitic antibodies. In vitro methods using the device of the invention are also part of the present invention.

BACKGROUND ART

Immunodiagnostic assays are methods that are available in several assay formats and are suitable for detecting antibodies, antigens, or combinations of both. Typically, these detection assays rely on the use of immobilized antigens, which capture any specific antibody present in the sample being analyzed, while the assays used for antigen detection consist of immobilized antibodies, which capture the specific antigen that is present in the sample.

These tests are widely used, for example, in immunohematology to detect infectious pathogens, etc., especially to ensure the safety of blood transfusions.

Blood transfusion consists of intravenous injection of concentrated red blood cell preparations (globular concentrates) obtained from donor blood. There are two main risks with blood transfusions.

The main risk is the possibility of combining the antibody and its erythrocyte antigen in the body of the recipient (the person receiving the transfusion). The consequences of such an immunological reaction can range from ineffective transfusion without clinical signs to a minor clinical reaction (anxiety, chills), a serious clinical reaction (shock, hemoglobinuria, renal failure) or a dramatic clinical reaction (shock, disseminated intravascular hemolysis) leading to death.

Blood transfusion is a possible source of disease transmission. A huge number of agents can potentially be transmitted through blood transfusions, including bacteria, viruses and parasites. Of these, bacteria are the most commonly transmitted and include species of the genus Treponema, Yersinia, Proteus, Pseudomonas, Escherichia, Klebsiella, Acinetobacter and Serratia, while Gram-positive organisms have been isolated as propionobacteria, staphylococcus, bacillus, clostridium and enterococcus. Viral agents that can be transmitted through blood transfusions include human immunodeficiency virus (HIV), hepatitis viruses, West Nile virus (WNV), cytomegalovirus (CMV), human T-cell lymphotrophic viruses (HTLV), and parvovirus B19. Another example is protozoa, which includes the Plasmodium species that cause malaria, and which can be transmitted through transfusions.

To ensure the safety of blood transfusion, on the one hand, it is required that the donor red blood cells are compatible with the recipient's blood, if the recipient does not have circulating antibodies directed against the donor's red blood cell antigens, and, on the other hand, that the donor red blood cells do not contain transfusion-transmitted infectious agents .

To ensure compatibility between donor red blood cells and recipients, tests are currently used to determine blood groups and the presence of antibodies to red blood cells in plasma/serum.

The purpose of immunohematological diagnosis is to determine and/or identify the immune reaction between a red blood cell antigen and an antibody specifically directed against such an antigen. To do this, it is necessary to have a means to detect antigens present on the surface of red blood cells. Their presence or absence determines the blood type. It is also possible to identify whether the recipient's blood contains one or more antibodies directed against known donor red blood cell antigens, the presence of an antibody indicating the possibility of incompatibility.

Among all the antigenic variants of the erythrocyte membrane antigen that make up blood groups, more than thirty antigenic systems of erythrocytes have been identified in humans to date: the ABO system with A (ABO1), B (ABO2), AB (AB03) and A1 (AB04), the Rh system with antigens D (RH1), E (RH3) or e (RH5) and C (RH2) or c (RH4), Kell system (K or KEL1, k or KEL2, ...), Duffy system (Fya or FY1, Fyb or FY2 , ...), the Kidd system (Jka or JK1, Jkb or JK2, ...), the MNS system (MNS1, MNS2, MNS3, MNS4 ...) or other less commonly studied systems that also exist, such as Lutheran, Lewis, etc. .d. Individuals with the same combination of red blood cell antigens belong to the same red blood cell blood group. Blood groups become even more complex and numerous when multiple antigenic systems are used.

Traditional techniques consist of searching for and identifying the presence or absence of red blood cell antigens on the surface of these cells and/or searching for and identifying the presence or absence of antibodies to blood group red blood cells in plasma or serum.

For example, for the ABO system, the Beth-Vincent assay determines the antigens carried by red blood cells, and the complementary Simonin-Michon or reverse blood grouping assay determines the antibodies circulating in the serum or plasma.

The Beth-Vincent assay combines an individual's red blood cells with antibody reagents of known specificity. In general, this analysis is made visible by observing the agglutination of red blood cells when antibody reagents recognize the corresponding red blood cell antigens.

In the Simonin-Michon assay, an individual's plasma or serum is combined with samples of red blood cells, each of which belongs to a specific ABO antigen group. This is an agglutination test of red blood cells from antibodies present in the plasma or serum of an individual. These antibodies, which are immunoglobulins of the IgM type, are capable of agglutinating red blood cells in vitro. They are called "conventional" antibodies because they are systematically present in the serum or plasma of individuals who do not carry the corresponding antigen on the surface of their red blood cells.

There is also a second class of antibodies called “transient” (or “immune”) antibodies. Their presence in serum or plasma is optional and they are directed against antigens of systems other than the ABO system. This usually involves IgG (sometimes IgM) arising during antigenic stimulation by foreign erythrocytes, for example, after immunization against one or more than one antigen following a blood transfusion, or during pregnancy through a maternal immune response directed against fetal erythrocyte antigens other than maternal blood group, or after allotransplantation.

Screening for these transient antibodies is called the indirect agglutinin test (IAT) or indirect Coombs test. This test is used to detect the presence or absence of IgG antibodies directed against various red blood cell antigens in an individual's blood, using human antiglobulin to facilitate agglutination, since IgG itself is not capable of spontaneously inducing agglutination in vitro. To achieve this, this assay aims to demonstrate the binding of these antibodies to test red blood cells of known antigenicity. This method is carried out simultaneously on several red blood cells with different known antigenicities, and comparison of the results makes it possible to identify the specificity of the IgG present.

The risk is greater for the most immunogenic antigens, such as Rh D (RH1), but also for other Rh types (E (RH3) more c (RH4) more e (RH5) more C (RH3)), Kell system antigen (KEL1 ), antigens of the Duffy system (FY1, FY2), antigens of the Kidd system (JK1, JK2), etc.

In practice, it is not possible to take all these antigens into account when performing a transfusion, since obtaining the correct blood type at the right time would not be possible, especially since some antigen combinations are extremely rare. In standard transfusions, only the ABO group and the presence or absence of antigen D (RH:1 or RH:-1) are taken into account. In situations where transient antibodies are present, a number of other systems are taken into account, namely the Rh system (C (RH2) or c (RH4) and E (RH3) or e (RH5)) and the Kell system, and sometimes other systems . Therefore, for these risk situations, it is important to ensure compatibility between the donor's blood group and the recipient's blood group, taking into account the presence or risk of these transient antibodies to red blood cells.

Thus, in recipient patients with transient anti-RBC antibodies or those in high-risk situations, such as those receiving multiple transfusions, it is important to select the unit doses of RBCs that are transfused so that the donor's RBCs are devoid of antigens against which the recipient's antibodies are directed or are likely to appear.

Tests to determine these blood groups and to search for and identify transient antibodies to red blood cells are mandatory in these patients and are used proactively in all recipient patients before the administration of red blood cells and in pregnant women to identify potential incompatibility in the mother-fetal system. Additional tests through direct compatibility testing with donor red blood cells in the presence of recipient serum or plasma are also mandatory to confirm compatibility. In these cases, neither an agglutination reaction nor a lysis reaction should be detected in the procedures used in the IAT.

The search for so-called transient antibodies entails identifying the presence or absence in the individual’s blood of immunoglobulins directed against various erythrocyte antigens. When antibodies have already been detected in vivo, a direct or direct Coombs assay is performed. In the case of searching for alloantibodies, the goal is to identify the fixation of these immunoglobulins on analyzes of red blood cells whose antigens are known, using the indirect Coombs technique.

In the field of immunohematology, there are a large number of methods and devices used to detect and/or identify erythrocyte antigens and to detect and/or identify antibodies to erythrocytes, but they have many disadvantages.

The slide method involves mixing on a glass slide or white porcelain substrate a drop of blood from the donor or recipient with anti-A antibody (anti-ABO1), anti-B antibody (anti-ABO2) and anti-D antibody (anti-RH1) separately. Agglutination can be observed visually, from which the ABO and Rh D blood systems can be determined. This analysis is completed in 5-10 minutes and is inexpensive. However, it is a non-sensitive method. This test cannot be performed for weakly or rarely reacting antigens, the results of which are difficult to interpret, and, in addition, a low titer of anti-A antibody (anti-ABO1) or anti-B antibody (anti-ABO2) could lead to false-positive or false-negative results . Because of its rapid results, it is very valuable in emergency situations for the preliminary determination of blood group matching.

However, it is not reliable enough to fully ensure safe transfusion.

Tube analysis consists of determination according to the analyzes of both Beth-Vincent and Simonin-Michon. In this method for determining Beth-Vincent groups, blood cells are placed in two tubes, along with saline as a dilution medium, and then one drop each of anti-A antibody (anti-ABO1) and anti-B antibody (anti-ABO2) separately added to these samples. These tubes are centrifuged for several minutes and then the resulting matrix is lightly shaken to observe agglutination. The purpose of centrifugation is to provide enhanced chemical interactions, especially for the reaction of weaker antibodies, thus leading to agglutination. Likewise, reverse Simonin-Michon grouping can be performed by treating serum against reactive red blood cell groups A1 and B (and possibly blood groups O and A2), and then monitoring the agglutination pattern. In general, the tube method is significantly more sensitive than the slide method, requires a small volume of reagents, and some unexpected antigens may also be detected. However, in infants, reverse grouping is somewhat difficult to perform because they do not produce sufficient amounts of detectable antibodies. Since this analysis is a manual method, its application for high-throughput analysis is very limited.

Among the classical methods, microplate technology consists of a large number of small tubes that contain several microliters (μL) of reagents that are processed against blood samples. After centrifugation and incubation, subsequent agglutination can be checked using an automatic reader. This method is a more sensitive and rapid analysis for blood typing and for the detection/identification of antibodies to red blood cells using small volumes of reagents and the possibility of automation for high-throughput analysis. However, since microplate techniques require a centrifugation phase followed by a shaking step, there is therefore a risk of canceling weak agglutination without success in resuspending strong agglutination. They should be carried out by visual inspection and special attention should be paid to the sticking phenomena of some reagents.

Gel filtration techniques are a standard procedure for quantitatively measuring cell agglutination. The column contains a gel matrix to capture agglutinates. For blood typing, red blood cells are mixed with anti-A (anti-ABO1), anti-B (anti-ABO2), and anti-D (anti-RH1) reagents or other antibodies against other blood groups in microtubes under controlled incubation and centrifugation. For reverse blood typing and for the detection/identification of antibodies to red blood cells, plasma or serum is mixed with red blood cells of known antigenicity under controlled incubation and centrifugation. The gel particles trap the agglutinates while allowing non-agglutinated blood cells to pass through the column. Analysis time can be reduced by using glass beads instead of gel material, since higher centrifugation speeds can be achieved with this method, leading to faster results. The technique is sensitive, direct and relatively easy to perform by less trained personnel. However, a major risk is the failure to detect some agglutinations, especially during ABO group plasma assays due to the dissociation of small agglutinates through shear forces as they pass through the gel.

All of these techniques also have a major disadvantage in that they require a centrifugation step to decant the red blood cells or the need to pass them through a gel - a limiting step that adds significant time and cost to the analysis, and which requires the use of volumetric centrifuges that are difficult to handle.

Other methods based on molecular imprinting (regarding the review: Mujahid, 2016, Sensors), on the technique of analyzing the surface of a chip with antibodies through plasmon resonance imaging (Houngkamhang, 2013, sensors; Pipatpanukul, 2018, Biosensors and Bioelectronics), on the analysis of quantum dots - magnetic beads (Xu, 2017, International Journal of Nanomedicine) or on microjets driven by capillaries or vacuum (Zhai, 2017, Lab on a Chip), and other methods have also been described in the related field.

WO 2012010666 uses magnetic immunodiagnostic methods to determine blood group and phenotype antibody/antigen complexes.

The immunofiltration method described, for example, in the application EP 2167967, consists of capturing the analyte present in the sample as it passes through a porous membrane carrying a capture element, and detecting its presence by means of a detection element. This method has significant challenges with sensitivity and specificity because as the sample is introduced, it is distributed throughout and absorbed into the porous membrane, and many analytes are lost in the dead volume of the porous membrane or escape from the capture region. The same applies to the revealing solution. Therefore, it is necessary to increase the size of the absorption system in order to be able to accommodate large volumes of samples to be analyzed and detection solution, preventing the performance of miniaturized kinetics-controlled assays that do not require the production of detection agents and are usable with a robotic pipette.

In an attempt to solve this problem and concentrate the signal emitted after the antigen/antibody reaction, CA 1312265 or WO 02022263 proposed the installation above and below a hydrophilic porous membrane of a hydrophobic structure pierced to force flow through the capture spot. However, with these devices there is still the problem of centrifugal diffusion from the spot, and detection elements that pass through the capture spot, and which are not fixed to it, will be able to centrifugally diffuse into the hydrophobic porous membrane and remain at the periphery of the spot.

Another important problem with existing immunofiltration devices today is the control of the speed of movement of the sample through the membrane and, in some cases, the preincubation time. This timing is important because the interaction between the capture element (often an antibody) and the analyte (often an antigen), and between the analyte and the detection element, has specific kinetics. Without a special system, passage through the hydrophobic membrane is rapid (500 µl/min).

It was proposed to use a valve to control the flow, in particular in application US 2008318342.

To control the preincubation time, WO 03016902 proposed the use of a device of two parts: an upper part containing a sample collection area and a porous membrane, and a lower part containing a porous membrane and an absorbent membrane.

Such mechanical approaches are difficult to implement using a robot, as they require the development and application of dedicated systems. They also expose professionals to any protrusions during handling.

Another method, described in EP 0334015, is to use an additional membrane located at the base of the first one to control the current, but the proposed device cannot solve the problems associated with the diffusion of the sample and the detecting element in the hydrophilic porous membrane.

Transfusions of platelets, also known as platelet mass, are used to prevent or treat bleeding in people with either a low platelet count or poor platelet function. Precautionary transfusion is often given to those whose platelet levels are less than 10 x 10 ⁹ /L. In those suffering from bleeding, transfusions are typically given in amounts less than 50 x 10 ⁹ /L. Platelets are given by injection into a vein. Platelets can be obtained either from whole blood or by apheresis. To date, 33 human platelet alloantigens (HPAs) have been identified on six functionally important platelet glycoprotein (GP) complexes. The largest number of recognized HPAs (20 of 33) are located on the GPIIb/IIIa complex, which serves as a receptor for ligands important in mediating hemostasis and inflammation. These include HPA-1a, the HPA most commonly implicated in FNAIT (fetal and neonatal autoimmune thrombocytopenia) and PTP in Caucasian populations. Other platelet GP complexes—GPIb/V/IX, GPIa/IIa, and CD109—express the remaining 13 HPA. Of the recognized HPAs, 12 occur as six serologically and genetically defined biallelic “systems,” with form “a” denoting the higher frequency allele and form “b” the lower frequency. Twenty-one other HPAs represent low-frequency or rare antigens for which the postulated higher-frequency “a” alleles have not yet been identified as antibody specificities. In addition to HPA markers, platelets also express ABO and human leukocyte antigen (HLA) antigens. Blood type matching (ABO, RH1) is typically recommended before platelets are given.

Accurate typing of patients for HPA antigens is required in several different clinical situations, and blood transfusion services need to maintain panels of HPA-typed apheresis platelet donors and whole blood donors to support HPA alloimmunized patients. The value of serologic HPA phenotyping is limited because too few platelets can often be obtained from thrombocytopenic patients, and reliable serotyping reagents for HPA antigens other than HPA-1a and HPA-5b are rare. In contrast to erythrocyte phenotyping, with the exception of HPA-1a, no monoclonal antibodies have been developed for HPA typing. However, several rapid screening methods using either polyclonal or recombinant antibodies against HPA-1a have recently been published. These phenotyping assays can complement genotyping assays to provide HPA-selected donor groups.

It is important to ensure compatibility between donor platelets and recipient platelets. However, inappropriate platelets are often used due to unavailability of appropriate platelets. Incompatibility can lead to alloimmune platelet disorders, including fetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP), and multitransfusion platelet refractoriness (MPR).

FNAIT, often known as fetal-maternal alloimmune thrombocytopenia (FMAIT), occurs as a consequence of maternal immunization against fetal platelet alloantigens inherited from the father. Maternal IgG alloantibodies then cross the placenta and cause immune destruction of platelets during fetal development, a situation analogous to hemolytic disease of the newborn.

Post-transfusion purpura (PTP) is a rare but serious condition that occurs approximately one week after the transfusion of any blood product containing platelets or platelet membranes. Patients are presumably sensitized by previous pregnancy or blood transfusion and respond to the second stimulus of incompatible platelets by producing high titres of anti-HPA (and often HLA) antibodies. The resulting immune destruction of transfused platelets may contribute to the transfusion reactions that commonly occur.

MPR is defined as an inadequate increase in platelet count following transfusion of random ABO-identical donor platelets. It is a common complication in patients receiving multiple platelet transfusions.

Although a wide range of techniques have been developed over the past 25 years to detect anti-HPA antibodies, four techniques or modifications thereof have become the most common: (1) platelet immunofluorescence assay (PIFT); (2) monoclonal antibody immobilization of platelet antigens (MAIPA) assay; (3) erythrocyte adhesion enzyme-linked assay; and (4) a range of ELISA (enzyme-linked immunosorbent assay) based techniques. However, effectiveness in detecting platelet antibodies depends on multiple factors, including techniques, cell populations, and operator experience, which can make some of the above techniques costly, difficult to perform, and less reliable.

To detect potential transfusion-transmitted infections, many diagnostic tests that use the immune response have been developed over the past four decades. The most commonly used assays are designed to either detect antigens of infectious agents, such as viral antigens, bacterial antigens, and parasitic antigens, or to detect antibodies directed against an infectious agent produced by immunization during infection. However, these assays are not suitable for all situations, and each assay has limitations that must be known and taken into account when selecting it. Immunodiagnosis is often based on the use of an antibody as a reagent.

There are different methods used in immunodiagnostic assays. Some of these methods are described below.

Immunoprecipitation is the simplest immunoassay that measures the amount of precipitate that forms after incubation of an antibody reagent with a sample and which reacts with its corresponding antigen to form an insoluble aggregate. Immunoprecipitation reactions can be qualitative or quantitative.

Particle-based immunoassays detect the presence of antibody or specific antigens in a sample and are analyzed using particles that are coated with either the antigen or the appropriate antibody. By binding multiple antibodies to a particle (gelatin or latex), the particle is capable of simultaneous binding to many molecules and facilitates agglutination. This significantly speeds up the speed of the visible reaction. The Treponema pallidum particle agglutination assay (also called TPPA assay) is another example of particle immunoassays used to detect antibodies to syphilis, a possible transfusion-transmitted infectious agent (Manavi et al. 2006 Int. J. STD AIDS). It is an indirect agglutination assay in which gelatin particles are sensitized with T. pallidum antigen. These particles aggregate to form clusters when the patient's serum is positive for syphilis, whereas a negative test does not demonstrate the formation of clusters of gelatin particles.

The immunonephelometric assay is another example of immunoassays designed to detect infections. When the immune complexes formed by combining antibody and antigen are too small to precipitate, the complexes can be measured using an instrument called a nephelometer, as they will scatter the incident light. The antigen concentration can be determined within min of reaction.

Radioimmunoassay (RIA) uses radioactive isotopes to label either an antigen or antibody. This isotope emits gamma rays, which are usually measured after the unbound radioactive tracer has been removed. The main advantage of RIA over other immunoassays is its higher sensitivity, easy signal detection, and well-established, rapid assays. The main disadvantages are the health and safety risks posed by the use of radiation and the time and cost associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in established clinical laboratory practice by enzymatic (EIA), fluorescent (FIA), or chemiluminescent (CLIA) immunoassays.

EIA, FIA, and CLIA are currently the most commonly used assays to detect viral, bacterial, and parasitic infections, especially transfusion-transmitted infections, in donor blood samples. One of the most commonly used EIA methods to detect infectious diseases is the enzyme-linked immunosorbent assay (ELISA). There are many patents and publications demonstrating the effectiveness of such assays for identifying infectious agents.

These methods are similar in principle and differ only in the method of detecting the resulting immune complexes: colorimetric for EIA and ELISA, and measuring the fluorescence and luminosity produced by the enzymatic/chemical reaction for FIA and CLIA. Fluorescence and chemiluminescence measurements are inherently more sensitive than colorimetric measurements. Therefore, these methods have greater analytical sensitivity than EIA methods that use absorbance measurements.

There are also quick/simple unit tests with no intermediate step and one-time use that are used only once and thrown away. Most of these tests are based on immunochromatography, in which a sample (blood, plasma or serum) is passed through an inert strip and reacted with reagents (antibodies or antigens) that have been previously immobilized on the strip. Any positive reaction is visualized as a spot or stripe appearing on that strip. These tests are easy to use and do not require additional reagents other than those included in the kit. They also give simple, high-quality results within a few minutes. However, these tests are not suitable for screening large numbers of samples.

In order to overcome the above-described disadvantages, the applicant has used an in vitro diagnostic device for detecting from a sample of blood or one of its components at least one reaction between an antigen and an antibody specifically directed against that antigen, comprising a hydrophobic porous membrane, wherein at least one hydrophilic the reaction region is intended to obtain the specified sample, has a less significant surface area than the surface of the hydrophobic porous membrane. This device is described, for example, in patent application WO 2013/186482. It corrects several shortcomings caused by the above-mentioned assays, in particular those caused by immunofiltration assays by:

- preventing detection agent returns responsible for false positive results

- increased sensitivity due to the use of smaller volumes

- miniaturization and automation of the system

- control of reaction kinetics without the need for mechanical manipulations with the device.

Despite the obvious advantages of this device, there is still a need to improve the sensitivity and specificity of the diagnostic performed when the diagnosis involves the use of a capture agent, and when the capture agent is an antibody (to detect the corresponding antigen in the sample) or an antigen (to detect the corresponding antibody in the sample). When using this device, a portion of the capture agent (eg, antibodies against blood cell antigens) located on the made hydrophilic reaction area during the diagnostic assay is adsorbed onto the porous membrane using the solution used to dilute the antibodies. Consequently, the specificity and sensitivity of this assay are reduced. To compensate for this “loss” of the capture agent, a large amount of the capture agent must be added, which can increase the cost of a given diagnostic assay.

To overcome this drawback, the present inventors have tested several technical solutions, such as using a thicker porous membrane or a less porous membrane, but without significant results.

Instead, while exploring another possibility for modulating the characteristics of the porous membrane or other structural elements of the device, the present inventors unexpectedly discovered that the addition of specially selected beads on the surface of which a capture agent (for example, an antibody or antigen) can be fixed or adsorbed onto the reaction region of the porous membrane, ensures that the capture agent is retained on the surface of the membrane and thus improves the emitted signal when the capture agent binds an analyte (eg, antigen or antibody). Therefore, the use of such beads provides an improvement in the specificity and sensitivity of the previously disclosed device without the use of a significant amount of capture agent.

A bead on the surface of which antibodies and/or antigens are fixed is well known in the art for the purpose of diagnosis. For example, international application WO 95/31731 discloses a method for detecting erythrocyte antigens, which involves adding a sample containing blood cells to a column containing particles on the surface of which specific antibodies to erythrocytes are fixed by means of protein ligands. However, the described method does not use a device containing a porous membrane made hydrophilic, and does not deal with the problem related to the absorption of the entrapment agent through the membrane.

Thus, the association of a partially hydrophilic porous membrane with beads on the surface of which antibodies and/or antigens are directly or indirectly fixed or adsorbed as a capture agent to carry out an in vitro diagnostic method having high specificity and sensitivity is unexpected in light of the state associated with this prior art.

SUMMARY OF THE INVENTION

As stated above, in order to improve the sensitivity and specificity of detection and/or identification of antigen and/or antibody and to reduce the number of analytes used, the inventors of the present invention have used a device similar to the device disclosed in patent application WO 2013/186482, which further comprises beads located on the reaction area where antibodies and/or antigens are fixed or adsorbed to the surface of the bead.

According to one aspect, the present invention thus relates to an in vitro diagnostic device for detecting and/or identifying an antigen and/or antibody from a sample of a biological fluid, preferably from a sample of blood or a sample of blood components, comprising:

- substrate and

- a hydrophobic porous membrane located on the support, containing at least one hydrophilic reaction region designed to receive the specified sample, and the surface of this hydrophilic reaction region, which is smaller than the surface of the hydrophobic porous membrane, contains at least one antibody and/or antigen, wherein said antibody and/or antigen is fixed or adsorbed to the surface of the bead.

The inventors of the present invention have conducted several assays and demonstrated that the in vitro diagnostic device of the invention can be used to detect and/or identify different types of antigens or antibodies. To do this, different types of antibodies and/or antigens can be fixed or adsorbed on the surface of the bead. These antibodies may be selected from antibodies directed against red blood cell antigens, antibodies directed against platelet antigens, antibodies against viral antigens, antibodies against bacterial antigens and antibodies against parasitic antigens or antibodies against immunoglobulins. These antigens may be selected from red blood cell antigens, platelet antigens, viral antigens, bacterial antigens and parasitic antigens.

According to another aspect, the present invention thus relates to the use of an in vitro device according to the invention for the determination and/or identification of:

- at least one antigen selected from the group consisting of erythrocyte antigens, including mixed field population, platelet antigens, viral antigens, bacterial antigens and parasitic antigens;

- at least one antibody selected from the group consisting of anti-red blood cell antibodies, anti-platelet antibodies, anti-viral antibodies, anti-bacterial antibodies and anti-parasitic antibodies from a sample of biological fluids, preferably from a sample of blood or a sample of blood components.

The present inventors have particularly demonstrated that the in vitro device of the present invention can be used in a method for in vitro detection and/or identification of red blood cell antigens and/or in vivo sensitized red blood cells and/or platelet antigens from a sample of biological fluids, in particularly from a sample of blood or blood components.

According to another aspect, the present invention thus relates to a method for in vitro detection and/or identification of erythrocyte antigens and/or in vivo sensitized erythrocytes and/or platelet antigens from a blood sample or a blood component sample, comprising the following steps:

- adding a solution containing said sample to the reaction area of the in vitro device according to this invention,

- reading before washing to obtain sample loading control

- applying the washing solution to the reaction area and

- determining the presence of specified antigens or specified blood cells by determining the presence of an antigen/antibody interaction if a red or pink spot appears in the reaction area, or determining the absence of specified antigens or specified blood cells by determining the absence of an antigen/antibody interaction if a colorless spot appears in the reaction area spot.

In addition, the inventors of the present invention have also demonstrated that the in vitro device of the present invention can be used in an in vitro method for detecting and/or identifying antibodies to red blood cells from a sample of a biological fluid, in particular from a sample of blood or blood components.

Another aspect of the present invention therefore relates to a method for in vitro detection and/or identification of antibodies to red blood cells from a blood sample or a blood component sample, comprising the following steps:

- incubation of the sample to be analyzed with buffer and red blood cell samples of known antigenicity or incubation of recipient plasma or serum with donor red blood cells,

- applying this mixture to the reaction area of the in vitro device according to this invention,

- applying the washing solution to the reaction area, and

- determining the presence of antibodies to red blood cells by determining the presence of an antibody/antigen interaction if a red or pink spot appears in the reaction area, or determining the absence of said antibodies by determining the absence of an antibody/antigen interaction if a colorless spot appears in the reaction area.

The in vitro device of the present invention can be used in an in vitro method for detecting and/or identifying anti-platelet antibodies from a sample of a biological fluid, in particular from a sample of blood or blood components.

Another aspect of the present invention therefore relates to a method for in vitro detection and/or identification of anti-platelet antibodies from a blood sample or a blood component sample, comprising the following steps:

- incubation of the sample to be analyzed with buffer and platelet samples of known antigenicity or incubation of recipient plasma or serum with donor platelets,

- applying this mixture to the reaction area of the in vitro device according to this invention,

- applying the washing solution to the reaction area, and

- determining the presence of antibodies to platelets by determining the presence of an antibody/antigen interaction if a red or pink spot appears in the reaction area, or determining the absence of said antibodies by determining the absence of an antibody/antigen interaction if a colorless spot appears in the reaction area.

The in vitro device of the invention can also be used in an in vitro method for detecting and/or identifying different types of antigens selected from the group consisting of viral antigens, bacterial antigens and parasitic antigens, or different types of antibodies selected from the group consisting of antiviral antibodies, antibacterial antibodies and/or anti-parasitic antibodies, wherein said antibodies are produced against viral, bacterial or parasitic antigens during infection.

Yet another aspect of the present invention thus relates to a method for in vitro detection and/or identification of antigens selected from the group consisting of viral antigens, bacterial antigens and parasitic antigens, from a sample of a biological fluid, preferably from a sample of blood or a sample of blood components, including the following stages:

- it is possible to incubate the sample to be analyzed with a buffer,

- adding a solution containing the specified sample or a mixture of sample and buffer to the reaction mixture of the in vitro device according to this invention,

- reading before washing to control sample loading is possible;

- it is possible to apply a washing solution to the reaction area, and

- determining the presence of the specified antigen by determining the presence of an antigen/antibody interaction if a colored spot appears in the reaction area, or determining the absence of the specified antigen by determining the absence of an antigen/antibody reaction if a colorless spot appears in the reaction area.

According to another aspect, the present invention also relates to a method for in vitro detection and/or identification of antibodies selected from the group consisting of antiviral antibodies, antibacterial antibodies and/or antiparasitic antibodies from a sample of a biological fluid, preferably from a sample of blood or a sample of blood components, comprising the following steps :

- it is possible to incubate the sample to be analyzed with a buffer,

- adding a solution containing the specified sample or a mixture of sample and buffer to the reaction area of the in vitro device according to this invention,

- reading before washing to control sample loading is possible;

- it is possible to apply a washing solution to the reaction area, and

- determining the presence of the specified antigen by determining the presence of an antigen/antibody interaction if a colored spot appears in the reaction area, or determining the absence of the specified antigen by determining the absence of an antigen/antibody reaction if a colorless spot appears in the reaction area.

The characteristics and advantages of the present invention will become apparent from the following detailed description, with examples showing certain embodiments of the present invention, also shown in the set of drawings accompanying this application.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Some of these definitions are referred to in the Background Art portion of this application and apply to the present invention. Specific definitions are also set forth below in the detailed description below.

• In vitro device according to the invention

As stated above, the inventors of the present invention have used an in vitro device to detect and/or identify antigen and/or antibody with high specificity and sensitivity.

In one aspect, the in vitro device of the present invention is an in vitro diagnostic device for detecting and/or identifying an antigen and/or antibody from a sample of a biological fluid, preferably from a sample of blood or a sample of blood components, comprising:

- substrate and

- a hydrophobic porous membrane located in the specified substrate containing at least one hydrophilic reaction region designed to receive the specified sample, and the surface of this hydrophilic reaction region, which is smaller than the surface of the hydrophobic porous membrane, contains at least one antibody and/or antigen, wherein said antibody and/or antigen is fixed or adsorbed on the surface of the bead.

The device of the present invention is suitable for detecting and/or identifying several types of antigens or antibodies. This detection and/or identification is accomplished by binding of an antigen to an antibody fixed or adsorbed on the surface of a bead, or by binding of an antibody to an antigen fixed or adsorbed on the surface of a bead. In this context, any antibody and/or antigen can be fixed or adsorbed on the surface of the bead in the device of the present invention, depending on the type of antigen or antibody to be detected and/or identified. The in vitro device of the invention thus not only serves to detect and/or identify specific antigens or antibodies (such as red blood cell antigens and anti-red blood cell antibodies), but can be adapted to detect and/or identify different types of antigens and antibodies. One skilled in the art will be able to tailor the in vitro device of the present invention, depending on the antibody or antigen of interest to be detected and/or identified, by fixing on the surface of the bead an antigen or antibody that binds to the antibody or antigen of interest, respectively.

Therefore, the term “antibody” is used herein in its broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies) of any isotype, such as IgG, IgM, IgA, IgD and IgE, polyclonal antibodies, multispecific antibodies, chimeric antibodies and antigen-binding antibodies. fragments. An antibody that reacts with a specific antigen can be produced by genetic engineering methods, such as the selection of libraries of recombinant antibodies in phage or similar vectors, or by immunization of an animal with the antigen or nucleic acid encoding the antigen.

A typical antibody contains two identical light chains and two identical heavy chains, which are connected by disulfide bonds. As used herein, the term “light chain” refers to a mammalian immunoglobulin light chain—lambda (λ) or kappa (κ)—having two consecutive domains: one constant domain and one variable domain.

The term "heavy chain" refers to the mammalian immunoglobulin chain designated α, δ, ε, γ and μ. Within these five classes, immunoglobulins can also be divided into subclasses (IgG1, 2, 3 or 4, IGA1 or 2) according to the heavy chain (γ1, γ2, γ3, γ4, α1 and α2). Each heavy chain has two regions: a constant region and a variable region. The constant region is identical in all antibodies of the same isotype. The variable region of each heavy chain consists of one Ig domain.

The term "variable region" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The heavy chain variable domain may be referred to as "VH". The light chain variable domain may be referred to as "VL". These domains are typically the most variable portions of an antibody, and they contain antigen binding sites. Each variable region contains three segments called “complementarity determining regions” (CDRs) or “hypervariable regions”, which are primarily responsible for binding the antigen epitope. The CDR thus controls the binding specificity of the antibody. They are usually called CDR1, CDR2 and CDR3, and are numbered sequentially from the N-terminus. The most highly conserved parts of the variable regions are called "framework regions".

In one embodiment of the present invention, the antibodies used in this invention, in particular, the antibodies fixed or adsorbed to the surface of the bead, are polyclonal antibodies. A "polyclonal antibody" is an antibody that is produced among or in the presence of one or more other non-identical antibodies. In general, polyclonal antibodies are produced from a B lymphocyte in the presence of several other B lymphocytes producing non-identical antibodies. Typically, polyclonal antibodies are obtained directly from the immunized animal.

According to another embodiment, the antibodies used in this invention, in particular, the antibodies fixed or adsorbed on the surface of the bead, are monoclonal antibodies.

The term "monoclonal antibody" as used herein refers to an antibody arising from a nearly homogeneous population of antibodies. More specifically, the individual antibodies of a population are identical except for a few possibly naturally occurring mutations which may occur in minimal proportions. In other words, a monoclonal antibody consists of a homogeneous population of antibodies arising from the growth of a single clone of cells (e.g., a hybridoma, a eukaryotic host cell transfected with a DNA molecule encoding a homogeneous antibody, a prokaryotic host cell transfected with a DNA molecule encoding a homogeneous antibody, and etc.), and is usually characterized by heavy chains of one and only one class and subclass and light chains of only one type. Monoclonal antibodies are highly specific and directed against a single antigen.

The term "chimeric antibody" as used herein is an antibody in which a constant region or part thereof is changed, replaced or exchanged such that the variable region binds to a constant region of a different species or belonging to a different class. or a subclass of antibody.

The term “antigen-binding fragments” as used herein refers to the region on an antibody that binds to antigens. It consists of one constant and one variable domain of each heavy and light chain.

According to one preferred embodiment of the present invention, the antibodies are selected from the group consisting of antibodies to red blood cells, antibodies to platelets, antibodies directed against viral antigens, antibodies directed against bacterial antigens and antibodies directed against parasitic antigens, preferably antibodies against red blood cells or antibodies to platelets.

The term “anti-platelet antibodies” or “antibodies to platelet antigens” as used herein refers to one or more antibodies directed against one or more than one platelet antigen. Preferably, the antibodies to platelet antigens used in the present invention may be a purified or semi-purified monoclonal antibody or a purified or semi-purified polyclonal antibody or antiserum.

The term “antibodies directed against viral antigens” as used herein refers to one or more antibodies directed against a viral antigen, which antibodies are produced upon infection of a subject. Preferably, the antibodies to viral antigens used in the present invention may be a purified or semi-purified monoclonal antibody, a purified or semi-purified polyclonal antibody, or an antiserum. The antiviral antibodies detected and/or identified by the in vitro device of the present invention may be selected from the group consisting of antibodies directed against antigens specific for chikungunya virus, cytomegalovirus, Dengue fever virus, Ebola, EBV (Epstein-Barr virus), encephalitis virus, feline leukemia virus, hantavirus, hepatitis A virus, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, HIV (human immunodeficiency virus), HTLV (human T-lymphotropic virus), Lasa fever virus, measles, mumps , norovirus, papillomavirus, parvovirus, rubella virus, SARS virus, varicella zoster virus, West Nile virus and Zika virus. In particular, detection of antibodies directed against HIV, HTLV, hepatitis B virus or hepatitis C virus is mandatory for the safety of blood transfusions. In particular, the in vitro device of the present invention is used for the detection and/or identification of HIV, hepatitis B virus and hepatitis C virus, more specifically the hepatitis B virus.

The term “antibodies directed against bacterial antigens” as used herein refers to one or more antibodies directed against a bacterial antigen that is produced upon infection of a subject. Preferably, the antibodies against bacterial antigens used in the present invention may be a purified or semi-purified monoclonal antibody, a purified or semi-purified polyclonal antibody or an antiserum. These antibodies may be polyclonal or monoclonal antibodies, in particular monoclonal antibacterial antibodies, well known to one skilled in the art. For example, such antibodies may be selected from gram-positive or gram-negative bacteria, in particular Borrelia, Chlamydia, Helicobacter pylori, Mycoplasma, S. typhi, Yersinia, Proteus, Pseudomonas, Escherichia, Klebsiella, Acinetobacter, Clostridium, Serratia, Propionibacterium, Staphylococcus, Bacillus, Treponema and Enterococcus. In particular, the antibacterial activity is directed against a specific Treponema antigen, in particular Treponema pallium, which is responsible for syphilis.

The term “antibodies directed against parasitic antigens,” as used herein, refers to one or more antibodies directed against a parasitic antigen that are produced upon infection of a subject. Preferably, the antibodies against parasitic antigens used in the present invention may be a purified or semi-purified monoclonal antibody, a purified or semi-purified polyclonal antibody or an antiserum. The anti-parasitic antibodies used in the present invention are selected from the group consisting of Toxoplasma, Trypanosoma, Plasmodium. Specifically, the antibody is directed against a specific antigen of Trypanosoma cruzi, which is responsible for Chagas disease. More specifically, the antibody is directed against Plasmodium, preferably against Plasmodium falciparum, which is responsible for malaria.

The term "anti-immunoglobulins" as used herein refers to antibodies directed against one or more than one immunoglobulin, such as IgG, IgM, IgA, IgD and IgE.

The term “anti-red blood cell antibodies” or “antibodies against red blood cell antigens” as used herein refers to one or more antibodies directed against one or more than one red blood cell antigen. Preferably, the antibodies to red blood cell antigens used in the present invention may be a purified or semi-purified monoclonal antibody, a purified or semi-purified polyclonal antibody, or an antiserum. Agglutinins or lectins can also be used. A non-limiting list of antibodies and antigens used in this invention follows:

Most of the antibodies listed above can be purchased from Diagast. According to one embodiment of the present invention, the antibodies fixed or adsorbed to the surface of the bead are anti-red blood cell antibodies selected from any of the antibodies in Table 1. For example, the anti-red blood cell antibodies can be selected from the group consisting of Anti-A, Anti-B, Anti-AB, Anti-D, Ahth-RH2, Ahth-RH3, Ahth-RH4, Ahth-RH5 and Anti-Kell, Anti-MNS (MNS1, 2, 3 and 4), Anti-JK (JK1, JK2), Anti-FY (FY1, FY2), Anti-LE (LE1, LE2), Anti-LU (LU1, LU2) and Anti-P1PK.

In the context of the present invention, the term "antigen" refers to a predetermined molecule to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, glycoprotein or glycolipid, or any other naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide, glycoprotein or glycolipid.

According to one preferred embodiment of the present invention, these antigens are selected from the group consisting of red blood cell antigens (as listed in Table 1), platelet antigens, viral antigens, bacterial antigens and parasitic antigens, more preferably anti-red blood cell antibodies or viral antigens. Preferably, the antigens used in the present invention may be a purified or semi-purified natural antigen, a recombinant or a synthetic antigen.

The term “viral antigen” as used herein refers to an antigen with multiple antigenicities that is strain specific and closely associated with the viral particle. The antigen may be a natural or recombinant protein. Viral antigens can be selected, for example, from the group containing antigens specific for chikungunya, cytomegalovirus, Dengue virus, Ebola virus, EBV, encephalitis virus, feline leukemia virus, hantavirus, hepatitis A virus, hepatitis B virus (HBsAg), hepatitis C (HCsAg), hepatitis D, hepatitis E, herpes, HIV, HTLV, Lassa fever virus, measles, mumps, norovirus, papillomavirus, parvovirus, rubella virus, SARS virus, varicella virus, West Nile virus and Zika virus. In particular, the present invention relates to the identification and/or detection of HBsAg and HCsAg antigens, as they are essential for the safety of blood transfusion, and more particularly to HBsAg.

The term "bacterial antigen" as used herein refers to a molecule that is found on the surfaces of bacterial organisms. The bacterial antigens may be selected from any of the bacterial strains, but are preferably selected from Borrelia, Chlamydia, Helicobacter pylori, Mycoplasma, S. typhi, Yersinia, Proteus, Pseudomonas, Escherichia, Klebsiella, Acinetobacter, Serratia, Clostridium, Propionibacterium, Staphylococcus, Bacillus and Enterococcus. In particular, a specific antigen of the bacterium Treponema pallidum, which is responsible for syphilis, can be detected and/or identified or fixed and/or adsorbed on the surface of the ball.

The term "parasitic antigen" as used herein refers to a molecule that is found on the surface of a parasite cell. This parasite may be as defined above. Preferably, the parasitic antigens are selected from the group consisting of antigens specific for Toxoplasma, Trypanosoma, Plasmodium, more preferably, antigens specific for Plasmodium, specifically for Plasmodium falciparum, which causes malaria.

A non-exhaustive list of antigens and antibodies directed against antigens of viruses, bacteria and parasites is given in Table 2 below:

The term "platelet antigen" as used herein refers to a platelet antigen related to all immunogenic molecules present on the surface of platelets where the ability to induce the production of antibodies directed against these molecules and/or provide their recognition. The platelet antigens may be selected, for example, from the group consisting of biallelic antigen HPA-1, -2, -3, -4, -5 and -15.

The term "red blood cell antigens", also known as blood group antigen, as used herein, refers to all immunogenic molecules present on the surface of red blood cells where the ability to induce the production of antibodies directed against these molecules is required, and/ or ensure their recognition. Red blood cell antigens include a mixed field population of antigens. A non-exhaustive list of red blood cell antigens is shown in Table 1.

The term "mixed field population" as used herein refers to two populations of red blood cells with different antigens that can be observed after transfusion of compatible red blood cells between a donor and recipient (O red blood cells transfused to patients A or B) or after bone marrow transplants. The in vitro device of the invention enables detection of a sample with a mixed field population.

The term "in vivo sensitized red blood cells" as used herein refers to red blood cells with immunoglobulin (IgG) or complement (C3d) bound in vivo to their membrane surface. In vivo sensitized red blood cells can appear in a variety of clinical hemolytic conditions, including hemolytic transfusion reactions, hemolytic disease of the fetus and newborn (HDFN), autoimmune hemolytic anemia (AIHA), and drug-induced antibodies in the patient.

According to another embodiment of the present invention, said antibody and/or antigen is directly or indirectly fixed or adsorbed to the surface of the bead.

There are currently several methods for attaching biological ligands to beads used as solid-phase supports in immunoassays and assays, including adsorption onto simple polymer beads and covalent attachment to surface-functionalized microspheres. The adsorption mechanism is based mainly on hydrophobic (van der Waals, London type) attractions between the hydrophobic parts of the adsorbed ligands and the polymer surface of the bead. This method mainly involves covalent binding to immobilize biomolecules where a very active and stable bead-based reagent is required, while indirect fixation is mediated by anti-immunoglobulin antibodies such as anti-human globulin (AHG) antibodies fixed covalently or adsorbed onto beads , which capture a second antibody directed against the antigen of interest.

In direct fixation or adsorption of the antibody and/or antigen on the surface of the bead, this is accomplished by bringing the beads into contact with the antibodies and/or antigens. For example, the solution containing antibodies and/or antigens may be a non-denaturing buffer containing a solution with a pH stabilized from pH 4 to pH 10, preferably from pH 6.5 to pH 7.8, even more preferably from pH 7 to pH 7.5. These antibodies can be either adsorbed or covalently linked.

Antibody solutions are selected from culture supernatants or supernatant concentrates, purified antibodies, semi-purified or enriched antibodies. Antigen solutions are selected from purified or semi-purified natural antigen, recombinant or synthetic antigen. These antibodies and/or antigens can be added to adjuvants designed to maintain their microbiological stability, such as sodium azide, antibiotics, or their conformational stability, such as sugars (sucrose, dextrose, trehalose), as well as any other agent known in the art in this area as performing these functions.

According to one embodiment, the surface of the ball contains at least one chemical group selected from aldehyde groups, chloromethyl group, NHS groups and carboxyl groups.

In particular, when the surface of the bead contains aldehyde groups, this group reacts with the amino group of the antibody, which allows direct fixation of the antibody directed to the antigen to be detected and/or identified, or antibodies used for indirect fixation (described below), on the surface of the bead. In the case of antigen fixation, this group interacts with the amino group of the antigen, which ensures direct fixation of the antigen, recognized by the antibodies to be detected and/or identified.

In another specific embodiment, the surface of the bead contains NHS groups or also referred to as N-hydroxysuccinimide (NHS) functional groups, preferably it contains NHS ester functional groups.

According to another embodiment of the present invention, a given antibody directed to an antigen to be detected and/or identified, or a known antigen, can be indirectly fixed to the surface of the bead by another antibody, for example an anti-immunoglobulin. Such an anti-immunoglobulin antibody is preferably directed against immunoglobulins, more preferably IgM or IgG. In this case, if the surface of the bead contains an aldehyde group, the amino group of the anti-immunoglobulin antibody interacts with the aldehyde group, thereby fixing the anti-immunoglobulin antibodies on the surface of the bead. The antibody directed to the antigen to be detected and/or identified is then fixed to the anti-immunoglobulin antibody through protein/protein interaction.

In yet another embodiment, an antibody directed to an antigen to be detected and/or identified, or a known antigen, can be indirectly fixed to the surface of the bead by a ligand selected from proteins with affinity for antibodies, such as protein A, protein G and/or L protein.

The beads applied to the porous membrane of the in vitro device according to the invention can be made of different types of materials, such as glass, polymer, in particular latex. Preferably, the beads are selected from the group of glass beads, COOH-functionalized polystyrene beads, Protein A/G and/or L-functionalized agarose beads, NHS-functionalized agarose beads, epoxy-functionalized beads, chloromethyl-functionalized latex beads, or aldehyde-functionalized beads. .

More preferably, the beads are latex beads, in particular aldehyde functionalized latex beads.

According to one embodiment, these beads are used in a concentration of 1% to 10%, preferably 1% to 5%, more preferably 2% to 6% or 1% to 3%, even more preferably 3% of the solution applied onto the hydrophilic reaction region of the porous membrane, wherein the applied volume of said solution is from 1 to 100 μl, preferably from 1 to 50 μl, more preferably from 5 to 50 μl and even more preferably from 1 to 10 μl or from 5 to 10 μl.

The size of the beads is selected as a function of the pore size of the hydrophobic porous membrane. The size of the beads must exceed the pore size of the porous membrane in order to avoid absorption of the beads into the porous membrane. However, the size of the beads should not be very significant in order to avoid the phenomenon of agglutination. A skilled person would be able to select the size of the beads depending on the pore size of the porous membrane.

In one preferred embodiment of the present invention, the size of the beads is from 1 μm to 130 μm, preferably from 5 μm to 20 μm, and more preferably 9 μm.

According to one embodiment of the in vitro device according to the invention, the porous membrane has a thickness of from 0.4 mm to 2 mm, preferably from 0.6 mm to 1.5 mm.

The hydrophobic porous membrane can contain any substance that is not changed by aqueous solvents. The substance may in particular be selected from natural polymers, whether or not chemically modified, such as, for example, nitrocellulose polymer, cellulose, or from synthetic polymers, such as, for example, polyethylene, high-density polyethylene (HDPE) or fluorinated polymers , such as polyvinylidene fluoride (PVDF), preferably polyethylene. These polymers may be functionalized or unfunctionalized with reagent groups capable of forming bonds with capture agents used later.

The hydrophobic porous membrane contains at least one hydrophilic reaction region onto which beads are applied. This reaction region has a surface area that is smaller than that of a hydrophobic porous membrane, that is, the membrane cannot be made completely hydrophilic.

The hydrophilic reaction region(s) of a given porous membrane have preferably been made hydrophilic by local addition of a surfactant, without modifying the chemical functions of the porous substrate by prior chemical or physical treatment of the hydrophobic porous membrane.

In the context of the present invention, the term "surfactant" means any hydrophilicizing agent, that is, any substance capable of rendering a hydrophobic membrane hydrophilic sufficiently to allow passage of a sample containing analytes by capillary forces.

The surfactant used may be selected from natural surfactants, natural surfactants modified chemically or obtained by chemical synthesis. Preferably it is a nonionic surfactant, such as Triton X-100, Tween 20 or saponin, polyoxyethylene or nonyl-β-O-glucoside.

This surfactant can be diluted in an aqueous solution or in an organic solvent such as ethanol at a concentration of 0.01 to 5% (w/v). Preferably, the surfactant used to make the membrane locally hydrophilic is used at a concentration of 0.1% to 3%, more preferably 0.5 to 2%, and even more preferably 0.5 to 1% (wt). ./about.).

In order to make a hydrophobic porous membrane hydrophilic, 0.1 to 5 μl, preferably 0.3 to 3 μl, more preferably 0.5 to 2 μl of a solution containing a surfactant is applied to the membrane.

Thus, according to one embodiment, the hydrophilic reaction region of the hydrophobic porous membrane is rendered hydrophilic by a surfactant, preferably used at a concentration of from 0.01 to 5% w/v, more preferably from 0.1% to 3 % w/v, even more preferably 0.5% to 2% w/v. solution, and wherein 0.1 to 5 μl, preferably 0.3 to 3 μl and more preferably 0.5 to 2 μl of said surfactant-containing solution is applied to the hydrophobic porous membrane.

The concentration of surfactant used, correlated with other membrane characteristics (particularly porosity and thickness), controls the rate of fluids that pass through the membrane. It is generally accepted that a maximum dosage of 0.1% for Triton X-100 and 0.05% for Tween does not need to be exceeded to produce a hydrophilic membrane designed to receive the capture element. But due to the particular characteristics of the membrane according to the invention, detergents capable of rendering the membrane locally hydrophilic can be used in a dose of up to 5%, preferably up to 3%, especially for Triton X-100 or Tween 20, without affecting the reactivity of the region. which facilitates the creation of hydrophilicity of this membrane.

The same hydrophobic membrane can contain several hydrophilic reaction regions, provided that these regions do not overlap.

These reaction areas can be in all geometric shapes, but preferably in the form of circles or spots with a diameter of 0.3 mm to 20 mm.

This reaction region may be hydrophilic throughout the thickness of the porous membrane and/or at the surface.

A given reaction region can have one degree of hydrophilicity, i.e. a given reaction region has the same degree of hydrophilicity.

According to one embodiment, the reaction region may contain several regions having different degrees of hydrophilicity. For example, the reaction region may contain two hydrophilic regions, with a greater degree of hydrophilicity provided at the center of the reaction region than at the periphery. These hydrophilic regions are preferably located only on the surface of the membrane.

The reaction region may also contain two hydrophilic regions with different degrees of hydrophilicity: one on the surface and the other in the depth.

In the case where the reaction region contains two hydrophilic regions, the reaction region has been made hydrophilic using two different surfactants without modifying the chemical functions of the porous substrate by prior chemical or physical treatment of the porous membrane.

Advantageously, a reaction region configuration with two regions having different degrees of hydrophilicity, particularly at the surface, is useful in identifying a mixed field population from a sample of blood or blood components. This configuration is also particularly suited for detecting and/or identifying specific antibodies and/or antigens in a sample to be analyzed.

The in vitro device of the invention according to one embodiment may comprise an absorbent layer (or also referred to as an absorbent membrane) located underneath the porous membrane. This layer absorbs liquids deposited at the level of reaction regions that are not retained by the porous membrane, in particular when the reaction region is hydrophilic throughout the thickness of the membrane.

The absorbent layer may comprise a material that provides passive absorption by capillary action, such as absorbent paper, cellulose, etc., or may be made of absorbent polymers. By way of example, the following products may be listed for use as the absorbent layer:

- MerckMillipore® C048, C068, C083, C248 (Merck)

- Whatman® CF3, CF4, CF10, grade 470, CF5, CF6, CF7, grade 900, grade 300

- Ahlstrom® grades 601, 642, 631, 238, 237, 222, 243, 320 (Munktell)

- Pall grades 111, 113, 133, 165, 197, 8975, 8964, 8301 (Pall Corporation), Accuwik® Ultra membrane for direct collection, storage and efficient rapid release of biological samples for clinical diagnostics

- superabsorbent layer Cleanis Gelmax®.

- MS Arlaid T-499, SCP-300-TCF, T-089, T-183-5 and SCP-200-TCF

The composition of the absorbent membrane and its dimensions must be chosen such that it can absorb all solutions used during the analysis (V _total in µl). Each membrane has a different absorption capacity (C in µl/ ^cm2 ), the membrane and its dimensions (D in ^cm2 ) are chosen to satisfy the following equation: D greater than V _total /C.

According to one embodiment, the in vitro device of the invention also comprises at least one further layer located between the porous hydrophobic membrane and the absorbent layer, said layer being made of a drainage substance. The drying agent is a substance such as cotton wool, NA7150PES, NT9610HY, NT9750HY NV170F, NV250F, NV340F, (Subrenat) or Packtex HY050B.

A porous membrane and possibly layers of absorbent and drainage are organized on the substrate of this device.

The support of the in vitro device according to the invention is preferably a solid support. For example, it can be a shell.

This support preferably contains a solid material that does not allow liquid to escape. These can be, in particular, plastics such as polypropylene, polyethylene, polystyrene, acronitrile butadiene styrene, polyethylene terephthalate, polycarbonate, polyamide, polyvinyl chloride, methyl polymethacrylate.

The substrate of the in vitro device according to the invention preferably contains two parts: a lower part on which the hydrophobic porous membrane, a drainage layer and an absorbent layer are located, and an upper part which covers the lower part.

Preferably, an absorbent layer is arranged on the surface of the lower part of the substrate, on which a hydrophobic porous membrane is located. More preferably, a drainage layer is provided between the absorbent layer and the porous membrane. Even more preferably, a layer of foam is arranged on the surface of the lower portion of the substrate, on which is applied an absorbent layer, then a drainage layer, on which is applied a hydrophobic porous membrane containing at least one hydrophilic reaction region, the surface of which is smaller than the surface of the membrane, and on this reaction region area, beads with antibodies fixed and/or adsorbed on their surface are applied (Fig. 1a and b).

The upper portion of the substrate covers the lower portion such that the hydrophobic porous membrane and any one or all other layers, when present on the substrate, can be embedded into the substrate.

The upper part of the substrate preferably contains at least one or more than one hole. This hole(s) corresponds to the collection area of the sample applied to the hydrophilic reaction area. The hydrophilic region may be identical in size to the size of the base of the hole, smaller or larger, provided that the two hydrophilic regions are always separated by a hydrophobic region on the membrane.

The collection area must be of such a size that it can contain at least the maximum volume of sample or reaction mixture to be analyzed applied to the reaction membrane.

According to one embodiment, the different parts of the in vitro device of the invention can be assembled by the user if they are supplied as a kit. This device can be supplied as a single unit.

As stated above, the in vitro device of the invention can also detect and/or identify an antibody.

According to one embodiment, detection of the antibody is accomplished by binding to a specific known antigen fixed and/or adsorbed on the surface of the bead. The antibody to be identified and/or detected in a biological fluid sample may specifically bind to a known antigen. Detection of this binding provides identification of an antibody specifically directed against the specified antigen.

According to another embodiment of the invention, detection of antibodies can be accomplished by fixing or adsorption of an anti-immunoglobulin (antibody) on the surface of a bead that is an anti-immunoglobulin antibody capable of binding another antibody (to be detected and/or identified) present in the sample to be analyzed. This second antibody will then be detected and/or identified using a detection agent, for example, a labeled antigen specifically bound by the antibody.

According to another embodiment of the invention, detection of antibodies can be accomplished by fixation or adsorption of a protein with affinity for antibodies on the surface of a bead that is capable of binding to the antibody (to be detected and/or identified) present in the sample to be analyzed. The antibody will then be detected and/or identified using a detection agent, eg, a labeled antigen specifically bound by the antibody.

In one embodiment, when detecting and/or identifying anti-red blood cell antibodies, the beads capture or adsorb an anti-immunoglobulin antibody (anti-IgG and/or anti-IgM) that will capture circulating anti-red blood cell antibodies in a blood sample, especially serum or plasma. Detection and identification of these antibodies can be carried out using red blood cells carrying the corresponding antigen.

In one embodiment, when detecting and/or identifying anti-red blood cell antibodies, the beads capture or adsorb an anti-immunoglobulin antibody (anti-IgG and/or anti-IgM) that will capture anti-red blood cell antibodies present on the surface of in vivo sensitized red blood cells. Detection and identification of these antibodies is carried out through the erythrocytes themselves sensitized in vivo.

In one embodiment, the beads capture or adsorb an anti-C3d antibody that will capture in vivo complement-sensitized red blood cells that are produced due to complement activation by capturing the in vivo IgM antibody to the red blood cells. Detection and identification of these antibodies is carried out through the erythrocytes themselves sensitized in vivo.

The same principle can be applied to antibodies directed against viral, bacterial or parasitic antigens that circulate in the blood of a test subject suspected of being infected, using, for example, a different detection system such as, for example, a second set of color beads associated with the corresponding antigens or an antibody conjugated to an enzyme (for example, horseradish peroxidase) or to colloidal gold nanoparticles.

The in vivo device of the present invention is intended to detect and/or identify an antigen and/or antibody from a sample of biological fluids.

In the context of the present invention, the term "biological fluid sample" refers to any sample obtained from bioorganic fluids produced by a living organism. These biological fluids are selected from the group consisting of extracellular fluids, intravascular fluids, interstitial fluids, lymphatic fluids and transcellular fluids. In particular, the biological fluid sample is selected from the group consisting of blood and blood components, urine, saliva, etc.

In a more preferred embodiment, the biological fluid sample is a blood sample or a blood component sample. The term “blood sample” or “blood component sample” means whole blood or one of its components selected, in particular, from the erythrocyte fraction, leukocyte fraction, platelet, plasma or serum.

In particular, when the in vitro device of the present invention is used to detect and/or identify red blood cell antigens and/or antibodies directed against these antigens, the sample is a blood sample or a blood component sample.

In particular, when the in vitro device of the present invention is used to detect and/or identify viral, bacterial or parasitic antigens and/or antibodies directed against these antigens, the sample is a blood sample or a blood component sample.

The sample, as described above, can be obtained from any organism, in particular from a mammal and, more particularly, from a human.

Preferred Embodiments

Various preferred specific characteristics corresponding to various typical elements of the in vitro device according to this invention have been described above in the section specifically related to this element. In the context of the present invention, each list of suitable characteristics for a particular element and each specific characteristic disclosed for a specified element can be combined with any other generic element, the list of suitable characteristics for the specified other element or any specific characteristic disclosed for the specified other element.

In particular, preferred embodiments of an in vitro device element according to this invention may be combined with any other exemplary element or with preferred embodiments of said other element.

Preferred embodiments, corresponding to embodiments in which at least one element is limited to the preferred embodiment, are listed in Table 3 below:

In vitro use of the device according to the invention

The in vitro device of the invention can be used to detect and/or identify an antigen and/or antibody from a sample of a biological fluid.

As stated above, this device is suitable for detecting and/or identifying antigen and/or antibody, depending on the antibody and/or antigen fixed or adsorbed on the surface of the bead.

The inventors of the present invention have conducted studies to demonstrate that the in vitro device of the present invention is suitable for use with antibodies fixed on the surface of beads having structural differences, such as antibodies directed against red blood cell antigens and antibodies directed against at least one antigen specific for a virus, bacterium or parasite. For example, the test antibody is an antibody directed against an antigen of HCV (HCsAg) or HBV (HBsAg), causing hepatitis C and B, respectively, and the antibody can be fixed or adsorbed on the surface of the bead in order to detect and/or identify the listed antigens, respectively. Preferably, said antibody is an antibody directed against an HBV antigen (HBsAg).

In particular, the inventors of the present invention have demonstrated that the in vitro device of the present invention is useful for detecting and/or identifying antigens and/or antibodies of red blood cells, viruses, bacteria and parasites from a biological fluid, in particular from a blood sample or a sample of components blood.

According to another aspect, the present invention thus relates to the use of an in vitro device according to the invention for the determination and/or identification of:

- at least one antigen selected from the group consisting of erythrocyte antigens, including mixed field population, platelet antigens, viral antigens, bacterial antigens and parasite antigens;

- at least one antibody selected from the group consisting of anti-red blood cell antibodies, anti-platelet antibodies, anti-viral antibodies, anti-bacterial antibodies and anti-parasitic antibodies from a sample of biological fluids, preferably from a sample of blood or a sample of blood components

from a sample of biological fluids, preferably from a sample of blood or a sample of blood components.

As stated above, different types of antigens or antibodies can be detected and/or identified by the in vitro device of the invention, depending on the type of antibodies or antigens fixed or adsorbed on the surface of the bead.

In particular, the in vitro device of the present invention is used to detect and/or identify antigens of the group containing red blood cell antigens, platelet antigens, viral antigens, bacterial antigens and parasitic antigens. More specifically, this device is used to detect and/or identify red blood cell antigens.

The in vitro device of the present invention can be used, in particular, for the detection and/or identification (or also phenotyping) of red blood cells, that is, the determination of antigens on their surface. Phenotyping red blood cells usually means determining and/or identifying blood groups. In the context of the present invention, the term "blood group" refers, for example, to groups selected from the systems ABO, D, Rh, Kell, MNS, JK (Kidd), FY (Duffy), LE (Lewis), LU (Lutheran), P1PK .

The in vitro apparatus of the present invention is also used to detect and/or identify antibodies selected from the group consisting of anti-red blood cell antibodies or also referred to as anti-blood cell antibodies, which include autoantibodies, alloantibodies, heteroantibodies and cold antiglobulin. In particular, these antibodies are detected and/or identified from a blood sample or a blood component sample.

The in vitro device of the present invention can be used in particular for the following:

- Simonin-Michon assay, which identifies the presence of antibodies against A (anti-ABO1) or against B (anti-ABO2);

- search or identification of antibodies directed against cellular antigens, in particular red blood cells, in terms of searching for alloantibodies, autoantibodies, heteroantibodies or even cold agglutinins by means of indirect antiglobulin analysis;

- search for compatibility between the donor’s red blood cells and the recipient’s blood through a blood group determination reaction;

- search for antibodies fixed in vivo on erythrocytes using direct antiglobulin analysis.

The term "alloantibody" as used herein refers to one or more antibodies produced by a subject that reacts with one or more antigens from a genetically different individual of the same species. These alloantibodies are produced by immunization after blood transfusion, during pregnancy, and after transplantation or transplantation.

The term "autoantibody" as used herein refers to one or more antibodies produced from a subject directed against one or more than one antigen from the same subject. Detection of autoantibodies is preferably performed after blood transfusion or in the case of hemolytic anemia (autoimmune, drug-induced or after blood transfusion).

The term “heteroantibody” as used herein refers to one or more antibodies produced from a subject directed against one or more than one antigens from another species.

The term "cold agglutinin" as used herein refers to antibodies that cause agglutination of red blood cells at low temperatures.

The term "blood typing test" as used herein refers to a test performed prior to blood transfusion to determine whether the donor's red blood cells are compatible with the intended recipient's blood or plasma.

Antiviral, antibacterial and antiparasitic antibodies, detected and/or identified by the device in vitro, are immune antibodies produced against viral, bacterial or parasitic antigens upon infection of a subject.

The in vitro device of the present invention can also be used to detect the presence of a pathogenic agent by detecting the presence of one or more than one viral, bacterial or parasitic antigen.

• Methods according to the invention

The in vitro device of the invention is particularly suitable for use in in vitro methods for detecting and/or identifying antigens selected from the group consisting of red blood cell antigens including mixed field population, in vivo sensitized red blood cells (also referred to as C3d positive cells), platelet antigens, viral antigens, bacterial antigens, parasitic antigens, or for the detection and/or identification of antibodies to red blood cells, including in vivo sensitized red blood cells (IgG-positive cells), and/or antibodies to platelets, and/or antibodies directed against viral, bacterial and parasitic antigens, which are produced in the blood against viral, bacterial or parasitic antigens when a subject is infected.

According to another aspect, the present invention thus relates to a method for in vitro detection and/or identification of red blood cell antigens, including a mixed field population and/or in vivo sensitized red blood cells (C3d-positive cells), and/or platelet antigens from a blood sample or sample blood components, including the following stages:

- adding a solution containing said sample to the reaction area of the in vitro device according to the invention and

- reading before washing to control sample loading;

- applying the washing solution to the reaction area and

- determining the presence of said antigens or blood cells by determining the presence of an antigen/antibody interaction if a red or pink spot appears in the reaction area, or determining the absence of said antigens or blood cells by determining the absence of an antigen/antibody interaction if a colorless spot appears in the reaction area.

According to one embodiment, before applying the sample to be analyzed to the reaction area containing the beads, it is possible to continue with the hydration of the reaction area, which has already been made hydrophilic, by means of a buffer solution. This buffer may contain a solution with a pH stabilized between pH 6 and pH 8.5, preferably between pH 6.5 and pH 7.8, in particular between pH 7 and pH 7.5, and an osmolarity between 250 mOsm and 800 mOsm , preferably from 300 mOsm to 600 mOsm. This solution may possibly contain a low concentration of surfactant (Tween 20 0.01 to 0.05% w/v), saturating agents (BSAs) (bovine serum albumin) and/or agents capable of potentiating antigen reactions -antibody. This buffer may possibly have protease activity, for example obtained by adding enzymes such as papain or bromelain. This buffer may optionally contain polycationic agents such as polybrene or polylysine to potentiate the reaction and maintain red blood cells in longer contact with the capture elements.

The in vitro method described above thus possibly includes the step of hydrating the porous membrane before adding the test sample.

According to another embodiment, the in vitro method described above also includes another possible step of diluting the red blood cells to be phenotyped in a buffer solution, in particular a buffer solution known to be favorable for immunohematological reactions, possibly containing additives such as, for example, a buffer containing solution with a pH stabilized from pH 6 to pH 8.5, preferably from pH 6.5 to pH 7.8, in particular from pH 7 to pH 7.5, and an osmolarity from 250 mOsm to 800 mOsm, preferably from 300 mOsm up to 600 mOsm. The solution may optionally contain low concentrations of detergents (eg Tween 20 0.01 to 0.05% w/v), saturating agents (eg BSA) and/or agents capable of potentiating antigen-antibody reactions. This buffer may possibly have protease activity, for example obtained by adding enzymes such as papain or bromelain. This buffer may optionally contain polycationic agents such as polybrene or polylysine to potentiate the reaction and maintain the globules in longer contact with the capture element.

According to yet another embodiment, the method of the invention described above optionally includes the additional step of incubating the test sample before applying it to the reaction area, preferably at a temperature of from 15 to 40°C, in particular from 18°C to 37°C, in over a period of from 2 s to 30 min and, in particular, from 1 min to 20 min.

Likewise, a buffer wash step must be applied to read the results. The wash buffer preferably contains PBS (Phosphate Buffer Saline), TBS (Tris Buffer Saline) or saline with a pH of 2 to 10, preferably 5 to 9. The osmolarity of this buffer must be controlled to avoid hemolysis of red blood cells. This buffer should be selected so as not to separate detection agents or colored analytes directly or indirectly fixed to the capture agents (antibodies fixed and/or adsorbed to the surface of the beads). Surprisingly, for in vitro use of the device according to the invention, it is preferable to use slightly hyperosmotic wash solutions (i.e., from 300 mOsm to 800 mOsm) obtained through the presence of saline agents such as NaCl, or nonionic osmolytes such as, for example, glycine or taurine This buffer may optionally be colored with dyes that contrast with the color of the detecting agent. For example, if the detection agents are red blood cells, the wash buffer solution may be blue or green. A low dose of surfactant can also be added to the wash solution to eliminate background noise. These surfactants are preferably non-ionic surfactants and in particular sugar esters, in particular polyoxyethylene sorbitan esters (Tween).

During in vitro use of the device, liquids can be applied, especially via a pipette system or via a high capacity capillary system.

In the specified in vitro method, ensuring the hydrophilicity of the reaction areas of the porous membrane of the in vitro device according to the invention can be ensuring hydrophilicity in thickness, i.e. hydrophilicity is ensured throughout the entire thickness of the porous membrane.

Hydrophilicity can be ensured by means of an aqueous solution of Triton X100 in a concentration of 0.01% wt./vol. up to 5% w/v, preferably from 0.1% w/v. up to 3% w/v, preferably from 0.5% w/v. up to 2% w/v in a volume of from 0.1 μl to 5 μl, preferably from 0.3 μl to 3 μl and more preferably from 0.5 μl to 2 μl.

In some applications, when using Triton, the hydrophilicity of the porous membrane may be partial, such that it does not occur throughout the entire thickness of the membrane.

If the red blood cells present in the sample carry an antigen (red blood cell antigens or antibodies fixed to in vivo sensitized red blood cells) recognized by a specific antibody fixed or adsorbed to the surface of the bead, these red blood cells remain immobilized on the surface of the bead located on the reaction area, despite washing , and the given reaction area remains red or pink. If the red blood cells present in the sample do not carry antigen recognized by the antibody fixed or adsorbed to the surface of the bead, these red blood cells are washed away by the washing solution and the reaction area remains colorless.

Reading of results can be visual or automatic.

Detection and/or identification of erythrocytes sensitized in vivo provides detection of autoantibodies or alloantibodies adsorbed on the surface of erythrocytes. In particular, this detection is based on the use of beads coupled with anti-immunoglobulin antibodies (anti-IgG) or anti-C3d antibody (for IgM), which will capture the red blood cells to be analyzed through IgG or C3d (the formation of which is induced by IgM) fixed on their membrane.

Advantageously, the device according to the invention requires neither centrifugation, nor shaking, nor the application of a vacuum, nor a specialized device. It can also be used completely autonomously by hand and easily automated by robots.

In addition, the inventors have also demonstrated that the in vitro device of the present invention can be used in an in vitro method for detecting and/or identifying anti-red blood cell antibodies (alloantibodies, autoantibodies and cold agglutinin) from a sample of a biological fluid, in particular from a sample of blood or blood components.

Another aspect of the present invention therefore relates to a method for in vitro detection and/or identification of antibodies to red blood cells from a blood sample or a blood component sample, comprising the following steps:

- incubation of the sample to be analyzed with a buffer and test erythrocytes of a known phenotype,

- applying this mixture to the reaction area of the in vitro device according to the invention,

- applying a washing solution to the reaction area and

- determining the presence of antibodies to red blood cells by determining the presence of an antibody/antigen reaction if a red or pink spot appears in the reaction area, or determining the absence of these antibodies by determining the absence of an antibody/antigen reaction if a colorless spot appears in the reaction area.

This incubation step can be carried out in the presence of an agent capable of aggregating red blood cells at a temperature of from 4 to 40° C. for a period of from 3 to 60 minutes, preferably from 5 to 30 minutes. A person skilled in the art would be able to determine this temperature depending on the antibody being detected and/or identified.

The buffer used in the incubation step is a low ionic strength buffer, such as LISS buffer (eg, containing less than 50 mM NaCl).

According to one embodiment of this method, an agent capable of aggregating red blood cells, for example a solution of hexadimethrine bromide, preferably hexadimethrine bromide in a concentration in solution of from 0.01 to 2% (w/v), can be added to the mixture obtained during the incubation step, even more preferably 0.05 to 0.5%. Hexadimethrine bromide stimulates erythrocyte aggregation.

According to another embodiment of the method for detecting and/or identifying antibodies to red blood cells, a human antiglobulin or anti-complement reagent can be applied to the reaction area after application of an agent capable of aggregating red blood cells.

The wash solution used to wash the reaction area may be, for example, a hypertonic saline solution with a pH stabilized between pH 6 and pH 8.5, preferably between pH 6.5 and pH 7.8, preferably between pH 7 and pH 7.5, and osmolarity from 300 mOsm to 800 mOsm. This solution may possibly contain a low concentration of detergents (eg Tween 20 0.01 to 0.05% w/v) and a color contrasting with the red (blue or green).

The sample to be applied may be plasma, serum or whole blood, or blood components, diluted or undiluted in a buffer containing a solution with a pH stabilized from pH 6 to pH 8.5, preferably from pH 6.5 to pH 7, 8, in particular from pH 7 to pH 7.5, and an osmolarity from 250 mOsm to 800 mOsm, preferably from 300 mOsm to 600 mOsm. The solution may optionally contain low concentrations of detergents (eg Tween 20 0.01 to 0.05% w/v), saturating agents (especially BSA) and/or agents capable of potentiating antigen-antibody reactions. This buffer preferably has a salt content of less than 50 mM due to NaCl.

If the plasma being tested contains antibodies directed against an antigen present in the detection agent (test red blood cells), a red (or pink) spot appears in the reaction area. A colorless center indicates the absence or undetectable dose of antibodies directed against the antigen present in the detecting agent (test red blood cells).

Reading of results can be visual or automatic.

Another aspect of the present invention further relates to a method for in vitro detection and/or identification of anti-platelet antibodies from a blood sample or a blood component sample, comprising the following steps:

- incubation of the sample to be analyzed with a buffer and test platelets of known antigenicity or incubation of recipient plasma or serum with donor platelets,

- applying this mixture to the reaction area of the in vitro device according to the invention,

- applying a washing solution to the reaction area and

- determining the presence of antibodies to platelets by determining the presence of an antibody/antigen interaction if a red or pink spot appears in the reaction area, or determining the absence of said antibodies by determining the absence of an antigen/antibody interaction if a colorless spot appears in the reaction area.

The in vitro device of the invention can also be used in an in vitro method for detecting and/or identifying another type of antigens selected from the group consisting of viral antigens, bacterial antigens and parasitic antigens.

Yet another aspect of the present invention thus relates to a method for in vitro detection and/or identification of antigens selected from the group consisting of viral antigens, bacterial antigens and parasitic antigens from a sample of a biological fluid, preferably from a sample of blood or a sample of blood components, comprising: the following stages:

- it is possible to incubate the sample to be analyzed with a buffer,

- adding a solution containing the specified sample or a mixture of this sample with a buffer to the reaction area of the in vitro device according to the invention,

- reading before washing to control sample loading is possible;

- it is possible to apply a washing solution to the reaction area and

- determining the presence of said antigen by determining the presence of an antigen/antibody interaction, preferably if a colored spot appears in the reaction region, or determining the absence of said antigens by determining the absence of an antigen/antibody reaction, preferably if a colorless spot appears in the reaction region.

The incubation step can be carried out in the presence of an agent capable of enhancing the reaction, at a temperature of from 4 to 40°C, for a period of from 3 to 60 minutes, preferably from 5 to 30 minutes. One skilled in the art would be able to determine the appropriate temperature depending on the antibody or antigens to be detected and/or identified.

The buffer used in the incubation step is a buffer with a molecule to enhance the reaction.

The sample to be applied may be a biological fluid, for example: plasma, serum or whole blood or blood components, diluted or undiluted in a buffer containing a solution with a pH stabilized from pH 6 to pH 8.5, preferably from pH 6.5 to pH 7.8, in particular from pH 7 to pH 7.5, and an osmolarity of from 250 mOsm to 800 mOsm, preferably from 300 mOsm to 600 mOsm. The solution may optionally contain low concentrations of detergents (eg Tween 20 0.01 to 0.05% w/v), saturating agents (especially BSA) and/or agents capable of potentiating antigen-antibody reactions. This buffer preferably has a salt content of less than 50 mM due to NaCl.

The wash solution used to wash the reaction area may be, for example, a hypertonic saline solution with a pH stabilized between pH 6 and pH 8.5, preferably between pH 6.5 and pH 7.8, preferably between pH 7 and pH 7.5, and osmolarity from 300 mOsm to 800 mOsm. This solution may possibly contain low concentrations of detergents (eg Tween 20 0.01 to 0.05% w/v).

Detection of antigen/antibody interactions can be accomplished preferably through the use of colored beads or HRP (horseradish peroxidase) coupled to antibodies or other molecule that interacts with the corresponding pathogen antigens.

One skilled in the art would be able to determine other labeling methods that provide detection and/or identification of said antigens.

If the analyzed plasma contains antigens directed against the antibody present in the detection agent, a colorless spot appears in the reaction area. A colorless center indicates the absence or undetectable dose of antigens directed against the antibody present in the detecting agent.

Reading of results can be visual or automatic.

As stated above, antibodies directed against antigens selected from the group of viral, bacterial or parasitic antigens can also be detected and/or identified using the in vitro device of the present invention. In fact, in most cases, when the presence or absence of a pathogen (virus, bacterium or parasite) is determined, for example during a blood transfusion, it is by detecting and/or identifying antibodies produced by the infected subject directed against said antigens. These antibodies are also referred to herein as immune antibodies produced against viral, bacterial or parasitic antigens upon infection of a subject. They can circulate in the blood of an infected subject.

Thus, according to yet another aspect, the in vitro device of the invention can also be used in an in vitro method for detecting and/or identifying various antibodies directed against viral antigens, bacterial antigens and parasitic antigens that are produced during infection of a subject.

Yet another aspect of the present invention therefore relates to a method for in vitro detection and/or identification of antibodies selected from the group consisting of antibodies directed against viral antigens, bacterial antigens and parasitic antigens, preferably said antibodies being produced during infection from a sample of a biological fluid preferably from a blood sample or a blood component sample, comprising the following steps:

- it is possible to incubate the sample to be analyzed with a buffer

- adding a solution containing said sample or a mixture of sample and buffer to the reaction area of the in vitro device according to the invention,

- reading before washing to control sample loading is possible;

- it is possible to apply a washing solution to the reaction area and

- determining the presence of said antibodies by determining the presence of an antigen/antibody interaction, preferably if a colored spot appears in the reaction region, or determining the absence of said antibody by determining the absence of an antigen/antibody reaction, preferably if a colorless spot appears in the reaction region.

The incubation step can be carried out in the presence of an agent capable of enhancing the reaction, at a temperature of from 4 to 40°C for a period of from 3 to 60 minutes, preferably from 5 to 30 minutes. One skilled in the art would be able to determine the appropriate temperature depending on the antibody or antigens to be detected and/or identified.

The buffer used in the incubation step is a buffer with a molecule to enhance the reaction.

The wash solution used to wash the reaction area may be, for example, a hypertonic saline solution with a pH stabilized between pH 6 and pH 8.5, preferably between pH 6.5 and pH 7.8, preferably between pH 7 and pH 7.5, and osmolarity from 300 mOsm to 800 mOsm. This solution may possibly contain low concentrations of detergents (eg Tween 20 0.01 to 0.05% w/v).

The sample to be applied may be a biological fluid, for example: plasma, serum or whole blood, or blood components, diluted or undiluted in a buffer containing a solution with a pH stabilized from pH 6 to pH 8.5, preferably from pH 6 .5 to pH 7.8, in particular from pH 7 to pH 7.5, and an osmolarity of from 250 mOsm to 800 mOsm, preferably from 300 mOsm to 600 mOsm. The solution may optionally contain low concentrations of detergents (eg Tween 20 0.01 to 0.05% w/v), saturating agents (eg BSA) and/or agents capable of potentiating antigen-antibody reactions. This buffer preferably has a salt content of less than 50 mM NaCl based.

If the plasma being analyzed contains antibodies directed against an antigen present in the detection agent, a colorless spot appears in the reaction area. A colorless center indicates the absence or undetectable dose of antibodies directed against the antigen present in the detecting agent.

Detection of the antibody/antigen reaction is preferably carried out through the use of colored beads or HRP (horseradish peroxidase) linked to antibodies or a molecule that would react with the corresponding antigens of the pathogens.

One skilled in the art would be able to identify other labeling methods that would enable detection and/or identification of the antibody.

Reading of results can be visual or automatic.

DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1: a. Diagram of a specific embodiment of an in vitro device according to the invention, considered in perspective, the said device contains a porous membrane including a reaction region on which are applied several beads having a size of 9 μm, on the surface of which antibodies or antigens are directly or indirectly fixed or adsorbed. Three layers are shown underneath the membrane: a drainage layer, an absorbent layer, and a foam layer. In FIG. Figure 1 also shows a scanning electron microscopy (SEM) view of a section of a porous membrane containing beads and antibodies fixed to their surface, b. A diagram of a specific embodiment of an in vitro device according to the invention, wherein said device comprises a support on which a porous membrane is located, including a reaction region made hydrophilic, onto which several beads are applied, on the surface of which antibodies are fixed. The same layers as those shown in Fig. 1a are shown under a porous membrane.

Fig. 2: comparative assays for the detection of erythrocyte antigens using Anti-ABO01, Anti-ABO02, Anti-ABO03, Anti-RH1, Ahth-RH2, Ahth-RH3, Ahth-RH4, Ahth-RH5, Anti-KEL1 and negative control using such a device in vitro as described in WO2013/186482 (Fig. 2a), and in vitro devices of the present invention (Fig. 2b).

Fig. 3: detection of erythrocyte antigens ABO01, ABO02 and ABO3 by means of IgM antibody indirectly fixed on latex beads through protein on protein A/G/L.

Fig. 4a and b: detection of erythrocyte antigen ABO01 by IgM antibody coupled to AHG (anti-human globulin antibody) bound to chloromethyl latex beads, with two different volumes of antibody-coupled beads loaded onto the membrane (a: 20 µl and b: 10 µl). end: detection of erythrocyte antigen ABO01 by IgG antibody coupled to AHG bound to chloromethyl latex beads, with two different volumes of antibody coupled beads loaded onto the membrane (c: 20 µl and d: 10 µl).

Fig. 5: Detection of red blood cell antigens using specific IgM antibody coupled to AHG bound to epoxy beads (antigens a: RH2; b: RH3; c: RH4; d: RH5; e: KEL1 and f: negative control).

Fig. 6: Detection of FY1 and RH1 erythrocyte antigens by specific IgG antibodies coupled to AHG (anti-human globulin antibody) coupled to NHS agarose beads under the conditions described in Example 3. a. b. and s. represent different detection techniques, and d. different reaction device and detection method.

Fig. 7: detection of ABO02 erythrocyte antigens and mixed field populations with different amounts (from 1 to 5%) of anti-ABO02 associated with 9 μm aldehyde latex beads loaded on a porous membrane.

Fig. 8: a. Detection of erythrocyte antigens ABO01, RH2, RH5 and KEL1 by varying amounts of specific antibodies bound to 9 μm aldehyde latex beads (from 0.125 to 4%) on a device containing a 1.5 mm thick membrane. b. Detection of erythrocyte antigens ABO01, RH2 and KEL1 using specific antibodies bound to 9 μm aldehyde latex beads (4%) on a device containing a 0.6 mm thick membrane.

Fig. 9: Detection of red blood cell antigen ABO1 by specific antibodies bound to 9 μm aldehyde latex beads loaded onto an in vitro device containing a 0.6 mm thick membrane made hydrophilic using a. Triton X-100, b. polyoxyethylene and s. nonyl-β-th-glucoside.

Fig. 10: a. Detection of erythrocyte antigen RH2 by specific antibodies bound to 9 μm aldehyde latex beads on a device containing T-499 (left) or SCP-300-TCF (right) as an absorbent. b. Detection of erythrocyte antigen RH2 by specific antibodies bound to 9 μm aldehyde/sulfate latex beads on a device containing T-099 (left) or T-499 (right) as an absorbent, p. Detection of erythrocyte antigen RH2 by specific antibodies bound to 9 μm aldehyde/sulfate latex beads on a device containing T-499 (left) or T-183-5 (right) as an absorbent, d. Detection of erythrocyte antigen RH2 by specific antibodies coupled to 9 μm aldehyde/sulfate latex beads on a device containing T-499 (left) or SCP-200-TCF (right) as an absorbent. For each image, the device is shown disassembled; from top to bottom: membrane on a plastic device and absorbent.

Fig. 11: a. Detection of erythrocyte antigen ABO02 by anti-ABO03 antibody bound to 9 μm aldehyde latex beads on a device containing cotton wool as a drainage layer. b. Detection of erythrocyte antigen ABO02 by anti-ABO03 antibody bound to 9 μm aldehyde/sulfate latex beads on a device containing NT 9750 HY as a drainage layer. With. Detection of erythrocyte antigen ABO02 by anti-ABO03 antibody bound to 9 μm aldehyde/sulfate latex beads on a device containing NT 9610 HY as a drainage layer. For each image, the device is shown disassembled; from top to bottom: membrane, drainage layer and absorbent.

Fig. 12: a. and s. Detection of erythrocyte antigens ABO01 or ABO02 using IgM antibody (anti-ABO01 and anti-ABO02) bound to 9 μm aldehyde/sulfate latex beads. b. and d. Signal interpretation to differentiate between mixed field populations.

Fig. 13: a. Detection of weak RH1 antigens: different types of weak RH1 erythrocytes (Df1, Df2, ...) are captured by anti-RH1 antibody (clones HM16 and ESD1) fixed to protein A/G/L bound to 9 μm aldehyde/sulfate latex beads. b. Detection of Duffy (FY1 and FY2), KIDD (JK1 and JK2) and MSN (MNS3 and MNS4 systems) antigens: positive red blood cells are captured by specific antibodies fixed to protein A/G/L bound to 9 μm aldehyde/sulfate latex beads.

Fig. 14: reverse group determination, test red blood cells of known phenotype (A1, A2, B and O) are incubated with plasma and captured with anti-human IgM antibody (CP6D4 clones) coupled to 9 μm aldehyde/sulfate latex beads.

Fig. 15: direct Coombs test: a. IgG-sensitized red blood cells are captured by anti-human globulin antibodies (clones SA6532, CPC5-1 and 188 33) bound to 9 μm aldehyde/sulfate latex beads. b. Positive red blood cells are captured by anti-human globulin antibody (SA6532) and/or anti-human C3d antibody (C7610) coupled to 9 μm aldehyde/sulfate latex beads, p. Positive red blood cells are captured by anti-human globulin antibody (SA6532) and/or anti-human C3d antibody (C7610) and captured on protein A/G/L bound to 9 μm aldehyde/sulfate latex beads.

Fig. 16: detection of antibodies specific for the MSP1 antigen of Plasmodium falciparum, a. Use of the device according to the invention with the detection method with HRP. b. Application of the device according to the invention with 1 μm red beads as a detection method, p. Application of the device according to the invention with colloidal gold nanoparticles as a detection method. For each example, negative and positive reactions are shown, as well as a quantitative measurement of signal intensity (n equals 3-5).

Fig. 17: Detection of antibodies specific for p15, p17 and p47 antigens of Treponema pallidum using the device of the invention with 1 μm red beads as a detection method. Negative and positive reactions are shown, as well as a quantitative measurement of signal intensity (n = 3).

Fig. 18: Detection of antibodies specific for the HBsAg antigen from the hepatitis B virus using the device of the invention with 1 μm red beads as a detection method. Negative and positive responses are shown, as well as a quantitative measurement of signal intensity (n = 3).

Fig. 19: identification of HBsAg antigen from hepatitis B virus a. Use of the device according to the invention with HRP (horseradish peroxidase) as a detection method (graph and photograph). b. Using a device according to the invention with 1 μm red beads as a detection method. For each example, negative and positive reactions are shown, as well as a quantitative measurement of signal intensity (n = 3).

EXAMPLES

EXAMPLE 1: typing and phenotyping of red blood cells in an in vitro device with direct and indirect binding of antibodies to red blood cells on beads via antibodies to immunoglobulins IgM and IgG

1. Protocol for obtaining a device in vitro, which contains antibodies to red blood cells on the surface of the reaction area, directly applied to the membrane.

1.1. Materials for obtaining the device in vitro:

- 0.6 mm hydrophobic membrane “POREX” with porosity from 9 to 12 microns; cotton;

- Cleanis;

- Buffer to ensure hydrophilicity (1% Triton, 1% green dye, phosphate-buffered saline).

- Wash buffer (TLA composition);

- Buffer for dilution (Chromasolcoombs);

- Antibodies (anti-ABO01, anti-ABO02, anti-ABO03, anti-RHI, anti-RH2, anti-RH3, anti-RH4, anti-RHS, anti-KEL1, negative control).

1.2. Method for preparing the reaction medium:

- add 1 µl of a solution ensuring hydrophilicity into each well;

- drying for 16 hours at 37°C and 10% humidity;

- add 2 μl of antibody per well;

- drying 72 hours at 37°C and 10% humidity.

The materials used to perform the assay are a reaction support, a wash buffer, and a dilution buffer.

1.3. Implementation of the analysis:

- dilute the red blood cell sediment to 20% in dilution buffer;

- add 20 µl of suspension into the wells;

- incubate for 3 minutes at room temperature;

- add 80 µl of washing buffer;

- incubate for 1 minute at room temperature;

- add 80 µl of washing buffer.

2. Protocol for obtaining a device in vitro with direct and indirect binding of antibodies to red blood cells on beads using antibodies against immunoglobulins IgM and IgG.

2.1. Protocol for direct binding of anti-ABO01 (anti-A), anti-ABO02 (anti-B) and anti-ABO03 (anti-AB) antibodies to latex beads.

To 1 ml of homogenized 4% beads (9 μm aldehyde/sulfate latex beads from Thermo Fisher, 4% w/v), add 100 μl of 20× phosphate buffered saline, and dissolve 2 ml of concentrated antibodies. The volume is adjusted to 4 ml with demineralized water. This solution is stirred by inversion overnight to ensure binding.

Excess active groups are blocked by suspending 150 μl of 1 M ethanolamine in an antibody solution on beads, followed by mixing for 2 hours at room temperature. The bound beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). Finally, the bead pellet is resuspended in the preservation buffer to obtain the injected bead solution mass.

2.2. Indirect binding protocol for anti-RH1, anti-RH2, anti-RH3, anti-RH4, anti-RH5 and anti-KEL1 antibodies

To 1 ml of homogenized 4% beads, add 200 µl of 20× phosphate-buffered saline and dissolve 250 µl 6D4 (anti-human IgM antibody, clone Diagast) at a concentration of 1 mg/ml or 100 µl C5-1 (anti-human IgG antibody, clone Diagast) at a concentration of 3.69 mg/ml. The volume is adjusted to 4 ml with demineralized water. This solution is stirred by inversion overnight to ensure binding.

Excess active groups are blocked by suspending 150 μl of 1 M ethanolamine in an antibody solution on beads, followed by mixing for 2 hours at room temperature. Beads with bound anti-human globulin (AHG) antibody are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). The bead pellet is resuspended with 2 ml of concentrated antibody plus 2 ml of phosphate-buffered saline. This solution is stirred by inversion overnight to ensure binding.

The resulting beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washing). Finally, the bead pellet is resuspended in the preservation buffer to obtain the injected bead solution mass. Negative control beads were directly saturated with ethanolamine.

The antibody clones used in the Examples are shown in Table 4 below:

The antibody clones in Table 4 are those obtained and marketed by the applicant (Diagast, France).

2.3. Preparation of the reaction device and detection method

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 μm, a drainage layer (NA7150 PES from Subrenat) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 0.5% Triton X-100; 0.5% green dye and 0.13 M sucrose) and potentially dried for 1 hour at 37°C, 10% humidity. 5 μl of antibody- or negative control-coupled beads are then spotted at 4% concentration and the device is dried for 1 hour at 37°C, 10% humidity before immediate use or long-term storage.

The assay is initiated by diluting red blood cells at a concentration of 5% in Chromasolcoombs (Diagast) and 10 µl injected into each well, followed by two 30 µl washes (phosphate buffered saline supplemented with 0.2% Tween 20 and 0.54% green dye).

In this example, the applicant also compared an in vitro device containing anti-RBC antibodies bound to blood typing and phenotyping beads obtained by the protocol described above with an in vitro device in which anti-RBC antibodies are directly applied as spots onto a porous membrane ( similar to the antibodies described in WO2013/186482).

3. Results

The obtained comparative results are shown in Fig. 2, where the presence of a dark gray spot (corresponding to the red spot in the color version) indicates the formation of an immune complex and therefore a positive reaction (presence of antigen on the surface of the red blood cell), whereas the presence of a colorless spot reflects a negative reaction (absence of antigen on the surface of the red blood cell) .

From Fig. 2 it is obvious that blood group antibodies anti-ABO01, anti-ABO02, anti-ABO03, anti-RH1, anti-RH2, anti-RH3, anti-RH4, anti-RH5 and anti-KEL1 are directly or indirectly fixed on the surface of the bead , specifically bind to the corresponding antigens present on the surface of the red blood cells of the analyzed samples. Indeed, the surface of the spot where the beads were applied remains well stained red, indicating that anti-RBC antibodies are not adsorbed by the porous membrane, allowing for specific and sensitive detection of RBC antigens (Figure 2b).

From Fig. 2a it can be seen that in the in vitro device without beads, reactions with some antibodies, in particular with anti-RH1, anti-RH3, anti-RH4, anti-RH5 and anti-KEL1 antibodies, have low sensitivities (very low dark gray intensity).

When comparing the results in Fig. 2b obtained with the in vitro device of the invention, with the results of FIG. 2a obtained with the in vitro device without the bead, it appears that the gray spots for anti-RH1, anti-RH3, anti-RH4, anti-RH5 and anti-KEL1 antibodies are significantly better visible in FIG. 2b than the spots of Fig. 2a, which demonstrates that the in vitro device of the invention allows for more sensitive detection of most red blood cell antigens.

EXAMPLE 2: indirect (mediated) binding of antibodies to red blood cells via antibody affinity proteins

1. Indirect (mediated) binding of antibodies

To 1 mL of homogenized 4% latex beads (9 μm latex aldehyde/sulfate from Thermo Fisher, 4% w/v), add 5 or 2.5 μg of AGL protein dissolved in 3 mL of 1× PBS. This solution is stirred by inversion overnight to ensure binding.

To 1 ml of centrifuged 4% beads (9 μm aldehyde/sulfate latex from Thermo Fisher, 4% w/v AGL protein bound), add 3 ml of 1× phosphate buffered saline, and dissolve 1 ml of anti-ABO01 antibody ( clone Diagast 91 13 D10) or antibodies against ABO02 (clone Diagast 96 21 A8), or against ABO03 (clone Diagast 152D12). This solution is stirred by inversion to ensure binding.

The bound beads are then centrifuged for 5 min at 2500 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the injected mass of bead solution.

2. Preparation of the reaction device and identification procedure

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610HY) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of beads with bound antibody at a concentration of 2% are applied as spots. Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting each well with 50 μl of red blood cells diluted at a concentration of 1.5% in 1× PBS supplemented with 0.00625% Tween 20.

The first image is acquired before washes. The reaction zone is then washed with 30 μl of phosphate-buffered saline supplemented with 0.2% Tween-20. An image is obtained after washing.

3. Results

In FIG. Figure 3 shows the resulting image, in which the presence of dark gray spots (corresponding to the red spot in the color image) is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction.

Example 3: binding of erythrocyte antibodies to beads having different chemical functional groups on their surface and different sizes under different reaction conditions

1. Chloromethyl group

1.2. Antibody binding

Add 1.5 mL of phosphate buffer to 500 μl of homogenized chloromethyl beads (chloromethyl latex beads from Thermo Fisher, 4% w/v, 2.0 μm), and the resulting solution is centrifuged at 3000 g for 20 min. The supernatant is discarded and the bead pellet is then resuspended with a mixture of 950 μl phosphate buffered saline and 50 μl concentrated CPC51 or CP6D4 (Diagast AHG). The antibody bead solution is incubated overnight at room temperature on a tube shaker.

After chemical binding of AHG, the bead solution is centrifuged at 3000 g for 15 min and the supernatant is discarded. The beads are washed with 1.5 ml of phosphate-buffered saline, and this solution is centrifuged at 3000 g for 10 minutes. The supernatant is discarded and excess active groups are blocked by adding 1 ml of 1 M ethanolamine before stirring for 20 min at room temperature. The solution of beads with bound AHG is centrifuged at 3000 g for 20 min before washing with 1.5 ml of phosphate-buffered saline. 1 ml of concentrated anti-A (anti-ABO1) antibodies (clones IgM 91 13 D10 or IgG 162 47 (3) E6, both clones from Diagast) are dissolved on the bead pellet, and this solution is stirred for two hours at room temperature before centrifugation and washing with 1.5 ml phosphate-buffered saline solution. Finally, the bead pellet is resuspended in preservation buffer to obtain the mass of the injected bead solution.

1.2. Preparation of the reaction device and detection method

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (cotton wool from Alan&co) and an absorbent layer (Cleanis) are assembled.

This porous membrane is rendered hydrophilic using 2.5 μL of surfactant solution (filtered water supplemented with 1% Triton X-100) and then spotted with 20 μL (FIGS. 4A and 4C) or 10 μL (FIGS. 4B and 4D) antibodies bound to beads at a concentration of 4%. Allow this device to stand for 30 minutes at room temperature before use.

The detection technique is initiated by introducing into each well 13 μl of a mixture of 30 μl of red blood cells at a concentration of 10% in chromasolcoombs (Diagast preparation) and 10 μl of hexadimethrine bromide solution.

The reaction zone is washed twice with 25 μl of phosphate-buffered saline supplemented with 0.1% Tween 20.

1.3. results

In FIG. Figure 4 shows the resulting image, in which the presence of a dark gray spot (corresponding to the red spot in the color image) is observed, indicating the formation of an immune complex (group A red blood cells) and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction ( red blood cells of group B).

2. Epoxy group

2.1. Antibody binding

0.5 g of epoxy resin (Biorad Profinity epoxy resin, supplied as a dry powder, particle size 45-90 micrometers) is weighed and allowed to swell in 50 ml of phosphate-buffered saline for 30 minutes with gentle shaking. Increase the volume of the bead solution to 5 ml, which gives a solution with a concentration of approximately 10%.

The beads are centrifuged at 2500 g for 5 minutes, the supernatant is discarded, and the pellet is resuspended in 4.5 ml of phosphate-buffered saline, then centrifuged again.

360 μl of concentrated anti-IgM AHG antibody (CP6D6 Diagast) is dissolved in 12 ml of phosphate-buffered saline, and this mixture is added to the binding bead pellet. The solution of antibody beads is incubated for 1 hour 30 minutes at room temperature on a rotary shaker.

After chemical binding of AHG, the bead solution is centrifuged at 2500 g for 5 min and the supernatant is discarded. The beads are washed with 4.5 ml of phosphate-buffered saline, and this solution is centrifuged at 2500 g for 5 min. The supernatant is discarded and excess active groups are blocked by adding 3 ml of 1 M ethanolamine before stirring for 45 min at room temperature. The solution of beads with bound AHG is centrifuged at 2500 g for 5 min before washing with 4.5 ml of phosphate-buffered saline.

300 µl of anti-RH2, anti-RH3, anti-RH4 or anti-RH5 (clone Diagast P3X255 13 G8, clone Diagast RH3 906, clone Millipore MS33 and clone Diagast P3GD C512, respectively) are diluted in 2200 µl of phosphate-buffered saline and added onto beads with bound AHG. For anti-Kell antibody, 1500 μl was diluted in 1000 μl phosphate-buffered saline, and 2500 μl phosphate-buffered saline was used as a negative control. The resulting solution was stirred for 45 minutes at room temperature before centrifugation and washing. Finally, the bead pellet is resuspended in phosphate-buffered saline with preservatives to obtain a 50% solution of bound beads.

2.2. Preparation of the reaction device and detection method

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (cotton wool from Alan&co) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 1 μl of surfactant solution (filtered water supplemented with 1% Triton X-100) and dried overnight at room temperature. Then 10 μl of beads with bound antibody are applied in the form of spots at a concentration of 50%. Allow this device to stand for 30 minutes at room temperature before use.

The detection technique is initiated by introducing 10 μl of undiluted red blood cells into each well. The reaction zone is washed twice with 40 μl of phosphate-buffered saline supplemented with 0.2% Tween-20 and preservatives.

2.3. results

In FIG. Figure 5 shows the resulting image in which the presence of dark gray spots (corresponding to the red spot in the color image) is observed to indicate the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction. Whatever the phenotype of the red blood cells, the negative control results in a colorless spot.

3. N-hydroxysuccinimide (NHS) group

3.1. Antibody binding

1 ml NHS beads (Pierce™ activated NHS agarose suspension) are diluted with 5 ml phosphate-buffered saline, centrifuged at 2500 g for 5 min, the supernatant is discarded and the pellet is resuspended in 4.5 ml phosphate-buffered saline for 3 additional washes .

2 ml of concentrated anti-IgG AHG antibodies (C51 Diagast) are dissolved in 3 ml of phosphate-buffered saline and this mixture is added to the binding bead pellet. The antibody solution with beads is incubated for one and a half hours at room temperature on a rotary shaker.

After chemical binding of AHG, the bead solution is centrifuged at 2500 g for 5 min and the supernatant is discarded. The beads are washed with 4.5 ml of phosphate-buffered saline, and this solution is centrifuged at 2500 g for 5 min. The supernatant is discarded and excess active groups are blocked by adding 3 ml of 1 M ethanolamine before stirring for 45 min at room temperature. The solution of beads with bound AHG is centrifuged at 2500 g for 5 min before washing with 4.5 ml of phosphate-buffered saline.

10 ml of anti-RH1 and anti-FY1 antibodies (clones HM16 and F655 from Diagast, respectively) were added to the AHG-bound beads. The resulting solution is stirred for 1 hour at room temperature before centrifugation and washing. Finally, the bead pellet is resuspended in phosphate-buffered saline supplemented with preservatives to obtain a 50% solution of bound beads.

3.2. Preparation of the reaction device and detection method

The device shown in Fig. 6 (A, B and C), is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (cotton wool from Alan&co) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 2.5 μl of surfactant solution (filtered water supplemented with 1% Triton X-100) and dried overnight at 37°C. Then 20 μl of beads with bound antibody at a concentration of 50% are applied as spots. Allow this device to stand for 30 minutes at room temperature before use.

Fig. 6A: the detection technique is initiated by introducing into each well 7 μl of red blood cells diluted to a concentration of 25% in a 0.25% hexadimethrine bromide solution. The reaction zone is washed with 20 μl plus 30 μl of phosphate-buffered saline supplemented with 0.01% Tween-20.

Fig. 6B: the detection technique is initiated by introducing into each well 5 μl of red blood cells diluted to a concentration of 25% in a 0.3% hexadimethrine bromide solution. The reaction zone is washed with 20 μl plus 30 μl of phosphate-buffered saline supplemented with 0.01% Tween-20.

Fig. 6C: the detection technique is initiated by introducing into each well 5 μl of red blood cells diluted to a concentration of 25% in a 0.4% hexadimethrine bromide solution. The reaction zone is washed with 20 μl plus 30 μl of phosphate-buffered saline supplemented with 0.01% Tween-20.

The device shown in Fig. 6D, is a molded plastic cassette in which a hydrophobic Lydall UHMWPE membrane with a thickness of 105 microns and a porosity of 12 microns (Solupor 70M01A), a drainage layer (cotton wool from Alan&co) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 2 μl of surfactant solution (filtered water supplemented with 1% Triton X-100) and dried for 1 hour 30 minutes at 37°C. Then 20 μl of beads with bound antibody at a concentration of 50% are applied as spots. Allow this device to stand for 30 minutes at room temperature before use.

The detection technique is initiated by introducing into each well 7 μl of red blood cells diluted to a concentration of 25% in a 0.25% solution of hexadimethrine bromide. The reaction zone is washed with 30 μl plus 50 μl of phosphate-buffered saline supplemented with 0.01% Tween-20 and an additional 20 μl of phosphate-buffered saline supplemented with 0.1% Tween-20 and 0.007% Triton X-100.

3.3. results

In FIG. 6 from the resulting image, the presence of dark gray spots (corresponding to the red spot in the color image) is observed, indicating the formation of an immune complex and therefore a positive reaction (detection of RH1 or FY1 antigen), while the presence of a colorless spot reflects a negative reaction.

EXAMPLE 4: bead concentration

The purpose of this experiment is to analyze different concentrations of beads to be applied to a porous membrane.

1. Antibody binding

To 1 ml of homogenized 4% beads (9 μm latex aldehyde/sulfate from Thermo Fisher, 4% w/v), add 100 μl of 20× phosphate-buffered saline, and dissolve 2 ml of affinity purified anti-ABO02 antibody (clone Diagast 96 21 A8). The volume is adjusted to 4 ml with demineralized water. This solution is stirred by inversion overnight to ensure binding.

Excess active groups are blocked by suspending 150 μl of 1 M ethanolamine in a solution of antibody beads and stirring for 2 hours at room temperature. The bound beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). Finally, the bead pellet is resuspended in preservation buffer to obtain the mass of the injected bead solution.

2. Preparation of the reaction device and detection method

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (cotton wool from Alan&co) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 1 μl of a surfactant solution (phosphate-buffered saline supplemented with 1% Triton X-100 and 1% green dye). Then 10 μl of beads with bound antibody are applied as spots at a concentration of 1 to 5%. This device is dried for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting each well with 50 μl of red blood cells diluted at a concentration of 0.15% in Chromasolcoombs supplemented with 0.0625% Tween-20. The reaction zone is washed once or twice with 30 μl of phosphate-buffered saline supplemented with 0.2% Tween-20.

3. Results

In FIG. 7 shows the resulting image, in which the presence of dark gray spots (corresponding to the red spot in the color image) is observed, indicating the formation of an immune complex and therefore a positive reaction (RBCs of the ABO02 group), while the presence of a colorless spot reflects a negative reaction. Specificity and sensitivity are maintained for bead concentrations ranging from 1 to 5% after one or two washes. Mixed field blood cells are detected by decreasing the intensity compared to the signal of a 100% positive population.

EXAMPLE 5: Porous hydrophobic membrane thickness

1. Antibody binding

1.1. Anti-ABO01 binding

To 1 ml of homogenized 4% beads (9 μm latex aldehyde/sulfate from Thermofisher, 4% w/v), add 100 μl of 20× phosphate buffer, and dissolve 2 ml of affinity purified anti-ABO01 antibody (clone Diagast 25 21 B8) . The volume is adjusted to 4 ml with demineralized water. This solution is stirred by inversion overnight to ensure binding.

Excess active groups are blocked by suspending 150 μl of 1 M ethanolamine in a solution of antibody beads and stirring for 2 hours at room temperature. The bound beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

1.2. Binding of anti-RH2, anti-RHS and anti-KEL1

To 1 ml of homogenized 4% beads, add 200 μl of 20× PBS, and dissolve 250 μl of 6D4 (anti-human IgM antibody) for anti-RH2 and anti-RH5 (clones Diagast P3x255 13 G8 and P3GD C512, respectively) at a concentration of 1 mg/ml or 100 µl C5-1 (anti-human IgG antibody) for anti-KEL1 (clone Diagast 601) at a concentration of 3.5 mg/ml. The volume is adjusted to 4 ml with demineralized water. This solution is stirred by inversion overnight to ensure binding.

Excess active groups are blocked by suspending 150 μl of 1 M ethanolamine in a solution of antibody beads and stirring for 2 hours at room temperature. The AHG-bound beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). The bead pellet is resuspended with 2 ml of concentrated antibody plus 2 ml of phosphate buffer. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then washed twice with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

1.3. Preparation of the reaction device and detection method

The device shown in FIG. 8a is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 1.5 mm and a porosity of 7 to 12 microns, a drainage layer (cotton wool from Alan&co) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 1 μl of a surfactant solution (phosphate buffered saline supplemented with 3% Triton X-100, 0.26 M sucrose and 1% green dye). This device is dried for the first time for 1 hour at 37°C, 10% humidity. Then 5 μl of beads with bound antibody are applied as spots at a concentration of 0.125 to 4%. This device is dried a second time for 1 hour at 37°C, 10% humidity before use.

The detection procedure is initiated by introducing 10 μl of red blood cells diluted at a concentration of 5% in saline into each well. The reaction zone is washed with 100 μl of phosphate-buffered saline supplemented with 0.1% Tween-20 and 0.007% Triton X-100.

The device shown in FIG. 8b is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 μm, a drainage layer (NA7300PES from Subrenat) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 0.5% Triton X-100, 0.13 M sucrose and 0.5% green dye). This device is dried for the first time for 1 hour at 37°C, 10% humidity. Then 5 μl of beads with bound antibody at a concentration of 4% are applied as spots. This device is dried a second time for 1 hour at 37°C, 10% humidity before use.

The detection technique is initiated by introducing into each well 10 μl of red blood cells diluted at a concentration of 5% in physiological solution. The reaction zone is washed with 100 μl of phosphate-buffered saline supplemented with 0.1% Tween-20 and 0.007% Triton X-100.

1.4. results

The presence of a dark gray spot (red spot) in FIG. 8a indicates the formation of an immune complex and therefore a positive reaction. Sensitivities are maintained for bead concentrations varying from 4 to 0.125%, with decreasing intensity.

The presence of a dark gray spot (red spot) in FIG. 8b indicates the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction.

EXAMPLE 6: Type and Concentration of Surfactant Used to Make a Porous Membrane Hydrophilic

1. Anti-ABO01 binding

To 1 ml of homogenized 4% beads (9 μm aldehyde/sulfate latex from Thermo Fisher, 4% w/v), add 100 μl of 20× phosphate buffered saline, and dissolve 1 ml of affinity purified anti-ABO01 antibody (clone Diagast 25 21 B8). The volume is adjusted to 4 ml with demineralized water. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 2500 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

2. Preparation of the reaction device and detection method

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (cotton wool from Alan&co) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 1 µl of a surfactant solution listed below:

A. 1.5% Triton X-100

b. 10% polyoxyethylene

c. 50 mM nonyl-β-D-glucoside

10 μl of antibody-coupled beads are then spotted at a concentration of 3%. The detection technique is initiated by introducing into each well 50 μl of red blood cells diluted at a concentration of 1.5% in phosphate-buffered saline supplemented with 0.00625% Tween-20. The reaction zone is washed with 30 μl of phosphate-buffered saline supplemented with 0.2% Tween-20.

3. Results

In FIG. Figure 9 shows the resulting image in which the presence of dark gray spots (corresponding to the red spot in the color image) is observed to indicate the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction. Specificity is maintained no matter what surfactant is used.

EXAMPLE 7: Absorbent and drainage material used in the device of the invention

1. Absorbent material

1.1. Antibody binding

To 1 ml of homogenized 4% beads, add 200 µl of 20× phosphate-buffered saline, and dissolve 250 µl of 6D4 (anti-human IgM antibody, clone from Diagast). The volume is adjusted to 4 ml with demineralized water. This solution is stirred by inversion overnight to ensure binding.

Excess active groups are blocked by suspending 150 μl of 1 M ethanolamine in a solution of antibody beads and stirring for 2 hours at room temperature. The AHG-bound beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). The bead pellet is resuspended with 2 ml of concentrated anti-RH2 antibody (clone Diagast P3X255 13 G8) plus 2 ml of phosphate-buffered saline. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). Finally, the bead pellet is resuspended in preservation buffer to obtain the mass of the injected bead solution.

1.2. Preparation of the reaction device and detection method

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns is assembled, a drainage layer (NA7150 PES from Subrenat) and an absorbent layer (links from Mc Arlaid below).

The adsorbent materials used are the following:

T-499 or SCP-300 TCF (Fig. 10a)

T-099 or T-499 (Fig. 10b)

T-499 or T-183-5 (Fig. 10c)

T-499 or SCP-200-TCF (Fig. 10d)

This porous membrane is made hydrophilic using 1 μl of a surfactant solution (phosphate-buffered saline supplemented with 0.5% Triton X-100; 1% green dye). 10 μl of beads with bound antibody or negative control are then spotted at a concentration of 1%, and the device is dried for 15 min at 37°C, 20% humidity before immediate use or long-term storage.

The assay is initiated by diluting red blood cells at a concentration of 2.5% in chromasolcoombs (Diagast preparation) and 50 µl injected into each well, followed by two 30 µl washes with buffer (phosphate-buffered saline supplemented with 0.2% Tween 20).

1.3. results

From Fig. 10a, b, c and d, it is evident that any of the absorbent layers analyzed could guarantee the detection of a PH2-positive red blood cell (presence of a red spot visible as dark gray spots). Specificity is maintained; an unstained spot reflects a PH2-negative erythrocyte.

2. Drainage materials

2.1 Antibody binding

1 ml of concentrated anti-AB antibody (clone 152 D12 from Diagast) is dissolved in 1 ml of homogenized 4% beads. The volume is adjusted to 4 ml with phosphate-buffered saline. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). Finally, the bead pellet is resuspended in preservation buffer to obtain the mass of the injected bead solution.

2.2. Preparation of the reaction device and detection method

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (links below) and an absorbent layer (Cleanis) are assembled.

The drainage materials are the following:

cotton wool (from Alan & co), shown in Fig. 11a

NT 9750 HY (from Subrenat), shown in FIG. 11b,

NT 9610 HY (from Subrenat), shown in FIG. 11s

This porous membrane is made hydrophilic with 1 μl of surfactant solution (phosphate-buffered saline supplemented with 1% Triton X-100; 1% green dye). 10 μl of antibody-bound or negative control beads are then spotted at 1% concentration and the device is dried for 15 min at 37°C, 10% humidity before immediate use or long-term storage.

The assay is initiated by diluting red blood cells at a concentration of 2.5% in chromasolcoombs (Diagast formulation) and 50 µl injected into each well, followed by two 30 µl washes (phosphate buffered saline supplemented with 0.2% Tween 20).

2.3. results

In FIG. 11 shows that any of the drying layers could ensure the detection of a B-positive erythrocyte (presence of a red spot, visible in this Fig. as a dark gray spot). Specificity is maintained; an uncolored spot is obtained with red blood cells O.

EXAMPLE 8: Group Definition and Identification of a Mixed Field Population Using a Device of the Invention

1. Antibody binding

To 1 ml of homogenized 4% beads (9 μm latex aldehyde/sulfate from Thermo Fisher, 4% w/v), add 100 μl of 20× phosphate-buffered saline, and dissolve 2 ml of anti-ABO01 antibody (clone Diagast 91 13 D10). The volume is adjusted to 4 ml with demineralized water. This solution is stirred by inversion overnight to ensure binding.

Excess active groups are blocked by suspending 150 μl of 1 M ethanolamine in a solution of antibody beads and stirring for 2 hours at room temperature. The bound beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). Finally, the bead pellet is resuspended in preservation buffer to obtain the mass of the injected bead solution.

2. Preparation of the reaction device and identification procedure

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (cotton wool from Alan&co) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 1 μl of a surfactant solution (phosphate-buffered saline supplemented with 1% Triton X-100 and 1% green dye). Then 10 μl of beads with bound antibody at a concentration of 3% are applied as spots. Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting each well with 50 µl of red blood cells diluted at a concentration of 0.15% in Chromasolcoombs supplemented with 0.00625% Tween 20.

Mixed field populations A and B were prepared by mixing, respectively, RBCs A and O or B and O, previously diluted at 0.15% in Chromasolcoombs at the desired concentrations (30% RBC O with 70% A or B, or 50% RBC O with 50% A or B).

The first image is acquired before washes. The reaction zone is then washed twice with 30 μl of phosphate-buffered saline supplemented with 0.2% Tween-20. An image is acquired after each wash.

3. Results

As shown in FIG. 12a and c, before washing, all spots turn out to be dark gray (or red). Specificity is achieved after the second wash: the presence of a red spot (visible as a dark gray spot) indicates the formation of an immune complex and therefore a positive reaction, whereas the presence of a colorless spot reflects a negative reaction.

In FIG. 12b and d show that discrimination between natural and mixed field populations is satisfied after interpreting the intensity of the acquired images (the signal before and after two washes is subtracted).

EXAMPLE 9: Identification of weak RH1 antigens and advanced phenotyping with indirect binding of anti-erythrocyte antibodies to beads via anti-antibody affinity proteins

1. AGL protein binding

To 1 ml of homogenized 4% beads, add 50 μg of AGL protein dissolved in 3 ml of 1× PBS. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 2500 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

2. Preparation of the reaction device and identification procedure

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610HY) and an absorbent layer (Cleanis) are assembled.

This porous membrane is rendered hydrophilic using 1 μl of AGL-coupled bead surfactant solution (1.5% Triton X-100; phosphate-buffered saline supplemented with 0.5% bovine serum albumin; 0.25 M sucrose and 5.1% of balls). Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting 50 µl of reagent into each well (weak RH1 or extended phenotyping); add 50 μl of red blood cells diluted at a concentration of 1.5% in 1× PBS supplemented with 0.00625% Tween 20.

The first image is acquired before washes. The reaction zone is then washed with 30 μl of phosphate-buffered saline supplemented with 0.2% Tween-20. The image is obtained after washing.

3 Results

In FIG. Figure 13a shows the resulting image of weak RH1, in which the presence of dark gray spots (corresponding to the red spot in the color image) is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction.

In FIG. 13b shows the resulting enhanced phenotyping image, in which the presence of dark gray spots (corresponding to the red spot in the color image) is observed to indicate immune complex formation and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction.

EXAMPLE 10: Reverse blood typing using 6D4 antibody bound to beads

1. Reverse determination of blood groups

1.1. Antibody binding

To 1 ml of homogenized 4% beads (9 μm latex aldehyde/sulfate from Thermo Fisher, 4% w/v), add 3 ml of 1× phosphate-buffered saline, and dissolve 100 μg of CP6D4 antibody. This solution is stirred by inversion overnight to ensure binding.

Excess active groups are blocked by suspending 150 μl of 1 M ethanolamine in a solution of antibody beads and stirring for 2 hours at room temperature. The bound beads are then washed extensively 2 times with 3 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). Finally, the bead pellet is resuspended in preservation buffer to obtain the mass of the injected bead solution.

1.2. Preparation of the reaction device and identification procedure

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (Subrenat 9610 HY) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 1.5% Triton X-100; 0.5% green dye). 5 μl of beads with bound antibody are spotted at a concentration of 1%, and the device is dried for 15 minutes at 37°C, 10% humidity before immediate use or long-term storage.

The assay is initiated by incubating 30 μl of plasma and 20 μl of red blood cells (A1; B; A2; O) for 5 minutes. 25 µl is injected into each well, followed by 30 µl of wash buffer (phosphate-buffered saline supplemented with 0.2% Tween 20).

1.3. results

In FIG. 14 shows the presence of dark gray spots (corresponding to red spots), which indicate the capture of IgM-sensitized red blood cells and therefore a positive reverse blood typing reaction, while the presence of a colorless spot reflects a negative reaction (non-sensitized red blood cells).

EXAMPLE 11: DAT (Direct Coombs Test) Analysis Using the Apparatus of the Invention

1. Red blood cells pre-sensitized with antibodies

1.1. Antibody binding

Dissolve 3 mL of anti-human globulin antibody solution into 3 mL of homogenized 4% beads (9 μm latex aldehyde/sulfate from Thermo Fisher, 4% w/v). The AHG tested are SA6532 (rabbit polyclonal antibody directed against human IgG), C5-1 (clone Diagast, mouse IgG directed against human IgG) and 188 33 (clone Diagast, mouse IgM directed against human IgG). This solution is stirred by inversion for one hour to ensure binding.

The bead suspension is centrifuged for 5 min at 2500 g. Excess of active groups is blocked by resuspending the bead sediment in 6 ml of 50 mM ethanolamine solution, followed by stirring for 30 min. The bound beads are then washed extensively 2 times with 9 ml of preservation buffer (centrifugation for 5 min at 2500 g before and after washes). Finally, the bead pellet is resuspended in preservation buffer to obtain the mass of the injected bead solution.

1.2. Preparation of the reaction device and identification procedure

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (Subrenat 9610 HY) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 1.5% Triton X-100; 0.5% green dye). 5 μl of beads with bound antibody are spotted at a concentration of 3%, and the device is dried for 15 minutes at 37°C, 10% humidity before immediate use or long-term storage.

RBCs are presensitized in vitro with IgG antibodies against FY, KEL1KEL1, or RH1 to stimulate direct Coombs positive RBCs.

The assay is initiated by diluting red blood cells (Coombs test negative or positive) at a concentration of 0.25% in chromasolcoombs (Diagast preparation) supplemented with 0.00625% Tween 20. 50 µl is injected into each well, followed by 30 µl wash buffer (phosphate saline solution supplemented with 0.2% Tween 20).

2. Direct antiglobulin test with anti-IgG antibody and anti-C3d antibody

2.1. Antibody binding

To 1 ml of homogenized 4% beads (9 μm aldehyde/sulfate latex from Thermo Fisher, 4% w/v), add 3 ml of 1× phosphate buffered saline, and dissolve 100 μg of SA6532 or C7610 antibody. This solution is stirred by inversion overnight to ensure binding. The bound beads are then centrifuged for 5 min at 2500 g. Finally, the bead pellet is resuspended in preservation buffer to obtain the mass of the injected bead solution.

2.2. Preparation of the reaction device and identification procedure

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (Subrenat 9610 HY) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 1.5% Triton X-100; 0.5% green dye). 5 μl of beads with bound antibody are spotted at a concentration of 4%, and the device is dried for 15 minutes at 37°C, 10% humidity before immediate use or long-term storage.

The assay is initiated by diluting red blood cells (DAT negative or positive) at a concentration of 1.5% in phosphate-buffered saline supplemented with 0.00625% Tween 20. 50 µl is injected into each well, followed by 30 µl of wash buffer (phosphate-buffered saline). buffer solution supplemented with 0.2% Tween 20).

3. DAT via antibody affinity proteins

3.1. AGL protein binding

For 1 mL of homogenized 4% beads, dissolve 50 μg of AGL protein in 3 mL of PBS 1× and add. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 2500 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

3.2. Preparation of the reaction device and identification procedure

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610PH) and an absorbent layer (Cleanis) are assembled.

This porous membrane is rendered hydrophilic using 1 μl of AGL-bound bead surfactant solution (1.5% Triton X-100; phosphate-buffered saline supplemented with 0.5% bovine serum albumin; 0.25 M sucrose and 5.1% beads). Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by incubating 20 μl of reagent (DAT) in the well; add 50 μl of red blood cells diluted in the well at a concentration of 3% in PBS 1x, supplemented with 0.00625% Tween 20; add 20 µl of IAT solution in buffer. Transfer 120 µl of the mixture into the well of the cartridge.

The first image is acquired before washes. The reaction zone is then washed with 40 µl of IAT solution in buffer. The image is obtained after washing.

4. Results

In FIG. 15a shows the presence of dark gray spots (corresponding to red spots), which indicate the capture of IgG-sensitized red blood cells, and therefore a positive direct Coombs reaction, while the presence of a colorless spot reflects a negative reaction (unsensitized red blood cells).

In FIG. 15b shows the presence of dark gray spots (corresponding to red spots), which indicate the capture of sensitized IgG and/or C3D red blood cells, and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction (non-sensitized red blood cells).

In FIG. 15c shows the resulting image, in which the presence of dark gray spots (corresponding to red spots in the color image) is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction.

EXAMPLE 12: detection of antibodies specific for malaria antigen using the device of the invention

1. Antigen binding

For 1 ml of homogenized 4% beads, dissolve 100 μl of Mal003 antigen in 3 ml of PBS 1× and add. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 2500 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the injected mass of bead solution.

2. Antibody binding for detection

For 1 ml of homogenized 4% 1 μm beads, dissolve 200 μg of 6D4 antibody in 3 ml of PBS 1× and add. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 5000 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

3. Reaction device preparation and detection procedure with HRP

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610PH) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of beads with the bound Mal003 antigen at a concentration of 4% are applied in the form of spots. Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting 100 µl of sample into each well (kit ab178649). Add 100 μl of anti-IgG and/or anti-IgM HRP conjugate (kit ab178649). The first image is acquired before washes. The reaction zone is then washed with 30 μl of phosphate-buffered saline supplemented with 0.2% Tween-20. The reaction zone is then washed with 30 μl of detection buffer (TMB (tetramethylbenzidine) substrate (kit ab178649)). The image is obtained after 15 minutes.

4. Preparation of the reaction device and detection procedure with beads

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610PH) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of beads with the bound Mal003 antigen at a concentration of 4% are applied in the form of spots. Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting 100 µl of sample into each well (kit ab178649). Add 30 μl of 1 μm beads with 6D4 antibody at a concentration of 0.1%. The first image is acquired before washes. The reaction zone is then washed with 120 μl of phosphate-buffered saline supplemented with 0.2% Tween-20. The image is obtained after washing.

5. Preparation of the reaction device and detection procedure with colloidal gold nanoparticles

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610PH) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 5 μl of beads with the bound Mal003 antigen at a concentration of 4% are applied in the form of spots. Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting 50 µl of sample into each well (kit ab178649). Add 10 μl of a 60 nm gold conjugate with an antibody against human IgA, IgG, IgM (AC-60-14-10). The first image is acquired before washes. The reaction zone is then washed with 50 μl of phosphate-buffered saline supplemented with 0.2% Tween-20. The image is obtained after washing.

6. Results

In FIG. Figure 16a shows the resulting image in which the presence of dark gray spots is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and a gray intensity plot.

In FIG. Figure 16b shows the resulting image, in which the presence of dark gray spots is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and a gray intensity plot.

In FIG. 16c shows the resulting image in which the presence of dark gray spots is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and a gray intensity plot.

EXAMPLE 13: Detection of antibodies specific for T. pallidum antigens using the device of the invention with 1 μm red beads as a detection method

1. Antigen binding

For 1 ml of homogenized 4% beads, 100 μg of T. pallidum p15, p17, p47 antigens (30-AT76) are dissolved in 3 ml of PBS 1× and added. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 2500 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

2. Antibody binding for detection

For 1 ml of homogenized 4% 1 μm beads, dissolve 200 μg of AGL protein in 3 ml of PBS 1× and add. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 5000 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

3. Preparation of the reaction device and identification procedure

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610HY) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of beads with bound antigens p15, p17, p47 of T. pallidum are applied in the form of spots at a concentration of 4%. Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting 10 μl of sample (anti-Treponema pallidum antibody 20-TR89) into each well. Add 30 μl of 1 μm beads with AGL protein at a concentration of 0.1%. The first image is acquired before washes. The reaction zone is then washed with 120 μl of phosphate-buffered saline supplemented with 0.2% Tween-20. The image is obtained after washing.

4. Results

In FIG. 17 shows the resulting image in which the presence of dark gray spots is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and a gray intensity plot.

EXAMPLE 14: Detection of antibodies specific for hepatitis B virus antigen (HbsAg) using a device according to the invention with a detection method with 1 μm red beads

1. Antigen binding

For 1 ml of homogenized 4% beads, dissolve 100 μg of HBs antigen (30-AH15) in 3 ml of PBS 1× and add. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 2500 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

2. Antibody binding for detection

For 1 ml of 4% homogenized 1 μm beads, dissolve 200 μg of AGL protein in 3 ml of PBS 1× and add. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 5000 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

3. Preparation of the reaction device and identification procedure

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610HY) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of beads with bound HBs antigen at a concentration of 4% are applied in the form of spots. Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting 10 µl of sample (anti-HBsAg antibody 10-H05G) into each well. Add 30 μl of 1 μm beads with AGL protein at a concentration of 0.1%. The first image is acquired before washes. The reaction zone is then washed with 120 μl of phosphate-buffered saline supplemented with 0.2% Tween-20. The image is obtained after washing.

4. Results

In FIG. 18 shows the resulting image in which the presence of dark gray spots is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and a gray intensity plot.

EXAMPLE 15: detection of hepatitis B virus antigen using a device according to the invention

1. Antigen binding

For 1 ml of homogenized 4% beads, dissolve 100 μg of anti-HBs antibody (10-H05G) in 3 ml of PBS 1× and add. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 2500 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

2. Antibody binding for detection

For 1 ml of 4% homogenized 1 μm beads, dissolve 200 μg of anti-HBs antibody (10-H05H) in 3 ml of PBS 1× and add. This solution is stirred by inversion overnight to ensure binding.

The bound beads are then centrifuged for 5 min at 5000 g. Finally, the bead pellet is resuspended in preservation buffer (phosphate-buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to obtain the mass of the injected bead solution.

3. Preparation of reaction device and detection procedure using HRP method as detection method

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610HY) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of beads with bound antibody against the HBs antigen (10-H05G), at a concentration of 4%, are applied as spots. Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting 50 µl of sample into each well (Kit 1701-12). Add 50 μl of HBs-HRP antigen antibody conjugate (kit 1701-12). The first image is acquired before washes. The reaction zone is then washed with 100 μl of detection buffer (TMB substrate (kit 1701-12)). The image is obtained after 15 minutes.

4. Preparation of the reaction device and detection procedure using the bead method

This device is a molded plastic cassette in which a hydrophobic Porex HDPE membrane with a thickness of 0.6 mm and a porosity of 9 to 12 microns, a drainage layer (9610HY) and an absorbent layer (Cleanis) are assembled.

This porous membrane is made hydrophilic using 0.5 μl of a surfactant solution (phosphate-buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of beads with bound anti-HBs antibody (10-H05G) at a concentration of 4% are applied as spots. Dry this device for 15 minutes at 37°C, 20% humidity before use.

The detection procedure is initiated by injecting 10 µl of HBs antigen sample (kit 1701-12) into each well. Add 100 μl of 1 μm beads with anti-HBs antibody (10-H05H) at a concentration of 0.025%. The first image is acquired before washes. The reaction zone is then washed with 120 μl of phosphate-buffered saline supplemented with 0.2% Tween-20. The image is obtained after washing.

5. Results

In FIG. 19a shows the resulting image in which the presence of dark gray spots is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and a gray intensity plot.

In FIG. Figure 19b shows the resulting image in which the presence of dark gray spots is observed, indicating the formation of an immune complex and therefore a positive reaction, while the presence of a colorless spot reflects a negative reaction and a gray intensity plot.

Claims (48)

1. An in vitro diagnostic device for detecting and/or identifying an antigen and/or antibody from a sample of biological fluid, preferably from a blood sample or a sample of blood components, containing: - substrate and - a hydrophobic porous membrane located on said support, containing at least one hydrophilic reaction region designed to receive said sample, wherein the surface of this hydrophilic reaction region, which is smaller than the surface of the hydrophobic porous membrane, contains beads on the surface of which are fixed or adsorbed at least one antibody or antigen, wherein said beads have a size greater than the pore size of the porous membrane and avoid absorption of the fixed or adsorbed at least one antibody or antigen by the hydrophobic porous membrane. 2. The in vitro diagnostic device according to claim 1, wherein the antibody is selected from the group consisting of antibodies to red blood cells, antibodies to platelets, antibodies to viral antigens, antibodies to bacterial antigens and antibodies to parasitic antigens, preferably from antibodies to red blood cells. 3. The in vitro diagnostic device according to claim 1, wherein the antigen is selected from the group consisting of erythrocyte antigens, platelet antigens, viral antigens, bacterial antigens and parasitic antigens. 4. An in vitro diagnostic device according to claim 2 or 3, wherein the antibodies to viral antigens and the viral antigens are derived from hepatitis B virus or hepatitis C virus, preferably from hepatitis B virus. 5. The in vitro diagnostic device according to claim 2 or 3, wherein the antibodies to bacterial antigens and the bacterial antigens are from the genus Treponema, preferably Treponema pallidum. 6. An in vitro diagnostic device according to claim 2 or 3, wherein the antibodies to parasitic antigens and the parasitic antigens are from the genus Plasmodium, preferably Plasmodium falciparum. 7. In vitro diagnostic device according to any one of claims. 1-6, in which the specified antibody or antigen is directly or indirectly fixed or adsorbed on the surface of the bead. 8. In vitro diagnostic device according to any one of claims. 1-7, wherein said antibody or antigen is indirectly fixed to the surface of the bead by another antibody, preferably an IgM or IgG immunoglobulin. 9. In vitro diagnostic device according to any one of paragraphs. 1-8, in which the specified antibody is indirectly fixed to the surface of the bead using a ligand selected from proteins with affinity for antibodies, such as protein A, protein G or protein L. 10. In vitro diagnostic device according to any one of paragraphs. 1-9, wherein said bead surface contains at least one chemical group selected from aldehyde groups, chloromethyl group, NHS groups and carboxyl groups. 11. In vitro diagnostic device according to any one of paragraphs. 1-10, in which the average size of the beads is from 1 μm to 130 μm, preferably from 5 μm to 50 μm, and most preferably 9 μm. 12. In vitro diagnostic device according to any one of paragraphs. 1-11, in which the beads are used in a concentration of 1% to 10%, preferably 1% to 5%, more preferably 2% to 6% or 1% to 3%, even more preferably 3% solution, in which said beads are suspended, and wherein the applied volume of said solution is from 1 to 100 μl, preferably from 1 to 50 μl, more preferably from 5 to 50 μl and even more preferably from 1 to 10 μl or from 5 to 10 μl. 13. In vitro diagnostic device according to any one of paragraphs. 1-12, in which the thickness of the hydrophobic porous membrane is from 0.4 mm to 2 mm, preferably from 0.6 mm to 1.5 mm. 14. In vitro diagnostic device according to any one of paragraphs. 1-13, wherein the hydrophilic reaction region of the hydrophobic porous membrane is rendered hydrophilic by a surfactant, preferably used at a concentration of from 0.01 to 5% w/v, more preferably from 0.1% to 3 % w/v, even more preferably 0.5% to 2% w/v. solution, and wherein 0.1 to 5 μl, preferably 0.3 to 3 μl and more preferably 0.5 to 2 μl of said surfactant-containing solution is applied to the hydrophobic porous membrane. 15. In vitro diagnostic device according to any one of paragraphs. 1-14, wherein said device further comprises at least one layer located underneath the porous membrane, which is an absorbent layer. 16. In vitro diagnostic device according to any one of paragraphs. 1-15, which further comprises at least one more layer located between the hydrophobic porous membrane and the absorbent layer, said layer being made of a drainage material. 17. Use of the device in vitro according to any one of paragraphs. 1-16 for definition and/or identification: - at least one antigen selected from the group consisting of erythrocyte antigens, platelet antigens, viral antigens, bacterial antigens and parasitic antigens; - at least one antibody selected from the group consisting of anti-red blood cell antibodies, anti-platelet antibodies, anti-viral antibodies, anti-bacterial antibodies and anti-parasitic antibodies from a sample of biological fluids, preferably from a sample of blood or a sample of blood components. 18. A method for in vitro detection and/or identification of erythrocyte antigens, and/or in vivo sensitized erythrocytes, and/or platelet antigens from a blood sample or a sample of blood components, including the following steps: - adding a solution containing the specified sample to the reaction area of the in vitro device according to any one of claims. 1, 2 and 7-16, - reading before washing to control sample loading; - applying the washing solution to the reaction area and - determining the presence of specified antigens or specified blood cells by determining the presence of an antigen/antibody interaction if a red or pink spot appears in a given reaction region, or determining the absence of specified antigens or specified blood cells by determining the absence of an antigen/antibody reaction if a red or pink spot appears in a given reaction region colorless spot. 19. A method for in vitro detection and/or identification of antibodies to red blood cells from a blood sample or a sample of blood components, including the following steps: - incubation of the sample to be analyzed with a buffer and test erythrocytes of a known phenotype, - applying this mixture to the reaction area of the in vitro device according to any one of claims. 1, 3 and 7-16, - applying a washing solution to the reaction area and determining the presence of antibodies to red blood cells by determining the presence of an antibody/antigen reaction if a red or pink spot appears in a given reaction region, or determining the absence of said antibodies by determining the absence of an antibody/antigen reaction if a colorless spot appears in a reaction region. 20. A method for in vitro detection and/or identification of antibodies to platelets from a blood sample or a sample of blood components, including the following steps: - incubation of the sample to be analyzed with a buffer and test platelets of known antigenicity or incubation of recipient plasma or serum with donor platelets, - applying this mixture to the reaction area of the in vitro device according to any one of claims. 1, 3 and 7-16, - applying a washing solution to the reaction area and - determining the presence of antibodies to platelets by determining the presence of an antibody/antigen interaction if a red or pink spot appears in a given reaction area, or determining the absence of said antibodies by determining the absence of an antigen/antibody interaction if a colorless spot appears in a reaction area. 21. A method for in vitro detection and/or identification of antigens selected from the group containing viral antigens, bacterial antigens and parasitic antigens from a sample of biological fluid, preferably from a blood sample or a sample of blood components, comprising the following steps: - it is possible to incubate the sample to be analyzed with a buffer, - adding a solution containing the specified sample or a mixture of sample and buffer to the reaction area of the in vitro device according to any one of claims. 1, 2 and 4-16, - reading before washing to control sample loading is possible; - it is possible to apply a washing solution to the reaction area and - determining the presence of said antigens by determining the presence of an antigen/antibody interaction if a colored spot appears in a given reaction region, or determining the absence of said antigens by determining the absence of an antigen/antibody reaction if a colorless spot appears in a reaction region. 22. A method for in vitro detection and/or identification of antibodies selected from the group containing antiviral antibodies, antibacterial antibodies and antiparasitic antibodies from a biological sample, preferably from a blood sample or a sample of blood components, comprising the following steps: - it is possible to incubate the sample to be analyzed with a buffer, - adding a solution containing the specified sample or a mixture of sample and buffer to the reaction area of the in vitro device according to any one of claims. 1, 3-16, - reading before washing to control sample loading is possible, - it is possible to apply a washing solution to the reaction area and - determining the presence of said antibodies by determining the presence of an antigen/antibody interaction if a colored spot appears in a given reaction region, or determining the absence of said antibodies by determining the absence of an antigen/antibody reaction if a colorless spot appears in a reaction region.